Gene Therapy & Molecular Biology Volume 12 Issue A by Niko Boulikas

GENE THERAPY & MOLECULAR BIOLOGY FROM BASIC MECHANISMS TO CLINICAL APPLICATIONS

Volume 12 Number 1 June 2008 Published by Gene Therapy Press

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work under a government grant by NIH (or EU/Japan government grant). If this is you case, please consult the NIH Manuscript Submission System http://www.nihms.nih.gov/. Editorial Office Teni Boulikas, Ph.D./ Maria Koutoudi, B.A. , M.A. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria.koutoudi@cancer-therapy.org The free electronic access to articles published in "GTMB" to a big general audience, the attractive journal title, the speed of the reviewing process, the no-charges for page numbers or color figure reproduction, the 25 complimentary reprints, the rapid electronic publication, the embracing of many fields in cancer, the anticipated high quality in depth reviews and first rate research articles and most important, the eminent members of the Editorial Board being assembled are prognostic factors of a big success for the newly established journal.

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Table of contents

Gene Therapy and Molecular Biology Vol 12 Number 1, June 2008

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

1-6

Research Article

Advantages of intracerebral versus systemic administration of a DNAbased vaccine in treatment of an intracerebral tumor

Terry Lichtor, Roberta P Glick, Goro Osawa, Julian Hardman, Lisa A Feldman2

7-14

Research Article

Pro-apoptotic gene enhances the immunogenicity of glycoprotein B gene of herpes simplex virus-1

Masoud Parsania, Zuhair Muhammad Hassan, Taravat Bamdad1, Maryam Kheirandish, Mohammad Hassan Pouriayevali, Rohollah Dorostkar Sari, Mohammad Nabi Sarbolouki, Abbas Jamali1, Mehdi Mahdavi

15-24

Research Article

Validation of the comparative quantification method of real-time PCR analysis and a cautionary tale of housekeeping gene selection

Richard D. McCurdy, John J. McGrath, Alan Mackay-Sim

25-30

Research Article

Health economics of nutrigenomics in weight management

31-38

Research Article

Antigenic epitopes of viral polyprotein: an approach for fragment based peptide vaccines from Papaya Ringspot virus

Brian Meshkin, Thomas JH Chen, Amanda LC Chen, Thomas JH Prihoda, Hayley Morrisette, Eric R. Braverman, Seth H. Blum, Kimberly Cassel, Lonna Williams, Roger L. Waite, B. Willliam Downs7, Howard Tung, Patrick Rhoades, Kenneth Blum Virendra S Gomase, Karbhari V Kale

39-44

Research Article

Activity and Integrating Expression of Human Endostatin Produced by Pichia pastoris

Chongbi Li, Minghai Zhou, Hongbin Cui3, Zhan Wang

45-68

Review Article

The impact of biomics technology and DNA directed anti-obesity targeting of the brain reward circuitry

69-76

Research Article

Hepatitis Virus Protein XPhenylalanine Hydroxylase fusion

Amanda LC Chen, Kenneth Blum, Thomas JH Chen, Jeffrey Reinking Roger L Waite, Bernard W. Downs, Eric R. Braverman, Vanassa Arcuri, Mallory Kerner, Alison Notaro, Kimberly Cassel, Seth H. Blum8, Debasis Bagchi, Manashi Bagchi, Ariel Robarge, Gilbert Kaats, David E. Comings, Patrick Rhoades, Lonna Williams, Howard Tung Jennifer E. Embury, Susan Frost, Catherine E. Charron, Emilio Madrigal, Omaththage Perera, Amy E. Poirier,

proteins identified in PKU mice treated with AAV-WPRE vectors Transgenomics

Andreas G. Zori, Russ Carmical, Terence R. Flotte, Philip J. Laipis Virendra S Gomase, Somnath Tagore

Prediction of MHC binder for fragment based viral peptide vaccines from cabbage leaf curl virus Development of MHC class nonamers from Cowpea mosaic viral protein The Homeless youth and their exposure to Hepatitis B and Hepatitis C among in Tehran, Iran

Virendra S Gomase, Karbhari V Kale

77-82

Review Article

83-86

Research Article

87-94

Research Article

95-100

Research Article

101-110

Research Article

Immunoresistant human glioma cell clones selected with alloreactive cytotoxic T lymphocytes: downregulation of multiple proapoptotic factors

111-128

Review Article

Aptamers in oncology: a diagnostic perspective

Huma Khan, Sotiris Missailidis

129-140

Research Article

Dopamine D2 Receptor Taq A1 allele predicts treatment compliance of LG839 in a subset analysis of pilot study in the Netherlands

Kenneth Blum, Thomas JH Chen, Amanda LC Chen, Patrick Rhoades, Thomas J Prihoda, B. William Downs, Debasis Bagchi, Manashi Bagchi, Seth H. Blum, Lonna Williams, Eric R. Braverman, Mallory Kerner, Roger L Waite, Brien Quirk1, Lisa White1, Jeffrey Reinking

Virendra S Gomase, Karbhari V Kale Fatemeh Fallah, Abdollah Karimi, Gita Eslami, Sedighe Tabatabaii, Hossein Goudarzi, Raheleh Radmanesh Arezou Moradi, Mohammad Malekan, Masoomeh Navidinia, Akram Golnabi, Zari Gholinejad, Maryam Golshani German G. Gomez, Michelle J. Hickey, Richard Tritz, Carol A. Kruse

GENE THERAPY & MOLECULAR BIOLOGY Addresses of Members of the Editorial Board OPEN ACCESS www.gtmb.org Editor

Editor Assistants Boulikas, Teni, Ph.D. Chairman of the Board, Regulon, Inc. Mt View CA 94043 and Regulon AE, Athens, Greece

Koutoudi, Maria M.A. Vougiouka, Maria, B.Sc. Kruit, Adrian, Ph.D. Bellimezi, M., Ph.D Katsoupi J, Mph, Tsogas I., Ph.D, Magkos, A., Ph.D, Christofis Petros., Ph.D, Leto Tziveleka., Ph.D

Associate Editors Missailidis, Sotiris, DPhil (York) Lecturer in Chemistry and Analytical Sciences, The Open University, UK

Roberts, Michael, Ph.D., Regulon A.E., Athens Greece

Berezney, Ronald, Ph.D., State University of New York at Buffalo, USA

Rossi, John, Ph.D., Beckman Research Institute of the City of Hope, USA

Crooke, Stanley, M.D., Ph.D., ISIS Pharmaceuticals, Inc, USA

Shen, James, Ph.D., Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China & University of California at Davis, USA.

Gronemeyer, Hinrich, Ph.D. I.N.S.E.R.M., IGBMC, France

Webb, David, Ph.D., Celgene Corporation, USA

Aguilar-Cordova, Estuardo, Ph.D., AdvantaGene, Inc., USA

Editorial Board Members Akporiaye, Emmanuel, Ph.D., Arizona Cancer Center, USA

Baldwin, H. Scott, M.D Vanderbilt University Medical Center, USA

Anson, Donald S., Ph.D., Women's and Children's Hospital, Australia

Barranger, John, MD, Ph.D., University of Pittsburgh, USA

Ariga, Hiroyoshi, Ph.D., Hokkaido University, Japan

Black, Keith L. M.D., Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, USA

Blum, Kenneth, Ph.D., Wake Forest University School of Medicine, USA

Eckstein, Jens W., Ph.D., Akikoa Pharmaceuticals Inc, USA

Bode, Jürgen, Gesellschaft für Biotechnologische Forschung m.b.H., Germany

Fisher, Paul A. Ph.D., State University of New York, USA

Bohn, Martha C., Ph.D., The Feinberg School of Medicine, Northwestern University, USA

Georgiev, Georgii, Ph.D., Russian Academy of Sciences, USA

Bresnick, Emery, Ph.D., University of Wisconsin Medical School, USA

Getzenberg, Robert, Ph.D., Institute Shadyside Medical Center, USA

Caiafa, Paola, Ph.D., Università di Roma “La Sapienza”, Italy

Ghosh, Sankar Ph.D., Yale University School of Medicine, USA

Cheng, Seng H. Ph.D., Genzyme Corporation, USA

Gojobori, Takashi, Ph.D., Center for Information Biology, National Institute of Genetics, Japan

Cole, David J. M.D., Medical University of South Carolina, USA

Harris David T., Ph.D., Cord Blood Bank, University of Arizona, USA

Crooke, Stanley, M.D., Ph.D. ISIS Pharmaceuticals, Inc. USA

Heldin, Paraskevi Ph.D., Uppsala Universitet, Sweden

Davie, James R, Ph.D., Manitoba Institute of Cell Biology, USA

Hesdorffer, Charles S., M.D., Columbia University, USA

DePamphilis, Melvin L, Ph.D., National Institute of Child Health and Human, National Institutes of Health, USA

Hoekstra, Merl F, Ph.D., Epoch Biosciences, Inc., USA

Hung, Mien-Chie, Ph.D., The University of Texas, USA

Kuroki, Masahide, M.D., Ph.D., Fukuoka University School of Medicine, Japan

Johnston, Brian, Ph.D., Somagenics, Inc, USA

Lai, Mei T. Ph.D., Lilly Research Laboratories USA

Jolly, Douglas J, Ph.D., Advantagene, Inc.,USA

Latchman, David S., PhD, Dsc, MRCPath

Joshi, Sadhna, Ph.D., D.Sc., University of Toronto Canada

Lavin, Martin F, Ph.D., The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, Australia

Kiyama, Ryoiti, Ph.D., National Institute of Bioscience and HumanTechnology, Japan

Lebkowski, Jane S., Ph.D., GERON Corporation, USA

Kotoku Kurachi, Ph.D., University of Michigan Medical School, USA

Li, Liangping Ph.D., MaxDelbr端ck-Center for Molecular Medicine, Germany

Kottaridis, Stavros D., Ph.D. Regulon Inc. USA

Lu, Yi, Ph.D., University of Tennessee Health Science Center, USA

Krawetz, Stephen A., Ph.D., Wayne State University School of Medicine. USA

Lundstrom Kenneth, Ph.D., Bioxtal/Regulon, Inc. Switzerland

University of London, UK

Kruse, Carol A., Ph.D., Sidney Kimmel Cancer Center. USA

MacDougald, Ormond A, Ph.D., University of Michigan Medical School, USA

Kuo, Tien, Ph.D., The University of Texas M. D. Anderson Cancer USA

Malone, Robert W., M.D., Aeras Global TB Vaccine Foundation, USA

Mirkin, Sergei, M. Ph.D., University of Illinois at Chicago, USA

Royer, Hans-Dieter, M.D., (CAESAR), Germany

Noteborn, Mathieu, Ph.D., Leiden University, The Netherlands

Rubin, Joseph, M.D., Mayo Medical School Mayo Clinic, USA

Paleos, Constantinos M., Ph.D. Institute of Physical Chemistry Demokritos. Greece

Saenko Evgueni L., Ph.D., University of Maryland School of Medicine Center for Vascular and Inflammatory Diseases, USA

Pomerantz, Roger, J., M.D., Tibotec, Inc., USA

Santoro, M. Gabriella, Ph.D., University of Rome Tor Vergata, Italy

Raizada, Mohan K., Ph.D., University of Florida, USA

Salmons, Brian, Ph.D., (FSGBiotechnologie GmbH), Austria

Razin, Sergey, Ph.D., Institute of Gene Biology Russian Academy of Sciences, USA

Sharrocks, Andrew, D., Ph.D., University of Manchester, UK

Robbins, Paul, D, Ph.D., University of Pittsburgh, USA

Smythe Roy W., M.D., Texas A&M University Health Sciences Center, USA

Rosenblatt, Joseph, D., M.D, University of Miami School of Medicine, USA

Srivastava, Arun Ph.D., University of Florida College of Medicine, USA

Rosner, Marsha, R., Ph.D., Ben May Institute for Cancer Research, University of Chicago, USA

Steiner, Mitchell, M.D., University of Tennessee, USA

Tainsky, Michael A., Ph.D., Karmanos Cancer Institute, Wayne State University, USA

White, Robert, J., University of Glasgow, UK

Taira, Kazunari, Ph.D., The University of Tokyo, Japan

White-Scharf, Mary, Ph.D., Biotransplant, Inc., USA

Thierry, Alain, Ph.D., National Cancer Institute, National Institutes of Health, France

Wiginton, Dan, A., Ph.D., Children's Hospital Research Foundation, CHRF , USA

Trifonov, Edward, N. Ph.D., University of Haifa, Israel

Yung, Alfred, M.D., University of Texas, USA

Van Dyke, Michael, W., Ph.D., The University of Texas M. D. Anderson Cancer Center, USA

Zannis-Hadjopoulos, Maria Ph.D., McGill Cancer Centre, Canada

Vournakis, John N., Ph.D. Medical University of South Carolina, USA

Zorbas, Haralabos, Ph.D., BioM AG Team, Germany

Chi-Un Pae, MD, PhD, Associate Professor, Department of Psychiatry The Catholic Universoty of Korea College of Medicine

Associate Board Members Falasca, Marco, M.D., University College London, UK

Hiroki, Maruyama, M.D., Ph.D., Niigata University Graduate School of Medical and Dental Sciences, Japan

Gao, Shou-Jiang, Ph.D., The University of Texas Health Science Center at San Antonio, USA

Kazunori, Aoki, M.D., Ph.D., National Cancer Center Research Institute, Japan

Gibson, Spencer Bruce, Ph.D., University of Manitoba, USA

Rigoutsos, Isidore, Ph.D., Thomas J. Watson Research Center, USA

Gu, Baohua, Ph.D., The Jefferson Center, USA

Priya, Aggarwal Ph.D., University of Pennsylvania

Morris, Kevin Vance, Assistant Professor, The Scripps Research Institute, La Jolla, CA

W. Todd Penberthy, PH.D.,

Romano, Gaetano Ph.D. Research Associate Professor; Temple University, Philadelphia, U.S.A.

Yuefei Yu Ph.D. Texas Tech University Health Science Center. Research Scientist. Head of the research group.

Assistant Professor, Department of Molecular Genetics,Biochemistry, and Microbiology,

Gene Therapy and Molecular Biology Vol 12, page 141 Gene Ther Mol Biol Vol 12, 141-146, 2008

Vaccinomics Review Article

Virendra S Gomase1,2,*, Somnath Tagore1 1

Department of Bioinformatics, Padmashree Dr. D.Y. Patil University, CBD Belapur, Navi Mumbai, 400614, India Department of Computer Science and Information Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431004 (MS), India 2

__________________________________________________________________________________ *Correspondence: Virendra S. Gomase, Department of Bioinformatics, Padmashree Dr. D.Y. Patil University, Plot No-50, Sector-15, CBD Belapur, Navi Mumbai, 400614, India; Tel- +91-22-27563600; Fax- +91-39286176; Mobile- +91-9226960668; Mailvirusgene1@yahoo.co.in Key words: Attenuation, Influenzae, Genetic Engineering, Toxoid, Vaccine Abbreviations: cysteine heart agar supplemented with 5% rabbit blood, (CHAB); diphtheria-pertussis-tetanus, (DPT); EGF receptor, (EGFR); Epidermal growth factor, (EGF); gas chromatography, (GC); glioblastoma multiforme, (GBM); Global Alliance on Vaccines, Immunization, (GAVI); hemoglobin, (Hb); high performance liquid chromatography, (HPLC); human leukocyte antigen, (HLA); Lipid transfer proteins, (LTPs); live vaccine strain, (LVS); Mass spectrometry, (MS); Meningitis Vaccine Project, (MVP); polymerase chain reaction, (PCR); Porcine Reproductive and Respiratory Syndrome Virus, (PRRSV); principal component analysis, (PCA); soft independent modeling of class analogy, (SIMCA); World Health Organization, (WHO)

Received: 12 June 2008; Revised:24 June 2008 Accepted: 25 June 2008; electronically published: August 2008

Summary Vaccinomics is the branch of omics, which deals with vaccine analysis; vaccine itself is used for boosting the immunity to diseases. Most of the vaccine reactions appear to be more common than vaccine-preventable diseases. Vaccinomics encompasses the fields of immunogenetics and immunogenomics as applied to understanding the mechanisms of heterogeneity in immune responses to vaccines and new drug targets. Recent advances in the fields of immunology, vaccines, genomics, proteomics and Human Genome Project allowed to discover and developed new vaccines.

composed of only the surface proteins of the virus. Fifthconjugate, linking the outer coats of certain bacteria that are poorly immunogenic to proteins, the immune system is able recognize the polysaccharide. E.g. vaccine developed against Haemophilus influenzae type B. Six- recombinant vector, combining physiology of one microbe and DNA of the other, immunity is created against diseases that have complex infection processes (Poland 2007; Smith et al, 2007).

I. Introduction The branch of omics, which deals with the vaccine analysis and development, is termed as Vaccinomics. A vaccine is used for boosting immunity to a disease. The term is derived from Edward Jenner's use of cowpox, as â&#x20AC;&#x2DC;vaccaâ&#x20AC;&#x2122; in Latin means cow. When administered to humans, it provided the protection against smallpox. The process of distributing and administrating vaccines is known as vaccination. Vaccines can be categorized into many types. One- those containing killed microorganisms i.e. previously virulent micro-organisms, which were killed with chemicals or heat. E.g. vaccines developed against flu and hepatitis A. Second- those containing live or attenuated microorganisms i.e. live micro-organisms that cultivated under conditions which can disable their virulent properties. E.g. vaccines developed against measles and mumps. Third- toxoids i.e. inactivated toxic compounds in cases where these cause illness. E.g. vaccines developed against tetanus. Fourth- subunit i.e. fragment of a micro-organism is used for creating an immune response. E.g. subunit vaccine against HBV is

II. History of Vaccinomics Vaccinomics is one of those branches of science that has contributed most to the relief of human misery and the spectacular increase in life expectancy. It has one of the only sciences that eradicated infectious diseases, like smallpox, which is responsible for 8-20% of all deaths in several countries in paste era. Some of the other disabling and lethal diseases that targeted for eradication are poliomyelitis and measles. According to global survey, it is estimated that vaccination saves the lives of 3 million children a year. The success of vaccines in controlling and 141

Gomase and Tagore: Vaccinomics eliminating diseases has the cause of a revival of the antivaccination movement, especially in developing countries. It has found that, many common infectious diseases such as diphtheria, tetanus, polio, measles and mumps need vaccination and which are very necessarily. Most of the vaccine reactions appear to be more common than vaccine-preventable diseases. Also, there are several developments, in the last two decades, which can essentially hampered vaccine usage and development in medicine. This has led to some erroneous conception that vaccines are expensive. One of the favorable trends for vaccinomics has fueled by recent major developments in the sciences of immunology, molecular biology, genomics, proteomics as well as computers. This has promised a bright future for prevention, not only of acute infectious diseases, but also treatment of conditions like chronic infections, allergy as well as cancer. Now-a-days, vaccines are made more user-friendly by the development of combined vaccines and less painful than the traditional syringe and needle. Some new initiatives, viz., Global Alliance on Vaccines, Immunization (GAVI), which are gathering new sources of funding for vaccination and predicted to be more beneficial for vaccinomics. Purification of microbial elements, genetic engineering allow direct creation of attenuated mutants, expression of vaccine proteins in live vectors, purification and even synthesis of microbial antigens through manipulation of DNA, RNA and proteins. Both noninfectious and infectious diseases are now within the realm of vaccinomics (Andre 2003; Plotkin 2005).

means that, once a critical percentage of the population vaccinated, the entire population is protected. The new meningitis vaccine given preventively to 1- to 29-yearolds and should provide protection for at least ten years. Through collaboration with Serum Institute of India Limited, MVP has helped limit the initial price of the vaccine to just 40 cents per dose (Ovsyannikova et al, 2006; Juhn et al, 2007).

IV. Vaccinomics strategies Peptide-based vaccine development is one of the most important strategies for developing new vaccines against pathogens. A variety of proteins giving rise too naturally process pathogen-derived antigenic peptides, representing B-cell and T-cell epitopes characterized. Numerous candidate vaccines consisting of synthetic peptides designed and evaluated. Mass spectrometry applied based on the isolation and identification of pathogen-derived peptides from the human leukocyte antigen (HLA) molecules is one of the major focuses of peptide-based vaccine development (Ovsyannikova et al, 2001). For conjugate vaccine, it has observed that Streptococcus pneumoniae is also one of the most common causes of invasive bacterial infections in children including bacteremia and meningitis. It is found that children less than two years of age suffer an increased incidence of invasive pneumococcal disease but fail to respond to the 23-valent polysaccharide vaccine because of the immaturity of the T-cell independent immune function. Also, covalently conjugating the polysaccharide antigen to a carrier protein improves the immune response by permitting the host to utilize a T-cell dependent immune response that is adequately mature in children less than two years of age. Immunogenicity studies of the currently licensed heptavalent conjugated polysaccharide vaccine demonstrated that infants vaccinated with three doses 2 months apart at 2, 4, and 6 months of age successfully developed antibodies to all 7 serotypes; booster doses at 12-15 months demonstrated an amnestic response for each serotype. An efficacy trial involving nearly 38,000 subjects demonstrated the vaccine's effectiveness in healthy children against invasive pneumococcal disease as well as against pneumonia and otitis media. Infants may receive the first dose as early as 6 weeks of age. The vaccine is also indicated for children 24 to 59 months of age who are at high risk for pneumococcal infection (Jacobson et al, 2002).

III. The Vaccine Project In annual dry season at sub-Saharan Africa, between December and June, Meningococcal meningitis rates increased enormously. Cough and sneeze, dry air and dusty winds damage their mucous membranes, allowing bacteria to invade and the deadly infection travels rapidly from one person to the next. About 450 million people in 21 countries live at risk. An international organization called PATH and the World Health Organization (WHO) determined to eliminate the threat of meningitis epidemics through a partnership called the Meningitis Vaccine Project (MVP). The partnership included development, testing, licensure, and widespread introduction of a vaccine that will not just halt outbreaks, but prevent them. Meningococcal meningitis is bacterial infection of the fluid surrounding the brain and spinal cord. The disease develops quickly, is highly contagious, and kills about one in ten people who get it. The antibiotics that can cure meningococcal meningitis are often unavailable in the poor countries that are most affected by the disease. Also, most of the current vaccines that exist are ‘polysaccharide’ vaccines. But, these have their limitations. They don’t work in children under two and thus leave the most vulnerable unprotected. And even for older children and adults, immunity lasts only two or three years. A new ‘conjugate’ vaccine targeted group A meningococcal meningitis, which caused most epidemics in Africa. Conjugate vaccines are shown to more immunogenic than polysaccharide vaccines, and they can give to infants. In addition, they shown to induce herd immunity, which

V. Vaccinomics Technology A. Analytical technologies: Separation techniques 1. Gas chromatography (GC) Capillary gas chromatography (GC) with flameionization detection for determining the cellular fatty acid profiles of Francisella tularensis. F. tularensis, the live vaccine strain (LVS) derived from holarctica and a novicida strain Utah 112 (U112), were used for comparing the extracted fatty acid methyl esters (FAMEs). A data set for the 2 subspecies was prepared using fatty acid profiles of bacteria grown on 2 types of media, Mueller-Hinton 142

Gene Therapy and Molecular Biology Vol 12, page 143 and cysteine heart agar supplemented with 5% rabbit blood (CHAB), and harvested at various time intervals (Day 1 through Day 4) with replicates prepared on different days. A total of 204 samples were analyzed. The results showed that these fatty acid quantitative profiles were unique for each of the subspecies and could be used as a fingerprint for the organism. Data analysis and determination of clustering were performed by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA). Also, both PCA and SIMCA showed clear separation of the LVS and U112 strain and would be useful for prediction of unknowns (Whittaker et al, 2007). It is found that Salmonella spp. infections transmitted by contaminated poultry and eggs represent a major global health burden. Enteritidis is one of the leading causes of human salmonellosis worldwide. The cell surface antigens of Salmonella Enteritidis play an important role in the host-pathogen interactions and as such represent potential candidates for subunit-vaccine development. Immunogenicity and protection studies against lethal Salmonella Enteritidis challenge are performed in BALB/c mice. Increased survival is observed in vaccinated mice as compared to a control group. The potential for mucosal vaccination suggests that HEnanoparticles may represent an important alternative to the conventional attenuated vaccines against Salmonella enteritidis (Ochoa et al, 2007).

IgG and AQP-4-autoantibodies (Beyer et al, 2007). Researchers investigated the safety and feasibility of autologous formalin-fixed tumor vaccines (AFTV) and the clinical responses to these vaccines by glioblastoma multiforme (GBM) patients. AFTV are prepared from formalin-fixed and paraffin-embedded tumor tissue obtained upon surgery and premixed with original adjuvant materials. The patients are given three five-site intradermal inoculations at weekly intervals. A delayedtype hypersensitivity test is performed before and after each vaccination. In addition, the tumor tissues are subjected to immunohistochemical analysis to determine whether MIB-1, p53, and major histocompatibility complex (MHC) class-I complex expression could predict the response to the treatment. Results show AFTV is safe and feasible, and could significantly improve the outcome of GBM (Ishikawa et al, 2007).

2. Mass spectrometry (MS) DNA-mediated immunization developed as an approach for prevention and treatment of various infectious diseases, viz., Hepatitis-B. An integrated multiple systems biology approach is undertaken for defining sets of serum protein and metabolite biomarkers that employed to determine the efficacy and safety of DNA vaccines on mice immunized with DNA vaccine, recombinant protein, plasmid vector, and phosphatebuffered solution. Their sera are analyzed by twodimensional electrophoresis and HPLC coupled with timeof-flight mass spectrometry. The results indicated that DNA vaccine stimulated hepatic sphingolipid synthesis, which may altered the structure of circulating lipoproteins and promoted atherogenesis (Yang et al, 2008). Functions of fungal wall glycoproteins include maintenance of cell wall integrity, homotypic and heterotypic adhesion, protein degradation and coping with oxidative stress. Viral transcriptome studies indicated that the expression levels of most cell wall glycoproteins could vary widely and were tightly controlled. However, owing to the complex and variable glycosylation, fungal wall glycoproteins were found to be difficult for analyzing using traditional proteomics approaches. Development in mass spectrometry-based proteomics is enabled rapid and sensitive identification and quantitation of fungal wall glycoproteins (Yin et al, 2008).

2. High performance liquid chromatography (HPLC) A fusion hemoglobin (Hb) results from a crossover between the (A)gamma- and beta-globin genes, is accompanied by an increased level of fetal Hb in adult life. They used cation exchange high performance liquid chromatography (HPLC) for identification of Hb F Kenya and of a polymerase chain reaction (PCR) method for confirmatory diagnosis. Data was taken from 584 children and 406 adults who were screened for eligibility for malaria vaccine trials at Kombewa, Western Kenya. They proposed that in the population studied, cation exchange HPLC was used and it was seen that there was an elevation of Hb F (>9.0%) and elevation of Hb A(2) (>9.2%), suggesting the presence of Hb Kenya (Kifude et al, 2007).

B. Analytical techniques

technologies:

Detection

C. Vaccinomics in practice Due to the increasing epidemiological data it is considered that norovirus is an important cause of acute gastroenteritis. Also, norovirus gastroenteritis is very difficult to control due to lack of a suitable antiviral agent or a vaccine. Despite the difficulty in cultivating noroviruses, significant advances in understanding the genomic structure, individual viral proteins, RNA replication strategy and virus-host interaction of the is made. These advancements provide new strategies in the development of antiviral agents against norovirus, including the inhibition of viral attachment to host cells through carbohydrate receptors, inhibition of viral protease and polymerase functions, and interference in viral replication (Tan et al, 2008). One of the causative agents

1. Magnetic resonance imaging (MRI) Recent clinical and laboratory findings are revealed that neuromyelitis optica (NMO) is a humorally mediated, autoimmune disorder. Scientists reported on a patient who suffered a first episode of transverse myelitis at the age of 6 months following diphtheria-pertussis-tetanus (DPT) vaccination. Fifteen years later, the further disease course revealed typical NMO meeting all diagnostic criteria. This development pointed to a broad clinical and temporal heterogeneity of NMO, with ADEM probably occurring in the context of a shared autoimmune diathesis. The findings challenged the pathogenic relevance of NMO-IgG and indicated a varying diagnostic value of testing for NMO-

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Gomase and Tagore: Vaccinomics of porcine high fever syndrome in China is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). This is also capable of genetic and antigenic mutations at a high frequency. An infectious cDNA clone of an attenuated strain of Type II PRRSV is generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. Based on the nucleotide sequencing results, the full-length cDNA clone is assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5' terminus of genome. To discern the rescued viruses from that of parental virus, a Mlu I restriction site is engineered into ORF5 coding region. The rescued viruses from the fulllength cDNA clones displayed the same virological and molecular properties. After this study, a valuable tool for development of chimeric PRRSV as vaccine candidate offering cross-protection to various genetically diversified PRRSV strains is proposed (Yuan et al, 2008).

D. Technology development Vaccinomics and approaches

vaccine preparations, significant protection is achieved in experimental animals against listeriosis. Being a robust bacterium capable of eliciting all aspects of cell-mediated immunity, L. monocytogenes is the potential to become an ideal vector for vaccine delivery against other infective agents (Liu 2006).

VI. Applications of Vaccinomics Vaccinomics is one of the peer branches of omics providing research in vaccine development, in order to provide best protection for children. Vaccines are developed against many diseases like hepatitis A, hepatitis B, polio, mumps, measles, rubella, diphtheria, pertussis, tetanus, HiB, chicken pox, rotavirus, influenza, meningococcal disease and pneumonia. It is noticed that vaccines do not guarantee complete protection from a disease. Sometimes this is because the host's immune system simply doesn't respond adequately or at all. This may be due to a lowered immunity in general (diabetes, steroid use, HIV infection) or because the host's immune system does not have a B-cell capable of generating antibodies to that antigen. Adjuvants used for boosting immune response. Most often aluminium adjuvants are used, but adjuvants like squalene are also used in some vaccines and more vaccines with squalene and phosphate adjuvants are tested. Vaccinomics is one of the pioneering fields for providing new ideas for vaccine development. This provided new research in this area, with development of new vaccines against various diseases such as meningitis. This is particularly useful in developing countries where large number of people suffer from theses disease (Assarsson et al, 2008).

1. Neuroimaging Structural and functional brain imaging are used recently for assisting the diagnosis of dementia for the increasing numbers of people with cognitive decline as they age. Structural imaging (CT or MRI) are used to identify space-occupying lesions and stroke. Functional methods, such as PET scanning of glucose metabolism, used to differentiate Alzheimer's disease from frontotemporal dementia. New neuroimaging methods that are currently developed and measure specific neurotransmitter systems, amyloid plaque and tau tangle concentrations, and neuronal integrity and connectivity. Successful codevelopment of neuroimaging surrogate markers and preventive treatments might eventually lead to brain-check scans for determining risk of cognitive decline (Small et al, 2008).

VII. Current Research Epidermal growth factor (EGF) might be a suitable immunotherapeutic target in non-small-cell lung cancer (NSCLC). Around eighty NSCLC patients are treated with first-line chemotherapy that is randomized to receive the EGF vaccine or supportive care. EGF concentration in sera, anti-EGF antibodies and their capacity to inhibit the binding between EGF/EGF receptor (EGFR), and the EGFR phosphorylation are measured. It is found that 78 % of vaccinated patients developed a good antibody response, whereas none of the controls did. In good antibody-responder patients, self EGF in sera is significantly reduced. In 58% of vaccinated patients, the post-immune sera inhibited EGF/EGFR binding; in the control group, no inhibition occurred. Post-immune sera inhibited the EGFR phosphorylation whereas sera from control patients did not have this capacity. It is confirmed that immunization with the EGF vaccine induced neutralizing anti-EGF antibodies were capable of inhibiting EGFR phosphorylation (Garcia et al, 2008). Cancer epitope landscape more importance due to the finding that individual cancers contain many mutant genes not present in normal. In silico-based epitope prediction algorithms and high throughput post hoc analysis to identify candidate tumor antigens. Also, analysis of 1,152 peptides containing missense mutations previously identified in breast and colorectal cancer revealed that individual cancers accumulate on average approximately

2. Mimotope mapping To map IgE mimotopes on the structure of Pru p 3 a Lipid transfer proteins (LTPs) i.e. major allergen of peach, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms and used for screening of a random dodecapeptide phage display library. Three-dimensional modeling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surfaces. Mimotopes are regularly used for studying the interaction between allergens and IgE. This is used for accelerating the process of designing new vaccines and new immunotherapy strategies (Chua et al, 2003).

3. Anti-infective vaccine strategies Human food-borne infections caused by Listeria monocytogenes, a Gram-positive intracellular bacterium and found that sublethal level of L. monocytogenes generates enduring immunity. Through use of killed, attenuated, naturally avirulent, subcellular and DNA

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Gene Therapy and Molecular Biology Vol 12, page 145 (CMV) coat protein. Asia Pacific Journal of Molecular Biology and Biotechnology, 11(2), 93-100. Garcia B, Neninger E, de la Torre A, Leonard I, Martinez R, Viada C, Gonzalez G, Mazorra Z, Lage A, Crombet T (2008) Effective inhibition of the epidermal growth factor/epidermal growth factor receptor binding by anti-epidermal growth factor antibodies is related to better survival in advanced non-small-cell lung cancer patients treated with the epidermal growth factor cancer vaccine. Clin Cancer Res 14, 840-846. Ishikawa E, Tsuboi K, Yamamoto T, Muroi A, Takano S, Enomoto T, Matsumura A, Ohno T (2007) Clinical trial of autologous formalin-fixed tumor vaccine for glioblastoma multiforme patients. Cancer Sci 98, 1226-1233. Jacobson RM, Poland GA (2002) The pneumococcal conjugate vaccine. Minerva Pediatr 54, 295-303. Juhn YJ, Kita H, Lee LA, Smith RW, Bagniewski SM, Weaver AL, Pankratz VS, Jacobson RM, Poland GA (2007) Childhood asthma and human leukocyte antigen type. Tissue Antigens 69, 38-46. Kifude CM, Polhemus ME, Heppner DG, Withers MR, Ogutu BR. Waitumbi JN (2007) Hb Kenya among Luo adults and young children in malaria holoendemic Western Kenya: screened by high performance liquid chromatography and confirmed by polymerase chain reaction. Hemoglobin 31, 401-408. Liu D (2006) Listeria-based anti-infective vaccine strategies. Recent Patents Anti-Infect Drug Disc 1, 281-290. Ochoa J, Irache JM, Tamayo I, Walz A, DelVecchio VG, Gamazo C (2007) Protective immunity of biodegradable nanoparticle-based vaccine against an experimental challenge with Salmonella Enteritidis in mice. Vaccine 25, 4410-4419. Ofstead CL, Tucker SJ, Beebe TJ, Poland GA (2008) Influenza Vaccination among Registered Nurses: Information Receipt, Knowledge, and Decision-Making at an Institution With a Multifaceted Educational Program. Influenza vaccination among registered nurses: information receipt, knowledge, and decision-making at an institution with a multifaceted educational program. Infect. Control Hosp Epidemiol 29, 99-106. Ovsyannikova IG, Dhiman N, Jacobson RM, Poland GA (2006) Human leukocyte antigen polymorphisms: variable humoral immune responses to viral vaccines. Expert Rev Vaccines 5, 33-43. Ovsyannikova IG, Johnson KL, Bergen HR Poland GA (2001) Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles. Clin Pharmacol Ther 19, 2692- 2700. Plotkin SA (2005) Vaccines: past, present and future. Nat Med 11(4 Suppl), S5-S11. Poland GA (2007) Pharmacology, vaccinomics, and the second golden age of vaccinolog. Clin Pharmacol Ther 82, 623626. Segal NH, Parsons DW, Peggs KS, Velculescu V, Kinzler KW, Vogelstein B, Allison JP (2008) Epitope landscape in breast and colorectal cancer. Cancer Res 68, 889-892. Small GW, Bookheimer SY, Thompson PM, Cole GM, Huang SC, Kepe V, Barrio JR (2008) Current and future uses of neuroimaging for cognitively impaired patients. Lancet Neurol 7, 161-172. Smith DI, Poland GA, Ovsyannikova IG, Jacobson RM (2007) Heterogeneity in vaccine immune response: the role of immunogenetics and the emerging field of vaccinomics. Clin. Pharmacol Ther 82, 653-664. Tan M, Jiang X (2008) Norovirus gastroenteritis, increased understanding and future antiviral options. Curr Opin Investig Drugs 9, 146-151.

10 and approximately 7 novel and unique HLA-A*0201 epitopes, respectively, including genes implicated in the neoplastic process. Thus, with appropriate manipulation of the immune system, tumor cell destruction in situ can provide a polyvalent tumor vaccine without a requirement for knowledge of the targeted antigens (Segal et al, 2008). To evaluate the receipt of information and knowledge about influenza virus, vaccination and influenza vaccination status, the study of vaccination, concerns about vaccine effectiveness and side effects, and dislike of injections are necessary. Strategies other than educational interventions are needed to increase influenza vaccination rates and thereby to ensure healthcare worker and patient safety (Ofstead et al, 2008).

VIII. Conclusion One of the major challenges in vaccine-related research is pathological condition and patients responses. This is due to the fact that many of the diseases most demanding a vaccine, including HIV, malaria exist principally in third world countries. Although pharmaceutical firms and biotech companies have little incentive to develop vaccines for disorders, because there is little revenue potential and number of vaccines actually administered are risen dramatically in recent decades. Intellectual property can also viewed as an obstacle to the development of new vaccines, because of the weak protection offered through the patent of the final product, the protection of the innovation regarding vaccines is often made through the patent of processes used on the development of new vaccines as well as the protection of secrecy. Also, vaccinations of animals are used both to prevent their contracting diseases and to prevent transmission of disease to humans. Both animals kept as pets and animals raised as stock are vaccinated. In some instances, wild populations may be vaccinated. This is sometimes accomplished with vaccine-laced food spread in a disease-prone area and used to attempt to control rabies. Vaccinomics are used as the science for generating large number of vaccines against various diseases. But due to several other problems like property rights and legal issues, it hasnâ&#x20AC;&#x2122;t developed as it was expected in this decade.

References Andre FE (2003) Vaccinology: past achievements, present roadblocks and future promises. Vaccine 21, 593-595. Assarsson E, Greenbaum JA, Sundstrom M, Schaffer L, Hammond JA, Pasquetto V, Oseroff C, Hendrickson RC, Lefkowitz EJ, Tscharke DC, Sidney J, Grey HM, Head SR, Peters B, Sette A (2008) Glycolipid activation of invariant T cell receptor+ NK T cells is sufficient to induce airway hyperreactivity independent of conventional CD4+ T cells. Proc Natl Acad Sci U S A 103, 2782-2787. Beyer AM, Wandinger KP, Siebert E, Zschenderlein R, Klehmet J (2007) Neuromyelitis optica in a patient with an early onset demyelinating episode: clinical and autoantibody findings. Clin Neurol Neurosurg 109, 926-930. Chua KH, Norzulaani K, Harikhrisna JA, Othman RY (2003) Synthesis of a soluble flag-tagged single chain variable fragment (scFv) antibody targeting Cucumber Mosaic Virus

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Gomase and Tagore: Vaccinomics Whittaker P, Day JB, Curtis SK, Fry FS (2007) Evaluating the use of fatty acid profiles to identify Francisella tularensis. J AOAC Int 90, 465-469. Yang F, Yan S, Wang F, He Y, Guo Y, Zhou Q, Wang Y, Zhang X, Zhang W, Sun S (2008) DNA immunization perturbs lipid metabolites and increases risk of atherogenesis. J Proteome Res 7, 741-748.

Yin QY de Groot, PW, de Koster CG, Klis FM (2008) Mass spectrometry-based proteomics of fungal wall glycoproteins. Trends Microbiol 16, 20-26. Yuan S, Wei Z (2008) Construction of infectious cDNA clones of PRRSV: separation of coding regions for nonstructural and structural proteins. Sci China C Life Sci 51, 271-279.

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Prediction of antigenic binders from c-terminal domain Human papillomavirus oncoprotein e7 Research Article

Virendra S Gomase1,2*, Somnath Tagore1, Krishnan Shyamkumar1 1

Received: 30 May 2008 Revised: 24 June 2008 Accepted: 25 June 2008; electronically published: August 2008

Summary Human papillomavirus (HPV) is one of the most common causes of sexually transmitted disease (STD). Human papillomavirus viral peptides are most suitable for subunit vaccine development because with single epitope, the immune response can be generated in large population. TAP is a transporter associated with MHC class I restricted antigen processing. The TAP is heterodimeric transporter belong to the family of ABC transporter, that uses the energy provided by ATP to translocate the peptides across the membrane. The subset of this transported peptide will bind MHC class II molecules and stabilize them. These MHC-peptide complexes will be translocated on the surface of antigen presenting cells (APCs). In this assay we predicted the binding affinity of Human papillomavirus oncoprotein e7 having 56 amino acids, which shows 49 nonamers. Small peptide regions found as 9-RHKILCVCC (score 6.186), 34-LRTLQQLFL (Score- 6.091), 31-AEDLRTLQQ (Score- 5.979), 8-QRHKILCVC (Score- 5.960), 45LSFVCPWCA (Score-5.604), known as oncoprotein e7 TAP transporter. Adducts of MHC and peptide complexes are the ligands for T cell receptors (TCR). These complexes elicit the immune response for clearing various intracellular infections. Prediction methods based on the specificity of TAP transporter will complement the wet lab experiments and speed up the knowledge discoveries on the basis of these two computational algorithms.

infected keratinocyte leaves the basal layer. Production of virus particles can occur only in highly differentiated keratinocytes; therefore, virus production only occurs at the epithelial surface where the cells are ultimately sloughed into the environment (Alani et al, 1998).

I. Introduction A. Human papillomavirus Papillomaviruses are highly species specific and do not infect other species, even under laboratory conditions. Humans are the only known reservoir for HPV. Papillomaviruses are nonenveloped viruses of icosahedral symmetry with 72 capsomeres that surround a genome containing double-stranded circular DNA with approximately 8000 base pairs. Papillomaviruses are thought to have 2 modes of replication. One is stable replication of the episomal genome in basal cells; the other is runaway, or vegetative, replication in more differentiated cells to generate progeny virus. Although all cells of a lesion contain the viral genome, the expression of viral genes is tightly linked to the state of cellular differentiation. Most viral genes are not activated until the

B. Molecular aspects The E7 oncoprotein from human Papillomavirus (HPV) mediates cell transformation in part by binding to the human pRb tumor suppressor protein and E2F transcription factors, resulting in the dissociation of pRb from E2F transcription factors and the premature cell progression into the S-phase of the cell cycle. This activity is mediated by the LXCXE motif and the CR3 zinc binding domain of the E7 protein (Liu et al, 2006).

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Gomase et al: Prediction of antigenic binders from c-terminal domain Human papillomavirus oncoprotein e7 exposed on the protein surface (Aboderin 1971; Bull et al, 1974; Chothia 1976; Manavalan et al, 1978; Janin 1979; Wilson et al, 1981; Wolfenden et al, 1981; Kyte et al, 1982; Fauchere et al, 1983; Sweet et al, 1983; Eisenberg et al, 1984a,b; Guy 1985; Miyazawa et al, 1985; Rose et al, 1985; Rao 1986; Abraham et al, 1987; Roseman 1988; Cowan et al, 1990; Black et al, 1991; Wilkins et al, 1999).

C. MHC Class-I binding peptides The new paradigm in vaccine design is emerging, following essential discoveries in immunology and development of new MHC Class-I binding peptides prediction tools. MHC molecules are cell surface glycoproteins, which take active part in host immune reactions. The involvement of MHC class-I in response to almost all antigens and the variable length of interacting peptides make the study of MHC Class I molecules very interesting. MHC molecules have been well characterized in terms of their role in immune reactions (Singh et al, 2002; Bhasin et al, 2003; Cui et al, 2006). They bind to some of the peptide fragments generated after proteolytic cleavage of antigen (Kumar et al, 2007). This binding acts like red flags for antigen specific and to generate immune response against the parent antigen. So a small fragment of antigen can induce immune response against whole antigen. Human papillomavirus viral peptides are most suitable for subunit vaccine development because with single epitope, the immune response can be generated in large population.TAP is a transporter associated with MHC class I restricted antigen processing. The TAP is heterodimeric transporter belong to the family of ABC transporter, that uses the energy provided by ATP to translocate the peptides across the membrane (Bhasin et al, 2004). The subset of this transported peptide will bind MHC class I molecules and stabilize them. These MHCpeptide complexes will be translocated on the surface of antigen presenting cells (APCs). This theme is implemented in designing subunit and synthetic peptide vaccines (Gomase et al, 2007).

E. Prediction of MHC Binding peptide MHC2Pred predicts peptide binders to MHCI and MHCII molecules from protein sequences or sequence alignments using Position Specific Scoring Matrices (PSSMs). In addition, we predicts those MHCI ligands whose C-terminal end is likely to be the result of proteosomal cleavage (Brusic et al, 1998; Bhasin et al, 2005; Gomase et al, 2008).

III. Result and interpretation A. The oncoprotein sequence is 56 residues long asGSHMAEPQRHKILCVCCKCDGRIELTVESSAED LRTLQQLFLSTLSFVCPWCATNQ.

B. Prediction of Antigenic peptides In these methods we found the antigenic determinants by finding the area of greatest local hydrophilicity. The Hopp-Woods scale was designed to predict the locations of antigenic determinants in a protein, assuming that the antigenic determinants would be exposed on the surface of the protein and thus would be located in hydrophilic regions (Figure 1). Its values are derived from the transfer-free energies for amino acid side chains between ethanol and water. Welling antigenicity plot gives value as the log of the quotient between percentage in a sample of known antigenic regions and percentage in average proteins (Figure 2). We also study B-EpiPred Server, Parker, Kolaskar and Tongaonkar antigenicity methods and the predicted antigenic fragments can bind to MHC molecule is the first bottlenecks in vaccine design (Figures 3-5).

II. Materials and Methods A. Protein Sequence analysis We analysed the oncoprotein sequence of Human papillomavirus oncoprotein e7 (Ohlenschlager et al, 2006).

B. Prediction of antigenicity This program predicts those segments from within viral oncoprotein that are likely to be antigenic by eliciting an antibody response (Nakagawa et al, 2004). Antigenic epitopes is determined using Gomase method in 2007, B-EpiPred Server, Hopp and Woods, Welling, Parker, Kolaskar and Tongaonkar antigenicity methods (Gomase 2006; Larsen et al, 2006; Hopp et al, 1981; Welling et al, 1985; Parker et al, 1986; Kolaskar et al, 1990). Predictions are based on a table that reflects the occurrence of amino acid residues in experimentally known segmental epitopes.

C. Secondary alignment The Robson and Garnier method predicted the secondary structure of pathogenicity protein. Each residue is assigned values for alpha helix, beta sheet, turns and coils using a window of 7 residues (Figure 6). Using these information parameters, the likelihood of a given residue assuming each of the four possible conformations alpha, beta, reverse turn, or coils calculated, and the conformation with the largest likelihood is assigned to the residue.

C. Prediction of protein secondary structure The important concepts in secondary structure prediction are identified as: residue conformational propensities, sequence edge effects, moments of hydrophobicity, position of insertions and Deletions in aligned homologous sequence, moments of conservation, auto-correlation, residue ratios, secondary structure feedback effects, and filtering (Robson et al, 1993).

D. Solvent accessible regions Solvent accessible scales for delineating hydrophobic and hydrophilic characteristics of amino acids and scales are developed for predicting potential antigenic sites of globular proteins, which are likely to be rich in charged and polar residues. It was shown that a oncoprotein is hydrophobic in nature and contains segments of low complexity and high-predicted flexibility (Figures 7-26).

D. Finding the location in solvent accessible regions For setting the solvent accessible regions in protein, type of plot determine the hydrophobic scale and it is utilized for prediction. This may be useful in predicting membrane-spanning domains, potential antigenic sites and regions that are likely

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Figure 1. Hopp & Woods hydrophobicity plot of oncoprotein e7.

Figure 2. Welling hydrophobicity plot of oncoprotein e7.

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Figure 3. B.cell epitopes are the sites of molecules that are recognized by antibodies of the immune system for the oncoprotein e7.

Figure 4. Parker HPLC hydrophobicity plot of oncoprotein e7.

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Figure 5. Kolaskar and Tongaonkar antigenicity are the sites of molecules that are recognized by antibodies of the immune system for the oncoprotein e7.

Figure 6. Secondary structure plot of pathogenicity protein.

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Figure 7. Sweet hydrophobicity plot of oncoprotein e7.

Figure 8. Kyte & Doolittle hydrophobicity plot of oncoprotein e7.

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Figure 9. Abraham & Leo hydrophobicity plot of oncoprotein e7.

Figure 10. Bull & Breese hydrophobicity plot of oncoprotein e7.

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Figure 11. Guy hydrophobicity plot of oncoprotein e7.

Figure 12.Miyazawa hydrophobicity plot of oncoprotein e7.

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Figure 13. Roseman hydrophobicity plot of oncoprotein e7.

Figure 14. Cowan HPLC pH7.5 hydrophobicity plot of oncoprotein e7.

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Figure 15. Rose hydrophobicity plot of oncoprotein e7.

Figure 16. Eisenberg hydrophobicity plot of oncoprotein e7.

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Figure 17. Manavalan hydrophobicity plot of oncoprotein e7.

Figure 18. Black hydrophobicity plot of oncoprotein e7.

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Figure 19. Fauchere hydrophobicity plot of oncoprotein e7.

Figure 20. Janin hydrophobicity plot of oncoprotein e7.

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Figure 21. Rao & Argos hydrophobicity plot of oncoprotein e7.

Figure 22. Wolfenden hydrophobicity plot of oncoprotein e7.

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Figure 23. Wilson HPLC hydrophobicity plot of oncoprotein e7.

Figure 24. Cowan HPLC pH3.4 hydrophobicity plot of oncoprotein e7.

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Figure 25.Rf mobility hydrophobicity plot of oncoprotein e7.

Figure 26. Chothia hydrophobicity plot of oncoprotein e7.

based on cascade support vector machine, using sequence and properties of the amino acids. The correlation coefficient of 0.88 was obtained by using jack-knife

E. Prediction of MHC Binding peptides These MHC binding peptides are sufficient for eliciting the desired immune response. The prediction is 161

Gomase et al: Prediction of antigenic binders from c-terminal domain Human papillomavirus oncoprotein e7 validation test. In this test, we found the MHCI and MHCII binding regions (Tables 1, 2). MHC molecules are cell surface glycoproteins, which take active part in host immune reactions and involvement of MHC class-I and MHC II in response to almost all antigens. In this assay we predicted the binding affinity of oncoprotein having 56 amino acids, which shows different nonamers (Tables 1, 2). For development of MHC binder prediction method, an elegant machine learning technique support vector machine (SVM) has been used. SVM has been trained on the binary input of single amino acid sequence. In this assay we predicted the binding affinity of Human papillomavirus oncoprotein e7 having 56 amino acids, which shows 49 nonamers. Small peptide regions found as 9-RHKILCVCC (score 6.186), 34-LRTLQQLFL (Score6.091), 31-AEDLRTLQQ (Score- 5.979), 8-QRHKILCVC (Score- 5.960), 45-LSFVCPWCA (Score-5.604), known as oncoprotein e7 TAP transporter (Table 1). We also found the SVM based MHCII-IAb peptide regions, 28ESSAEDLRT, 2-SHMAEPQRH, 7-PQRHKILCV, 4MAEPQRHKI, (optimal score is 0.869); MHCII-IAd peptide regions, 37-LQQLFLSTL, 42-LSTLSFVCP, 25LTVESSAED, 39-QLFLSTLSF, (optimal score is 0.466); MHCII-IAg7 peptide regions , 2-SHMAEPQRH, 46SFVCPWCAT, 17-CKCDGRIEL , 24-ELTVESSAE, (optimal score is 1.207); and MHCII- RT1.B peptide regions, 26-TVESSAEDL, 29-SSAEDLRTL, 36TLQQLFLST, 35-RTLQQLFLS, (optimal score is 0.938) which represented predicted binders from oncoprotein (Table 2). The predicted binding affinity is normalized by the 1% fractil. The MHC peptide binding is predicted using neural networks trained on C terminals of known epitopes. In analysis predicted MHC/peptide binding is a log-transformed value related to the IC50 values in nM

units. These MHC binding peptides are sufficient for eliciting the desired immune response. Predicted MHC binding regions in an antigen sequence and there are directly associated with immune reactions, in analysis we found the MHCI and MHCII binding regions.

IV. Discussion Gomase (2007) method, B-EpiPred Server, Hopp and Woods, Welling, Parker, Kolaskar and Tongaonkar antigenicity scales were designed to predict the locations of antigenic determinants in Human papillomavirus oncoprotein. Oncoprotein shows beta sheets regions, which are high antigenic response than helical region of this peptide and shows highly antigenicicity (Figures 1-5). We also found the Sweet hydrophobicity, Kyte & Doolittle hydrophobicity, Abraham & Leo , Bull & Breese hydrophobicity, Guy, Miyazawa hydrophobicity, Roseman hydrophobicity, Cowan HPLC pH7.5 hydrophobicity, Rose hydrophobicity, Eisenberg hydrophobicity, Manavalan hydrophobicity, Black hydrophobicity, Fauchere hydrophobicity, Janin hydrophobicity, Rao & Argos hydrophobicity, Wolfenden hydrophobicity, Wilson HPLC hydrophobicity, Cowan HPLC pH3.4, Rf mobility hydrophobicity, Chothia hydrophobicity scales. Theses scales are essentially a hydrophilic index, with apolar residues assigned negative values (Figures 7-26). In this assay we predicted the binding affinity of Human papillomavirus oncoprotein e7 having 56 amino acids, which shows 49 nonamers. Small peptide regions found as 9-RHKILCVCC (score 6.186), 34-LRTLQQLFL (Score6.091), 31-AEDLRTLQQ (Score- 5.979), 8-QRHKILCVC (Score- 5.960), 45-LSFVCPWCA (Score-5.604), known

Table 1. TAP Peptide binders of oncoprotein e7. Peptide Rank 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Start Position 9 34 31 8 45 5 7 39 17 37 46 29 42 40 14 4 30 10 1 33

Sequence RHKILCVCC LRTLQQLFL AEDLRTLQQ QRHKILCVC LSFVCPWCA AEPQRHKIL PQRHKILCV QLFLSTLSF CKCDGRIEL LQQLFLSTL SFVCPWCAT SSAEDLRTL LSTLSFVCP LFLSTLSFV CVCCKCDGR MAEPQRHKI SAEDLRTLQ HKILCVCCK GSHMAEPQR DLRTLQQLF

*Optimal Score for given MHC binder in Mouse.

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Score 6.186 6.091 5.979 5.960 5.604 5.598 5.429 5.326 5.205 4.263 4.062 4.059 3.951 3.770 3.733 3.456 3.435 3.276 3.174 3.155

Predicted Affinity High High Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate

Gene Therapy and Molecular Biology Vol 12, page 163 as oncoprotein e7 TAP transporter. Adducts of MHC and peptide complexes are the ligands for T cell receptors (TCR) (Table 1). MHC molecules are cell surface glycoproteins, which take active part in host immune reactions and involvement of MHC class-I and MHC II in response to almost all antigens (Table 2). Kolaskar and Tongaonkar antigenicity are the sites of molecules that are recognized by antibodies of the immune system for the oncoprotein e7, analysis shows epitopes present in the Human papillomavirus the desired immune response (Table 3). The region of maximal hydrophilicity is likely to be an antigenic site, having hydrophobic characteristics, because C- terminal regions of oncoprotein is solvent accessible and unstructured, antibodies against those regions are also likely to recognize the native protein. For the prediction of antigenic determinant site of oncoprotein, we got eighteen antigenic determinant sites in the sequence. The highest pick is recorded between sequence of AA in the region are ‘10-HKILCVCCKCD-20, 24ELTVESS-30’ (Table 3). We also found the SVM based MHCII-IAb peptide regions, 28-ESSAEDLRT, 2SHMAEPQRH, 7-PQRHKILCV, 4-MAEPQRHKI, (optimal score is 0.869); MHCII-IAd peptide regions, 37LQQLFLSTL, 42-LSTLSFVCP, 25-LTVESSAED, 39QLFLSTLSF, (optimal score is 0.466); MHCII-IAg7 peptide regions , 2-SHMAEPQRH, 46-SFVCPWCAT, 17CKCDGRIEL , 24-ELTVESSAE, (optimal score is 1.207); and MHCII- RT1.B peptide regions, 26TVESSAEDL, 29-SSAEDLRTL, 36-TLQQLFLST, 35RTLQQLFLS, (optimal score is 0.938) which represented predicted binders from oncoprotein (Table 2). The

average propensity for the oncoprotein is found to be above 1.0350 (Figure- 5). All residues having above 1.0 propensity are always potentially antigenic (Table 3). The predicted segments in oncoprotein are ‘10HKILCVCCKCD-20, 24-ELTVESS-30’. Fragment identified through this approach tend to be high-efficiency binders, which is a lagers percentage of their atoms are directly involved in binding as compared to larger molecules.

V. Conclusion Human papillomavirus oncoprotein involved multiple antigenic components to direct and empower the immune system to protect the host from infection. MHC molecules are cell surface proteins, which take active part in host immune reactions and involvement of MHC class in response to almost all antigens and it give effects on specific sites. Predicted MHC binding regions acts like red flags for antigen specific and generate immune response against the parent antigen. So a small fragment of antigen can induce immune response against whole antigen. This theme is implemented in designing subunit and synthetic peptide vaccines. The sequence analysis method is allows potential drug targets to identify active sites, which form antibodies against or plant diseases. The method integrates prediction of peptide MHC class binding; proteosomal C terminal cleavage and TAP transport efficiency. Antigenic epitopes of oncoprotein are important antigenic determinants against the various toxic reactions and viral infections.

Table 2. Peptide binders to MHCII molecules of oncoprotein e7. Prediction method ALLELE: I-Ab ALLELE: I-Ab ALLELE: I-Ab ALLELE: I-Ab ALLELE: I-Ad ALLELE: I-Ad ALLELE: I-Ad ALLELE: I-Ad ALLELE: I-Ag7 ALLELE: I-Ag7 ALLELE: I-Ag7 ALLELE: I-Ag7 ALLELE: RT1.B ALLELE: RT1.B ALLELE: RT1.B ALLELE: RT1.B

Rank 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

Sequence ESSAEDLRT SHMAEPQRH PQRHKILCV MAEPQRHKI LQQLFLSTL LSTLSFVCP LTVESSAED QLFLSTLSF SHMAEPQRH SFVCPWCAT CKCDGRIEL ELTVESSAE TVESSAEDL SSAEDLRTL TLQQLFLST RTLQQLFLS

Residue No. 28 2 7 4 37 42 25 39 2 46 17 24 26 29 36 35

Peptide Score 0.869 0.683 0.415 0.337 0.466 0.357 0.330 0.327 1.207 1.176 0.961 0.808 0.938 0.696 0.563 0.503

*Optimal Score for given MHC II peptide binder in Mouse.

Table 3. Antigenic epitopes from oncoprotein e7. No. 1 2

Start position 10 24

End position 20 30

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Peptide HKILCVCCKCD ELTVESS

Peptide length 11 7

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Hopp TP, Woods KR (1981) Prediction of Protein Antigenic Determinants from Amino Acid Sequences. Proc Natl Acad Sci USA 78, 3824-3828. Janin J (1979) Surface and inside volumes in globular proteins. Nature 277, 491-492. Kolaskar AS, Tongaonkar PCA (1990) semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276, 172-174. Kumar M, Gromiha MM, Raghava GP (2007) Identification of DNA-binding proteins using support vector machines and evolutionary profiles. BMC Bioinformatics 8, 463. Kyte J, Doolittle RF (1982) A Simple Method for Displaying the Hydropathic Character of a Protein. J Mol Biol 157, 105132. Larsen JEP, Lund O, Nielsen M (2006) Improved method for predicting linear B-cell epitopes. Immunome Res. 2, 2. Liu X, Clements A, Zhao K, Marmorstein R (2006) Structure of the Human Papillomavirus E7 Oncoprotein and Its Mechanism for Inactivation of the Retinoblastoma Tumor Suppressor. J Biol Chem 281, 578-586. Manavalan P, Ponnuswamy PK (1978) Hydrophobic character of amino acid residues in globular proteins. Nature 275, 673674. Miyazawa S, Jernigen RL (1985) Estimation of Effective Interresidue Contact Energies from Protein Crystal Structures: Quasi-Chemical Approximation. Macromolecules 18, 534-552. Nakagawa M, Kim KH, Anna-Barbara M (2004) Different Methods of Identifying New Antigenic Epitopes of Human Papillomavirus Type 16 E6 and E7 Proteins. Clin Diagn Lab Immunol 11, 889-896. Ohlenschlager O, Seiboth T, Zengerling H, Briese L, Marchanka A, Ramachandran R, Baum M, Korbas M, Meyer-Klaucke W, Durst M, Gorlach M (2006) Solution structure of the partially folded high-risk human papilloma virus 45 oncoprotein E7. Oncogene 25, 5953-5959. Parker JMR, Guo D, Hodges RS (1986) New Hydrophilicity Scale Derived from High-Performance Liquid Chromatography Peptide Retention Data: Correlation of Predicted Surface Residues with Antigenicity and X-rayDerived Accessible Sites? Biochemistry 25, 5425-5431. Rao MJK, Argos P (1986) A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta 869, 197-214. Robson B, Garnier J (1993) Protein structure prediction. Nature 361, 506. Rose GD, Geselowitz AR, Lesser GJ, Lee RH, Zehfus MH (1985) Hydrophobicity of amino acid residues in globular proteins. Science 229, 834-838. Roseman MA (1988) Hydrophilicity of polar amino acid sidechains is markedly reduced by flanking peptide bonds. J Mol Biol 200, 513-522. Singh H, Raghava GPS (2002) Matrix Optimization Technique for Predicting MHC binding Core. Biotech Software and Internet Report 3, 146. Sweet RM, Eisenberg D (1983) Correlation of sequence hydrophobicities measures similarity in three-dimensional protein structure. J Mol Biol 171, 479-488. Welling GW, Weijer WJ, Van der Zee R, Welling-Wester S (1985) Prediction of sequential antigenic regions in proteins. FEBS Lett 188, 215-218. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF (1999) Protein identification and

References Aboderin AA (1971) An empirical hydrophobicity scale for alpha-amino-acids and some of its applications. Int J Biochem 2, 537-544. Abraham DJ, Leo AJ (1987) Extension of the fragment method to calculate amino acid zwitterions and side chain partition coefficients. Proteins 2, 130-152. Alani RM, Munger K (1998) Human papillomaviruses and associated malignancies. J Clin Oncol 16, 330-337. Bhasin M, Raghava GP (2005) Pcleavage: an SVM based method for prediction of constitutive proteasome and immunoproteasome cleavage sites in antigenic sequences. Nucleic Acids Res 33, W202-207. Bhasin M, Raghava GPS (2004) Analysis and prediction of affinity of TAP binding peptides using cascade SVM. Protein Science 13, 596-607. Bhasin M, Singh H, Raghava GPS (2003) MHCBN: A comprehensive database of MHC binding and non-binding peptides. Bioinformatics 19, 666â&#x20AC;&#x201C;667. Black SD, Mould DR (1991) Development of Hydrophobicity Parameters to Analyze Proteins Which Bear Post- or Cotranslational Modifications. Anal Biochem 193, 72-82. Brusic V, Rudy G, Honeyman G, Hammer J, Harrison L (1998) Prediction of MHC class II-binding peptides using an evolutionary algorithm and artificial neural network. Bioinformatics 14, 121-30. Bull HB, Breese K (1974) Surface tension of amino acid solutions: A hydrophobicity scale of the amino acid residues. Arch Biochem Biophys 161, 665-670. Chothia C (1976) The nature of accessible and buried surfaces in proteins. J Mol Biol 105, 1-14. Cowan R, Whittaker RG (1990) Hydrophobicity indices for amino acid residues as determined by HPLC. Peptide Research 3, 75-80. Cui J, Han LY, Lin HH, Tang ZQ, Jiang L, Cao ZW, Chen YZ (2006) MHC-BPS: MHC-binder prediction server for identifying peptides of flexible lengths from sequencederived physicochemical properties. Immunogenetics 58, 607-613. Eisenberg D, Schwarz E, Komaromy M, Wall R (1984a) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Bio. 179, 125-142. Eisenberg D, Weiss RM, Terwilliger TC (1984b) The hydrophobic moment detects periodicity in protein hydrophobicity. Proc Natl Acad Sci USA 81, 140-144. Fauchere JL, Pliska VE (1983) Hydrophobic parameters of amino-acid side-chains from the partitioning of N-acetylamino-acid amide. Eur J Med Chem 18, 369-375. Gomase VS (2006) Prediction of Antigenic Epitopes of Neurotoxin Bmbktx1 from Mesobuthus martensii. Curr Drug Discov Technol 3, 225-229. Gomase VS, Changbhale SS, Kale KV (2008) Insilico analysis of nucleocapsid protein from groundnut ringspot virus. Advancements in Information Technology and Internet Security, Shroff Publisher and Distributors, Pvt. Ltd. ISBN10:81-8404-427-5, 370-378. Gomase VS, Kale KV, Chikhale NJ, Changbhale (2007) SS Prediction of MHC Binding Peptides and Epitopes from Alfalfa mosaic virus. Curr Drug Discov Technol 4, 117121. Guy HR (1985) Amino acid side chain partition energies and distributions of residues in soluble proteins. Biophys J 47, 61-70.

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Gene Therapy and Molecular Biology Vol 12, page 167 Gene Ther Mol Biol Vol 12, 167-174, 2008

Retrovirally-mediated genetic correction of mesenchymal stem cells from patients affected by mucopolysaccharidosis type II (Hunter Syndrome) Research Article

Carla Corradi-Perini1, Thomas D. Southgate2, Guy T Besley3, Alan Cooper3, John A Deakin4, J Ed Wraith3, Leslie J. Fairbairn2, Robert F. Wynn1, Ilaria Bellantuono5,* 1

Stem Cell Research Group, Royal Manchester Children's Hospital, Manchester CRUK Gene Therapy Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust; Manchester, UK 3 Willink Unit, Royal Manchester Children's Hospital, Manchester 4 Medical Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust; Manchester, UK 5 Academic Unit of Bone Biology, University of Sheffield, Sheffield, UK 2

__________________________________________________________________________________ *Correspondence: Ilaria Bellantuono, Room DU19, Academic Unit of Bone Biology, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, UK; Tel: +44 114 2711798; Fax: +44 114 2711711; e-mail: I.Bellantuono@sheffield.ac.uk Key words: Marrow stromal cells, Iduronate-2-Sulphatase, MPSII Abbreviations: Bone marrow transplantation, (BMT); central nervous system, (CNS); colony-forming-unit fibroblast, (CFU-F); Dulbeccoâ&#x20AC;&#x2122;s modified Eagleâ&#x20AC;&#x2122;s medium, (DMEM); enhanced green fluorescent protein, (eGFP); enzyme replacement therapy, (ERT); fetal calf serum, (FCS); glycosaminoglycans, (GAGs); graft versus host disease, (GvHD); Human MSC, (hMSC); iduronate-2-sulphatase, (IDS); internal ribosome entry site, (IRES); mannose-6-phosphate receptor, (M-6-P); Mesenchymal stem cells, (MSC); Mucopolysaccharidosis type II, (MPSII); population doublings, (PDs)

No competing financial interests exist Received: 6 May 2008; Revised: 28 June 2008 Accepted: 1 July 2008; electronically published: September 2008

Summary Mucopolysaccharidosis type II (MPSII) is an X-linked metabolic storage disorder due to the deficiency of iduronate-2-sulphatase (IDS) and accumulation of glycosaminoglycans (GAGs). Clinically it presents as a multisystem disorder with developmental delay, bone and joint disease and in the severe forms progressive mental retardation. At present little therapeutic options are available. Bone marrow transplantation is no longer recommended due to the severe side effects and lack of proven efficacy in correcting central nervous system and bone disease. Enzyme replacement therapy is under assessment and it requires weekly, expensive administration for the lifespan of the individual. Mesenchymal stem cells (MSC) are bone marrow-derived cells capable of differentiation into tissue such as bone and have been shown to contribute to bone repair. They are amenable to gene manipulation and therefore provide an excellent target for the correction of MPSII disease, especially with regard to bone disease. In this study we tested whether MSC from MPSII patients (hMSCMPSII) could be corrected with a retroviral vector expressing the IDS gene. Following transduction hMSCMPSII maintained the capacity to differentiate into osteoblasts and adipocytes and showed levels of IDS enzyme over 10 fold higher than those detected in MSC from healthy donors. This led to normalization of GAGs storage in hMSCMPSII. Such transduced cells were able to cross-correct MPSII fibroblasts by uptake of the IDS enzyme via the mannose-6-phosphate receptor. This study suggests that correction of autologous hMSCMPSII by retroviral gene transfer is effective and may be amenable for the improvement of the skeletal features of the disease.

to the deficiency of iduronate-2-sulphatase (IDS; EC 3.1.6.13) activity. This results in incomplete degradation of the glycosaminoglycans (GAGs) dermatan and heparan sulphate, which accumulate in the cells and interfere with

I. Introduction Mucopolysaccharidosis type II (MPSII), or Hunter syndrome, is an X-linked systemic metabolic disorder due

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Corradi-Perini et al: Gene therapy for MPSII with the Helsinski Declaration of 1995). Human MSC (hMSC) were isolated and cultured as previously described (Bruder et al, 1997). Briefly, mononuclear cells (MNCs) were seeded at a concentration of 8 x 105 MNC/cm2 in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (FCS; StemCell Technologies, London, UK). The medium was changed 24-48 h later, and cells were fed twice a week. When cultures reached confluence, hMSC were detached using 0.05% trypsin 5-mM EDTA (Invitrogen), counted using a hemocytometer; then one third of the cells were replated, and the remainder were used for differentiation assays. The number of hMSC at the start of the culture was determined by the colony-forming-unit fibroblast (CFU-F) assay, as previously described (Friedenstein et al, 1976) and was used to determine the number of population doublings that cells have undergone to reach primary confluence. Thereafter, the number of population doublings was calculated by dividing the log N by log 2, where N equals the total number of cells divided by the initial seeding number of cells. All hMSC cultures were stained with anti-CD45, anti-CD34 antigen and showed no expression when compared to cells stained with an isotype matched control. They were found positive for CD105 (Barry et al, 1999).

their functions (Lim et al, 1974; Wraith et al, 1991; Hopwood et al, 1993). Clinically, the disorder is a spectrum and at the severe end is characterized by mental retardation, skeletal deformities, joint stiffness, organomegaly, airway obstruction and premature death (usually in their teens). The attenuated end of the clinical spectrum has no central nervous system (CNS) involvement and death may occur in early to midadulthood (Young et al, 1982). Bone marrow transplantation (BMT) and enzyme replacement therapy (ERT) are two treatments available for patients with MPS disease. However, despite some success with BMT in MPS types I and VI, this is not recommended for MPSII due to difficulties in engraftment, the severe side effects, including graft versus host disease (GvHD) (Peters and Krivit, 2000), and because it had limited efficacy in delaying progressive CNS deterioration (Peters and Steward, 2003). ERT is under evaluation but it is a costly treatment and has to be administered throughout the lifespan of the patient (Muenzer et al, 2006). Genetic manipulation of stem cells is an attractive therapeutic option. Stem cells are defined by their ability to self renew and differentiate into multiple tissues for the lifespan of the individual. Therefore genetic manipulation of stem cells would guarantee lifelong enzyme production capability. Mesenchymal stem cells (MSC) can be easily isolated from bone marrow, expanded several fold and efficiently gene modified with minimal manipulation (Prockop, 1997). They are capable of differentiation into osteoblasts and chondrocytes (Pittenger et al, 1999), they have been shown to integrate and participate to bone repair in fractures (Quarto et al, 2001) and to partially correct inherited disorders of the skeleton, such as those described in osteogenesis imperfecta (Horwitz et al, 1999). Thus, they may act as a suitable enzyme delivery system to difficult tissues such as bone. Moreover as cells from the patients’ bone marrow could be isolated, corrected by gene modification in vitro and reinfused into the patient they would not suffer from the same limitations as allogeneic bone marrow transplant including donor identification and the severe morbidity associated with this procedure. Previously we have shown that hMSC could be isolated and genetically modified to correct deficiency of !-L-Iduronidase in patients affected by MPSI (Baxter et al, 2002). In this study we have shown that autologous hMSC can be isolated from MPSII patients, efficiently gene modified by retroviral vectors to express supernormal levels of IDS enzyme and correct GAGs accumulation. Moreover IDS enzyme is secreted in sufficient amount to provide uptake by the surrounding cells and correction of GAGs accumulation via the mannose-6-phosphate receptor (M-6-P).

B. Differentiation Human MSCs were plated at 1 x 104 and 3 x 104 per well in 6-well plates in growth medium with osteogenic or adipogenic supplements, respectively and as previously described (Pittenger et al, 1999). To detect differentiation to the osteogenic lineage alkaline phosphatase expression was assessed using an alkaline phosphatase cytochemical staining kit (Sigma Chemical Corp., Poole, Dorset, UK) according to manufacturer’s instruction. Oil Red O staining was used to identify differentiation to the adipogenic lineage (Baxter et al, 2004).

C. Recombinant retroviral vector expressing IDS and transduction of hMSC The retroviral vector used in this study (Figure 1) is based upon the pSF!91 backbone (Hildinger et al, 1998) that is optimised for initiation of transcription in haemopoietic cells, (kind gift of Christopher Baum, Hannover, Germany) modified to include an internal ribosome entry site (IRES) (Jang et al, 1988), and enhanced green fluorescent protein (eGFP) sequences (Clontech, Palo Alto, USA). The IDS cDNA was cloned into the unique NotI and BamHI sites in the retroviral vector to form pSF!91 IDS IRES eGFP that thus contains a cassette coexpressing IDS and eGFP whose translation is driven by the encephalomyocarditis virus IRES. GP+envAM12 packaging cells (Markowitz et al, 1988) were transfected with plasmids encoding the retroviral vectors described above using Transfast (Promega, Southampton, UK) as per manufacturers instructions. One week after transfection, cells harbouring a stable expressing integrant were isolated using a FACSVantage based upon GFP fluorescence. GP+envAM12 were cultured in DMEM containing 10% newborn calf serum and non essential amino acids (Invitrogen). hMSCMPSII (30% - 40% confluent) were transduced by incubating for 8 hours on 2 consecutive days with 2ml cellfree retroviral supernatant supplemented with 4µg/mL polybrene (Sigma Chemical Corp., Poole, UK), followed by replacement of supernatant by fresh culture medium. Seventy-two hours posttransduction, the cells were analysed for eGFP expression by flow cytometry analysis using Becton Dickinson FACScalibur equipment and the acquired data were analysed by CellQuest software.

II. Materials and methods A. Isolation and culture of human MSCs Human MSC were isolated from bone marrow obtained from the posterior iliac crest of two MPSII patients (hMSCMPSII) and two healthy donors (hMSCN) aged 46 and 181 months after parental consent (in accordance with the ethical committee and

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Figure 1 SF!91-IDS vector. Schematic representation of the vector used in this study based on the SF!91 backbone. ", packaging signal; IDS, Iduronate-2-Suplhatase; IRES, internal ribosome entry site; eGFP, enhanced green fluorescent protein.

expansion capacity of hMSC from two MPSII patients and age matched controls were analysed. Although the number of MSC progenitors (assessed by CFU-F assay) was in the lower range when compared to the age matched controls (Figure 2A) the kinetic of expansion was similar in the two groups (Figure 2B). Both hMSCN and hMSCMPSII primary cultures reached primary confluence in 9-14 days. On replating, MSC from both groups decreased their proliferation at similar rates. Cultures were examined up to 27-30 population doublings (PDs) with no noticeable differences.

D. Determination of IDS activity hMSCN, hMSCMPSII and retrovirally transduced hMSCMPSII were tested for IDS activity using the tritium-labelled disulphated disaccharide derived from heparin sulphate basically as described by (Lim et al, 1974). Cell pellets were suspended in 120 µl distilled water, disrupted by sonication, briefly centrifuged and the supernatants dialysed against 0.15M-NaCl overnight at 4ºC. Aliquots (30 µl) were incubated with acetate buffered substrate for 2h at 37ºC. Following termination with 0.1M-Na2HPO 4 the monosulphated product was separated on Ecteola cellulose (Sigma, Cambridge, UK) by elution in 0.6M- sodium formate and the radioactivity counted. For reference, the lysosomal enzyme #-galactosidase, was also assay by a standard fluorimetric method (Galjaard, 1980).

E. 35SO4-GAG sequestration assay Confluent hMSCN, hMSCMPSII and retrovirally transduced hMSCMPSII were exposed to 35S-labeled Na2SO 4 (NEN Life Science Products, Boston, MA) at 20 µCi/mL (0.74 MBq/mL) in Dulbecco modified Eagle medium plus 10% fetal calf serum for 24 hours and subsequently cultured for 1 week. Cells were then trypsinized and washed in phosphate-buffered saline to remove external GAGs. Following centrifugation at 800g for 10 minutes, cells pellets were solubilized in 2 mL of 6 M urea/0.15 M sodium phosphate, pH 7.0, containing 1% Triton X-100 at 4ºC for 1 hour. Extracts were filtered before application to a fast protein liquid chromatography Mono-Q HR 5/5 anion exchange column (Pharmacia, St Albans, UK). Nonincorporated 35SO 4 was removed by washing through with 0.15 M NaCl/20 mM phosphate, pH 7.0, containing 1% Triton X-100. Bound 35Slabeled material was eluted using a 60mL linear gradient of 0.15 to 1.5 M NaCl in 20 mM phosphate, pH 7.0, containing 1% Triton X-100 at a flow rate of 1 mL/min and collecting 1 mL fractions. The 35S content of fractions was determined by liquid scintillation counting.

F. Cross-correction To assess whether transduced IDS was secreted, fibroblasts from patients affected by MPSII were fed with IDS-conditioned medium (0.133mls/cm 2) from transduced hMSCMPSII at 20-24 PD. This medium was collected and filtered with a 0.45µM filter after an overnight incubation on an 80-90% confluent MSC monolayer. IDS activity was measured as described above. Medium collected from normal fibroblasts (80-90% confluent) was used as control. To assess whether recombinant IDS was endocytosed via the mannose-6-phosphate (M-6-P) receptor, fibroblasts were incubated with or without 5mM M-6-P or with the structural analog 5mM glucose-6-phosphate. Cell lysates were then analyzed for total protein content and IDS activity as described above.

Figure 2 Bone marrow of patients affected by MPSII contains MSC with similar expansion capacity to age matched healthy individual. (A) The number of colonyforming-unit fibroblast (CFU-F) in patients affected by MPSII (black bars) and age matched healthy controls (white bars). Results are expressed per 106 mononuclear cells. (B) Growth kinetics of hMSC culture from patients affected by MPSII (black triangle) at the age of 46 months and 180 months; and 2 healthy donors (black square) at the age of 46 months and 181 months. The curves represent the cumulative number of population doubling (PD) versus time in culture.

III. Results A. Isolation and culture of hMSC In order to assess whether hMSCMPSII had similar characteristics to hMSCN, the bone marrow frequency and

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Corradi-Perini et al: Gene therapy for MPSII hMSC from both groups were tested for their ability to undergo osteogenic and adipogenic differentiation after appropriate induction at two different times in culture (1820 and 23-25 PDs). After 2 weeks in osteogenic medium, both hMSCMPSII and hMSCN cultures exhibited upregulation of alkaline phosphatase expression (Figure 3A and C, respectively) and mineralized deposits visualized by Von Kossa staining (data not shown). In contrast alkaline phophasphatase was very low (Figure 3D) and presence of Von Kossa precipitates (data not shown) were not observed in cultures not exposed to the osteogenic supplements. Induction of adipogenic differentiation was apparent by the accumulation of lipidrich vacuoles which were stained with Oil-Red-O. No differences in the pattern of differentiation was observed between hMSCMPSII and hMSCN (Figure 3E and G, respectively). As expected no formation of adipocytes was

observed in the cultures, which were not exposed to adipogenic supplements (Figure 3H).

B. Transduction and enzyme reconstitution of hMSCMPSII Both hMSCMPSII cultures (hMSCMPSIIa and hMSCMPSIIb) were transduced at 17.9 and 20.5 population doublings respectively. The transduction efficiency was 52.8% and 70.5% in hMSCMPSIIa and b, respectively. Cultures maintained a growth rate similar to the untransduced hMSCMPSII cultures (Figure 4) and ability to differentiate into osteoblasts and adipocytes (Figure 3B and F) suggesting that the transduction procedure did not have detrimental effects on the proliferation and differentiation capacity of hMSC.

Figure 3. hMSCMPSII showed multipotent differentiation potential similar to hMSCN. A representative example of hMSC MPSII before (A) and after (B) transduction, and hMSC N (C) exposed to osteogenic supplements and stained for the expression of alkaline phosphatase. hMSCMPSII before (E) and after (F) transduction, and hMSCN (G) exposed to adipogenic supplements for two weeks and staining with Oil-Red-O. All hMSC were also cultured in medium without osteogenic or adipogenic supplements and stained for the expression of alkaline phosphatase (D) and for Red Oil O (H). Panels D and H show one representative example of a culture not exposed to differentiation supplements.

Figure 4. hMSCMPSII cultures retain their expansion capacity after retroviral transduction. Growth kinetics of MSC culture from patients affected by MPSII at the age of 46 months (triangle) and 180 months (square) before (close shapes) and after (open shapes) transduction with the retroviral vector containing the IDS gene.

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Gene Therapy and Molecular Biology Vol 12, page 171 As expected very low levels of IDS enzyme activity were found in the untransduced hMSCMPSII cultures whilst IDS enzymes levels were more than 10 fold higher than those detected in hMSCN following transduction (Figure 5A). In order to determine whether expression of supranormal levels of IDS enzyme affected the expression of other lysosomal enzymes #-galactosidase activity was assessed in both transduced and untrasduced hMSCMPSII cultures and compared to the levels in hMSCN cultures. All cultures showed levels of #-galactosidase in the normal range (Figure 5B) suggesting that overexpression of IDS enzyme did not alter the expression of other lysosomal enzymes. In addition, in order to evaluate the capability of the recombinant enzyme to correct the metabolic defect, GAGs levels were assessed by measurement of 35SO4 incorporation. As expected untransduced hMSCMPSII showed significant amounts of heparan and dermatan sulphate. In contrast transduced hMSCMPSII cultures, showed levels of GAGs similar to those found in hMSCN from unaffected individuals, indicating that the recombinant IDS enzyme was produced in adequate amount and was functional (Figure 5C).

C. Cross correction of MPSII skin fibroblasts In order to investigate the cross-correction potential of the IDS recombinant enzyme cell-free conditioned medium was obtained from transduced hMSCMPSII cultures following an overnight incubation and used to feed two MPSII fibroblast cultures (MPSII fibroblast a and b) (Figure 6A). As expected, no enzyme activity was found when MPSII fibroblasts were incubated with medium from untransduced hMSCMPSII (IDS enzyme activities of 0.1 and 0.01 nmol/h/mg protein, for a and b cultures, respectively, equivalent to 2.1 and 2.9% of IDS activity in normal fibroblasts). Conditioned medium from hMSCN only modestly increased IDS enzyme levels (0.35 and 0.04 nmol/h/mg, for a and b cultures, respectively, equivalent to 7.5 and 11.7% of IDS activity in normal fibroblasts). In contrast the medium from transduced hMSCMPSII cultures conferred high IDS levels (38.9 and 4.42 nmol/h/mg, for a and b cultures, respectively, equivalent to 837 and 1300% of IDS activity in normal fibroblasts). This was well above levels observed in normal skin fibroblasts. Crosscorrection was inhibited by mannose-6-phosphate, but not by the structural analogue glucose-6-phosphate, confirming that uptake was dependent on the mannose-6phosphate receptor.To test whether the endocytosed IDS enzyme was capable of degrading GAGs storage in MPSII fibroblasts, cultures were exposed to 35SO4 to label glycosamonoglycans (Figure 6B). Fibroblasts from MPSII patients showed significant amounts of 35SO4 incorporation due to its accumulation in the GAGs. MPSII fibroblasts exposed to transduced hMSCMPSII conditioned medium, however, showed levels of 35SO4 incorporation similar to those in fibroblasts from unaffected individuals, showing that transduced hMSC MPSII can secrete the IDS enzyme in a form capable of cross-correction. Surprisingly despite the modest enzyme levels obtained following cross-correction of MPSII fibroblasts with cell free conditioned medium from MSCN these were sufficient to degrade intracellular GAGs to normal levels.

Figure 5. Following retroviral transduction hMSCMPSII express supernormal levels of IDS enzyme capable of degrading GAGs to normal levels. (A) IDS activity in hMSCN (white bars, n=2), hMSCMPSII (n=2) before (black bars) and after (grey bars) transduction. (B) Beta galactosidase activity in hMSCN (white bars, n=2), hMSCMPSII (n=2) before (black bars) and after (grey bars) transduction. Columns represent levels of enzymes expressed as nmol/h/mg. (C) Levels of GAGs accumulation in hMSC cells measured by sulphate sequestration assay (a representative example). Black diamond represents levels of GAGs in untransduced hMSCMPSII, gray triangle in transduced hMSCMPSII and black square in hMSC N

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Corradi-Perini et al: Gene therapy for MPSII immune response to the IDS enzyme, which is seen in patients with a total absence of protein expression. Limited studies are available on the feasibility of gene therapy for the correction of Hunter syndrome (Friso et al, 2005; Cardone et al, 2006). Very recently excellent results have been obtained in a murine model of MPSII. AAV2-IDS viral particles were administered intravenously to adult MPSII mice (Cardone et al, 2006). The plasma and tissue IDS activities were completely restored in all of the treated mice with normalization of the GAGs levels and correction of the skeletal malformation, suggesting that the gene therapy approach has the potential for the systemic treatment of MPSII. However clinically, there are safety concerns in directly injecting viral particles at high titre. Moreover it is known that transduction of murine cells not always reflect what is observed in human cells (Heim and Dunbar, 2000). In contrast data are limited with regard to ex vivo correction of human cells from patients affected by Hunter syndrome. Ex vivo gene modification of lymphocytes has been attempted. However efficiency was poor as lymphocytes are notoriously difficult to transduce (Stroncek et al, 1999). Moreover little is known about their lifespan and therefore it is unknown whether they would provide long term cure. In this study we propose hMSC as a delivery system for the IDS enzyme. Stem cells have the ability to selfrenew and differentiate into multiple tissues, contributing to tissue repair and maintenance. Correction of stem cells by genetic modification can potentially lead to a definitive cure for the disease. Human MSC are easy to isolate from the patient bone marrow, can be expanded in vitro and efficiently transduced by a variety of viral vectors. Due to their ability to differentiate in several cell types including osteoblasts, they can directly participate to regeneration of tissues such as bone. Bone malformations are present in MPSII patients and are difficult to correct with other therapeutic modalities. Indeed, so far hMSC have been shown to be able to home to several organs including bone (Pereira et al, 1998; Devine et al, 2001) following intravenous infusion in both animal models and a phase I clinical trial with some benefits. Here we show that hMSC can be efficiently trasduced with a retroviral vector using a simple two days transduction protocol. This does not alter the expansion and differentiation capacity of hMSC and result in supranormal levels of enzyme that is functionally active being capable of degrading the accumulated GAGs and cross-correct the surrounding cells. Of interest is that we observed very low enzyme uptake following exposure of fibroblasts obtained from patients affected by MPSII to conditioned medium from hMSC derived from normal donors. Even if very low levels of enzyme activity seem to be sufficient to completely correct GAGs accumulation in the cells in vitro this may not be the case in vivo where the circulating enzyme may be diluted in the circulation. These data emphasize how a gene therapy approach may be particularly beneficial in the case of MPSII, where overexpression of the IDS gene may be required to prevent poor cross-correction.

Figure 6. hMSC MPSII transduced to express supranormal levels of IDS enzyme secrete the IDS enzyme and is capable to cross-correct GAGs storage in fibroblasts from MPSII patients. (A) IDS activity expressed as percentage of IDS activity in normal fibroblasts. White bars represent IDS activity in fibroblast obtained from healthy donors (NFB); grey bars represent fibroblasts from patients affected by MPSII (HF) exposed to cell free conditioned medium from hMSC culture from a healthy donor (NSN), from a donor affected by MPSII (HSN), from a donor affected by MPSII and transduced with the IDS expressing retroviral vector (IDSSN) in presence of mannose-6-phosphate (M6P) or glucose-6-phosphate (G-6-P). (B) A representative experiment of 35SO4-GAG sequestration assay. Levels of 35S-GAG accumulation in fibroblasts from healthy donors (black triangle), from MPSII patients exposed to cell-free conditioned medium from hMSCN (black diamond), IDS transduced hMSCMPSII (black star) and untransduced hMSCMPSII (grey square) were measured.

IV. Discussion There is a strong rational in using gene modification of mesenchymal stem cells for the therapy of MPSII. In contrast to MPSI, the two main treatments currently available, BMT and ERT, are unlikely to result in long term benefit in patients with severe MPSII. Moreover, BMT requires a suitable donor and has often side effects such as GvHD; ERT is a lifelong and expensive treatment. Therefore the possibility of correcting the patientsâ&#x20AC;&#x2122; autologous cells overcome some of the problem of BMT such as finding a suitable donor and the severe side effects associated to GvHD. MSC have also been shown to have immunosuppressive properties, reducing the risk of 172

Gene Therapy and Molecular Biology Vol 12, page 173 following implantation of encapsulated recombinant myoblasts. J Gene Med 7, 1482-1491. Galjaard H (1980) Genetic Metabolic Diseases: early diagnosis and prenatal analysis. (Elsevier, Amsterdam/New York). Heim DA, Dunbar CE (2000) Hematopoietic stem cell gene therapy: towards clinically significant gene transfer efficiency. Immunol Rev 178, 29-38. Hildinger M, Eckert HG, Schilz AJ, John J, Ostertag W, Baum C (1998) FMEV vectors: both retroviral long terminal repeat and leader are important for high expression in transduced hematopoietic cells. Gene Ther 5, 1575-1579. Hopwood JJ, Bunge S, Morris CP, Wilson PJ, Steglich C, Beck M, Schwinger E, Gal A (1993) Molecular basis of mucopolysaccharidosis type II: mutations in the iduronate-2sulphatase gene. Hum Mutat 2, 435-442. Hopwood JJ, Bunge S, Morris CP, Wilson PJ, Steglich C, Beck M, Schwinger E, Gal A (1999) Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 5, 309-313. Jang SK, Krausslich HG, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer E (1988) A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 62, 2636-2643. Lim TW, Leder IG, Bach G, Neufeld EF (1974) An assay for iduronate sulfatase (Hunter corrective factor). Carbohydr Res 37, 103-109. Markowitz D, Goff S, Bank A (1988) Construction and use of a safe and efficient amphotropic packaging cell line. Virology 167, 400-406. Muenzer J, Wraith JE, Beck M, Giugliani R, Harmatz P, Eng CM, Vellodi A, Martin R, Ramaswami U, GucsavasCalikoglu M, Vijayaraghavan S, Wendt S, Puga AC, Ulbrich B, Shinawi M, Cleary M, Piper D, Conway AM, Kimura A (2006) A phase II/III clinical study of enzyme replacement therapy with idursulfase in mucopolysaccharidosis II (Hunter syndrome). Genet Med 8, 465-73. Pereira RF, O'Hara Md, Laptev AV, Halford KW, Pollard MD, Class R, Simon D, Livezey K, Prockop DJ (1998) Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta. Proc Natl Acad Sci U S A 95, 1142-1147. Peters C, Krivit W (2000) Hematopoietic cell transplantation for mucopolysaccharidosis IIB (Hunter syndrome) Peters C, Steward CG (2003) Hematopoietic cell transplantation for inherited metabolic diseases: an overview of outcomes and practice guidelines. Bone Marrow Transplant 31, 229239. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71-74. Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, Kon E, Marcacci M (2001) Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344, 385-386. Stroncek DF, Hubel A, Shankar RA, Burger SR, Pan D, Mccullough J, Whitley CB (1999) Retroviral transduction and expansion of peripheral blood lymphocytes for the treatment of mucopolysaccharidosis type II, Hunter's syndrome. Transfusion 39, 343-350.

In vivo studies with long term follow up are now required to demonstrate the establishment of hMSC longterm residence within the BM, bone and possibly other tissues in sufficient number to contribute to the correction of the systemic accumulation of GAGs; to improve bone regeneration for the life-span of the individual with no adverse effects such as clonal transformation due to insertional mutagenesis. This may be difficult to achieve in murine models as murine MSC display marked differences from human MSC (Aguilar et al, 2007). Immunocompromised models and/or non human primate models may be required to assess successful engraftment of human cells and long term follow up.

Acknowledgements We are very grateful to Margaret Thornley for technical assistance in the IDS enzyme activity assay. CCP is funded by Mucopolysaccharidosis society USA, IB by the Jeans for Genes appeal and the Mucopolysaccharidosis society UK, LJF, JD and TDS by Cancer Research UK. This paper is dedicated to LJF who died prematurely during the preparation of this manuscript. He is greatly missed.

References Aguilar S, Nye E, Chan J, Loebinger M, Spencer-Dene B, Fisk N, Stamp G, Bonnet D, Janes SM (2007) Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung. Stem Cells 25, 1586-1594. Barry FP, Boynton RE, Haynesworth S, Murphy JM, Zaia J (1999) The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochem Biophys Res Commun 265, 134-139. Baxter MA, Wynn RF, Deakin JA, Bellantuono I, Edington KG, Cooper A, Besley GT, Church HJ, Wraith JE, Carr TF, Fairbairn LJ (2002) Retrovirally mediated correction of bone marrow-derived mesenchymal stem cells from patients with mucopolysaccharidosis type I. Blood 99, 1857-9. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I (2004) Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 22, 675-682. Bruder Sp, Jaiswal N, Haynesworth SE (1997) Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 64, 278-294. Cardone M, Polito VA, Pepe S, Mann L, D'Azzo A, Auricchio A, Ballabio A, Cosma MP (2006) Correction of Hunter syndrome in the MPSII mouse model by AAV2/8-mediated gene delivery. Hum Mol Genet 15, 1225-1236. Devine SM, Bartholomew AM, Mahmud N, Nelson M, Patil S, Hardy W, Sturgeon C, Hewett T, Chung T, Stock W, Sher D, Weissman S, Ferrer K, Mosca J, Deans R, Moseley A, Hoffman R (2001) Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol 29, 244-255. Friedenstein AJ, Gorskaja JF, Kulagina NN (1976) Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 4, 267-274. Friso A, Tomanin R, Alba S, Gasparotto N, Puicher EP, Fusco M, Hortelano G, Muenzer J, Marin O, Zacchello F, Scarpa M (2005) Reduction of GAG storage in MPS II mouse model

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Corradi-Perini et al: Gene therapy for MPSII Wraith JE, Cooper A, Thornley M, Wilson PJ, Nelson PV, Morris CP, Hopwood JJ (1991) The clinical phenotype of two patients with a complete deletion of the iduronate-2sulphatase gene (mucopolysaccharidosis II--Hunter syndrome). Hum Genet 87, 205-6. Young ID, Harper PS, Newcombe RG, Archer IM (1982) A clinical and genetic study of Hunter's syndrome. 2. Differences between the mild and severe forms. J Med Genet 19, 408-411.

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Gene Therapy and Molecular Biology Vol 12, page 175 Gene Ther Mol Biol Vol 12, 175-180, 2008

A characterization of genetic haplotypes in BRCA1 identifies linkage disequilibrium with a novel polymorphism in intron 7 Research Article

Josefa Salgado1,*, Carmen Gil1, Maitane Robles1, Cristina Gutierrez1, Carmen Reyna2, JesĂşs GarcĂa-Foncillas1,2 1 2

Clinical Genetics Unit Department of Oncology, University Clinic of Navarra (CUN). Avda. Pio XII, 36. 31008, Pamplona. Navarra. Spain

__________________________________________________________________________________ *Correspondence: Dr. Josefa Salgado, Clinical Genetic Unit, University Clinic of Navarra, Avda. Pio XII, 36, 31008 Pamplona, Navarra, Spain; Tel: + 34-948-255400 Ext: 1104; Fax: + 34-948-296795; e-mail: jsalgadog@unav.es Key words: BRCA1-novel polymorphism, BRCA1-haplotypes, breast cancer, linkage disequilibrium Abbreviations: Breast Cancer Information Core (BIC) Received: 27 June 2008; Revised: 22 July 2008 Accepted: 23 July 2008; electronically published: September 2008

Summary Inherited mutations in the BRCA1 gene are known to confer a predisposition to breast and ovarian cancer. Mutations, and low-frequency variants, have been invariably detected on any of the two major haplotypes H1 and H2, albeit more frequently on the dominant haplotype H1. Deleterious mutations, detected in our patients, were studied in relation with different haplotypes. A group of 77 patients with hereditary and familiar breast and/or ovarian cancer have been studied. BRCA1 gene analysis was done by direct sequencing. A seven polymorphic site cassette is used to define the BRCA1-haplotypes in our population. The frequency of a novel polymorphism found was studied in a control population of 100 unrelated healthy volunteers. Two new haplotypes (H2+H4 and H2+H5) not defined before have been found. We have first characterized a novel polymorphism (IVS7+16(TTC)nTTTTC) at intron 7 of BRCA1 gene. The IVS7+16(TTC)nTTTTC polymorphism shows a significant linkage disequilibrium with a cassette of seven polymorphisms that define H2 haplotype in this population. Seven out of eleven patients with deleterious mutations show the polymorphic site cassette. The (TTC)7/7 and (TTC)6/7 genotypes in the intron 7 could be markers for H1 and H2 haplotypes respectively. Larger patient population studies would be needed to study the association between BRCA1 mutations and the presence of the cassette.

major haplotypes H1 and H2, albeit more frequently on the dominant haplotype H1 (Frost et al, 2005). During BRCA1-haplotypes characterization we have found two new haplotypes (H2+H4 and H2+H5), and a novel polymorphism in BRCA1-intron 7 that shows significant linkage disequilibrium with a seven polymorphic site cassette. Deleterious mutations, detected in our patients, have been studied in relation to the haplotypes and the novel polymorphism.

I. Introduction Mutations in the tumor suppressor breast/ovarian cancer susceptibility gene BRCA1 have been identified in patients with high incidence of familial breast and ovarian cancer (Peto et al, 1999; Frank et al, 2002). According to BIC (Breast Cancer Information Core) database, more than 1500 distinct BRCA1 mutations and sequence variants have been reported. Approximately 10% of these variants reside within introns, or close to exon junctions, and may impact RNA stability and/or splicing. On the other hand, 10 different BRCA1-haplotypes were described (ShattuckEidens et al, 1997). Since then, other haplotypes have been described according to the population studied and the SNPs used (Freedman et al, 2005). Mutations, and lowfrequency variants, have been detected on any of the two

II. Materials and Methods A. Patients 77 cases that fulfilled familiar hereditary breast cancer criteria (Eccles et al, 2000) were selected. In addition, 100 healthy blood donors were also studied. All participants gave their informed consent prior to blood sample extraction.

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Salgado et al: A characterization of genetic haplotypes in BRCA1 Results showed frequencies of 44% (TTC)7/7, 46% (TTC)6/7 and 10% (TTC)6/6 in the 100 healthy donors. The statistical analysis indicated that the distribution was in agreement with that predicted by the Hardy-Weinberg equilibrium, with no statistical differences between patients and healthy donors (p=0.8). We studied the distribution of the IVS7+16(TTC)nTTTTC genotype among the different BRCA1 haplotypes (Table 2) in the patients. Given the fact that some haplotypes were present in a very low frequency we defined the “H1-like” haplotype (H1, H4 and H5, all of them lacking the cassette) and the “H2-like” haplotype (H2, H6, H2+H4 and H2+H5, all of them carrying the cassette). Results indicated that (TTC)7/7 and (TTC)6/7 polymorphisms were preferentially, and respectively, associated to “H1like” and “H2-like haplotypes; while (TTC)6/6 polymorphism was present in the two groups. We could establish that the (TTC)7/7 genotype segregated with the absence of the cassette, and the (TTC)6/7 genotype was associated with the presence of the cassette, with a statistically significance of p<0.001. Moreover, 11 out of 77 patients studied had deleterious variants (Table 3), and 7 out of these 11 patients carried the seven polymorphic site cassette (Table 2).

B. DNA isolation and sequencing Genomic DNA was automatically extracted (MagNaPure, Roche) from peripheral blood. Direct sequencing of the BRCA1 gene was done on an automated sequencer ABI PRISM® 3130XL (Applied Biosystems). Genetic variants were detected by comparison with the consensus wild-type sequence (Genbank Nº U14680), and were confirmed by repeated analysis, including reverse-primer sequencing. BRCA1 gene variants were used to define the BRCA1 haplotype, as described by Frost and colleagues in 2005.

C. Statistical Analysis The !2 or Fischer’s two-tailed exact test was used to compare the observed genotype distributions with those expected by the Hardy-Weinberg equilibrium. Linkage disequilibrium between BRCA1-intron 7 polymorphism and BRCA1-haplotypes was also evaluated.

III. Results BRCA1 haplotypes detected fell into two major (H1 and H2), two minor (H4 and H6) and three low-frequency (H5, H2+H4 and H2+H5) groups (Table 1). The most common haplotype (H1), which corresponds to the consensus sequence ( Miki et al, 1994; Smith et al, 1996) was found in the 36% of the alleles. The second most common haplotype H2 (28%) was characterized by a seven polymorphic site cassette (S694S, L771L, P871L, E1038G, K1183R, S1436S, S1613G). The H4 (10%) and H5 (3%) haplotypes contained respectively the sequence variants Q356R and S1040N, when compared to H1 haplotype. The H6 haplotype (17%) was identical to H2 except for the one additional variant D693N. Finally, two first described haplotypes were present: H2+H4 (4%) and H2+H5 (2%). A novel triplet deletion (TTC) in the intron 7 of the BRCA1 was identified in our patients, in a (TTC)7TTTTC region. Resulting genotypes were (TTC)7/7, (TTC)6/7 and (TTC)6/6 (Figure 1). The frequencies were 47% (TTC)7/7, 41% (TTC)6/7 and 12% (TTC)6/6 These variant alleles were not described before and, therefore, there were no information about its distribution in normal population. We studied the segregation of these alleles in the 77 patients and in 100 healthy volunteers, in order to compare if there were a particular (TTC)n genotype associated to disease. We could not find any association.

IV. Discussion The seven polymorphic site cassette identified in this study, S694S, L771L, P871L, E1038G, K1183R, S1436S and S1613G, have been reported previously to have no significant differences in allele frequencies between familial breast/ovarian cancer patients and the general population (Peto et al, 1999). Although these polymorphisms are actually part of the same haplotype, they are usually reported individually. In so doing, the reported degree of variation in the BRCA1 sequence may appear to be more substantial than it is. Shattuck-Eidens and coworkers described in 1997 a total of 10 haplotypes among 1590 alleles from a US population. Their reported haplotypes included those identified in our study (H1, H2, H4, H5 and H6), and up to ten low-frequency haplotypes absent among our 77 patients. It has been suggested that haplotypes H1 and H2 represent common chromosomes on which mutations and other variants occur.

Table 1. BRCA1-haplotypes found.

Sequence Variant c.1186A>G(p.Q356R) c.2196G>A(p.D693N) c.2201C>T(p.S694S) c.2430T>C(p.L771L) c.2731C>T(p.P871L) c.3232A>G(p.E1038G) c.3238G>A(p.S1040N) c.3667A>G(p.K1183R) c.4427T>C(p.S1436S) c.4956A>G(p.S1613G) Haplotypes frequency

Exon 11 11 11 11 11 11 11 11 13 16

HAPLOTYPES H1 H2 H4 + + + + + + + + 36% 28% 10%

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H5 + 3%

H6 + + + + + + + + 17%

H2+H4 + + + + + + + + 4%

H2+H5 + + + + + + + + 2%

Gene Therapy and Molecular Biology Vol 12, page 177

Figure 1. Direct sequencing showing the IVS 7 + 16(TTC)nTTTTC region where a TTC deletion has been found. The resulting variant alleles (TTC)7/7, (TTC)6/7 and (TTC)6/6 are shown in the figure.

Table 2. Distribution of the IVS7+16(TTC)nTTTTC genotype among the different BRCA1 haplotypes found.

Intron 7 genotypes IVS7+16 (TTC) 7/7 IVS7+16 (TTC) 6/7 IVS7+16 (TTC) 6/6

Intron 7 7 7

HAPLOTYPES H1 H2 H4 26* 8 18* 2* 4

H5 2

H2+H4

H2+H5

10* 3

* Deleterious mutations have been found in patients of these groups.

Table 3. Deleterious mutations found in 11 patients. Patient 1 2 3 4 5 6 7 8 9 10 11

Haplotype H1 H1 H1 H1 H2 H2 H2 H6 H6 H6 H6

IVS7 variant IVS7+16 (TTC) 6/6 IVS7+16 (TTC) 6/6 IVS7+16 (TTC) 7/7 IVS7+16 (TTC) 7/7 IVS7+16 (TTC) 6/7 IVS7+16 (TTC) 6/7 IVS7+16 (TTC) 6/7 IVS7+16 (TTC) 6/7 IVS7+16 (TTC) 6/7 IVS7+16 (TTC) 6/7 IVS7+16 (TTC) 6/7

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Mutation type Arg71Gly Arg71Gly Ala1708Glu Ala1708Glu 4314delAC 4156-7delAA Tyr261Stop codon Arg71Gly Arg71Gly Arg71Gly 1191delC

Salgado et al: A characterization of genetic haplotypes in BRCA1 Haplotype H3 would be related to haplotypes H1 and H2 by a recombination event between exons 11 and 13. Haplotypes H4 through H10 occur at low frequency and would require more recombination events to be related to the two common haplotypes (Shattuck-Eidens et al, 1997). Similarly, in our study, haplotypes H1 and H2 appear to be made up of ancient alleles that have independently acquired additional variants. In this way, H4 and H5 haplotypes on one hand, and H6 haplotype on the other, are identical to H1 and H2 respectively except for the one additional variant acquired on each case. Frequencies of our haplotypes vary compared to bibliographic data (Shattuck-Eidens et al, 1997; Frost et al, 2005), markedly for the minor haplotypes H4 and H6, suggesting a variety of ancestral populations. We have found two new lowfrequency haplotypes (H2+H4 and H2+H5) that resemble H2 haplotype but acquiring the variants that define H4 and H5 respectively. All these less frequent haplotypes could be expected to originate from different ancestral populations or they could have been transferred from one haplotype to the other by gene conversion. We have detected deleterious mutations in 11 of our patients. Considering the seven polymorphic site cassette, H2 and H6 haplotypes carry this cassette while it is absent in H1. Seven out of these 11 patients with deleterious mutations (64%) belong to the H2 or H6 haplotypes. Although larger patient population studies would be needed, there seems to exist a weak association between BRCA1 mutations and the presence of the cassette. To our knowledge, no triplet deletion in the intron 7 of the BRCA1 has yet been published. Analysis of intron 7 among breast cancer patients revealed size variations in the IVS7+16 (TTC)7TTTTC region due to a triplet deletion, being the variant alleles (TTC)7/7, (TTC)6/6 and (TTC)6/7. We have study the segregation of these alleles in the 77 patients and in 100 healthy volunteers, in order to compare if there is a particular (TTC)n genotype associated to disease. We could not find any association. Moreover, the frequency of this intron 7 variant among the healthy donors suggests that it would be a polymorphic site. TTC repeats are one of the most ubiquitous short tandem repeats, among 10 possible trinucleotide sequences, in the human genome ( Subramanian et al, 2003a,b) with several important biological functions in vivo. In Mycoplasma GAA-TCC repeats are responsible for the regulation of gene expression (Glew et al, 1998; Liu et al, 2000). Large expansions of the GAA-TTC sequence have been found in human genes like FXN, in association with the autosomal recessive FriedreichÂ´s ataxia (Pandolfo, 2000). A TTC deletion in intron 5 of the gene CYP19 encoding the P450 aromatase protein has been described, although without clear evidence of its relationship with breast cancer (Probst-Hensch et al, 1999). Biochemical studies suggest that GAA-TCC repeats per se are preferred sites for possible intramolecular recombinations. Although the frequency of recombination between any two homologous sequences, in the vast majority of cases, increases upon lengthening the recombining DNA fragment, GAA-TCC sequences demonstrated an exactly reverse relationship (Napierala et al, 2004). The role of the newly identified TTC deletion

would deserves further investigation in this context. We have studied the relevance of these size variations within the different haplotypes detected in our patients. From table 2 we can say that (TTC)6/6 genotype is detected on any of the two major haplotypes, H1 or H2, while (TTC)7/7 and (TTC)6/7 are in linkage disequilibrium invariably associated to the H1 haplotype (or its variants H4 and H5) and H2 haplotype (or its variants H6, H2+H4 and H2+H5), respectively. From the point of view of the seven polymorphic site cassette, (TTC)6/7 segregates with this cassette, opposite to (TTC)7/7 genotype that never carries this cassette. The (TTC)7/7 and (TTC)6/7 genotypes in the intron 7 could be markers for H1 and H2 haplotypes respectively.

Acknowledgments We are grateful to the patients are their families for their participation in our research project. We thank other members of the Clinical Genetics Unit for their support and encouragement.

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Smith TM, Lee MK, Szabo CI, Jerome N, McEuen M, Taylor M, Hood L, King MC (1996) Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1, Genome Res 6, 1029-1049. Subramanian S, Madgula VM, George R, Mishra RK, Pandit MW, Kumar CS, Singh L (2003a) Triplet repeats in human genome: distribution and their association with genes and other genomic regions, Bioinformatics 19, 549-552. Subramanian S, Mishra RK, Singh L (2003b) Genome-wide analysis of microsatellite repeats in humans: their abundance and density in specific genomic regions, Genome Biol 4, R13.

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Gene Therapy and Molecular Biology Vol 12, page 181 Gene Ther Mol Biol Vol 12, 181-188, 2008

Molecular targets in medullary thyroid carcinoma Review Article

Sam W Moore Division of Paediatric Surgery, University of Stellenbosch, Cape Town, South Africa

__________________________________________________________________________________ *Correspondence: Sam W Moore, Division of Paediatric Surgery, Faculty of Medicine, University of Stellenbosch, P.O. Box 19063, 7505, Tygerberg, South Africa; Tel: +27-21 9389428; Fax: +27-21 9337999; E-mail: swm@sun.ac.za Key words: medullary thyroid carcinoma, RET structure, RET activation, Molecular targets, prophylactic thyroidectomy Abbreviations: epidermal growth factor receptor, (EGFR); Familial Medullary thyroid carcinoma, (FMTC); GDNF-family receptoralpha, (GFR!); glial cell line-derived neurotrophic factor, (GDNF); Hirschsprung’s disease, (HSCR); kinase, (KD); Multiple Endocrine Neoplasia, (MEN); papillary thyroid carcinoma, (PTC); REarranged during Transfection, (RET); receptor tyrosine kinases, (RTK); serum VEGF, (sVEGF-C); vascular endothelial growth factor receptors, (VEGF) Received: 26 June 2008; Revised: 15 July 2008 Accepted: 21 July 2008; electronically published: September 2008

Summary A high percentage of thyroid cancer has been associated with a number of oncogenic genetic variations. The RET proto-oncogene (REarranged during Transfection; RET) is thought to play an important role in the etiology of thyroid tumours and is clearly implicated in Medullary thyroid carcinoma. RET variations which confer oncogenic gain of function, appear to result in the development of cancer due to uncontrolled cellular proliferation as well as failure to undergo normal differentiation and the loss of apoptotic functions. Understanding the biological role of the RET proto-oncogene in thyroid carcinoma is an important area of biomedical research which leads not only to better understanding of the pathophysiological changes involved in oncogenesis but also determines the optimal management and identifies molecular targets allowing the development of novel therapeutic approaches. This review looks at the structure and function of the RET proto-oncogene, its understood method of activation and important molecular targets identified to date. It also studies agents used for novel molecular targeting in treatment of thyroid carcinomas.

important role in the etiology of thyroid tumours because of the numbers of rearrangements frequently present in thyroid tumour cells (Porter and Vaillancourt, 1998) and its clearly defined role in Medullary thyroid carcinoma (Machens and Dralle, 2007a). RET variations which confer oncogenic gain of function, appear to result in the development of cancer due to uncontrolled cellular proliferation as well as failure to undergo normal differentiation and the loss of apoptotic functions. The RET proto-oncogene plays a pivotal role in at least 4 clinical syndromes (Donis-Keller et al, 1993; Mulligan et al, 1993). Multiple Endocrine Neoplasia (MEN) Type 2 A and B and Familial Medullary thyroid carcinoma (FMTC) (Mulligan et al, 1993; Carlson et al, 1994; Eng et al, 1995) and Hirschsprung’s disease (HSCR) (Luo et al, 1993; Edery et al, 1994; Takahashi et al, 1985), in a unique “switch on”/ “switch off” manner (van Heyningen V, 1994).

I. Introduction Thyroid cancer has been associated with a number of oncogenic genetic abnormalities which are fairly unique due to the fairly high percentage relating to genetic rearrangements and chromosomal translocations (eg RET proto-oncogene, RET/PTC, NTRK, ras mutation, Braf mutation, met genes and Pax 8/PPARgamma etc). In addition, tumor suppressor gene malfunction (eg RB1 and p53) appear to be associated with anaplastic thyroid tumors (Moretti et al, 2000) and other genes (eg miR) are thought to further modulate the oncogenic response (eg to RET/ PTC (Jazdzewski et al, 2008). Study of these genetic variations is important to not only understand the fundamental mechanisms of oncogenesis and the development of molecular target therapies but to also identify those at risk with resultant prophylactic protocols. Recent research has shown that receptor tyrosine kinases (RTK) are vital oncogenic agents as RTK activation promotes cellular angiogenesis and increases cellular proliferation, invasion and metastases. The RET proto-oncogene (REarranged during Transfection; RET) is regarded as a proto-type RTK and is thought to play an

II. The structure of the RET protooncogene Since the initial association of the RET protooncogene (10q 11.2) with MEN2 syndromes in 1993 181

Moore: Molecular targets in medullary thyroid carcinoma (Donis-Keller et al, 1993; Mulligan et al, 1993), exploration of its oncogenic role has resulted in the rapid advancement of knowledge as to its structure and possible mechanisms of action. The RET (Rearranged during Transfection) protooncogene consists 21 exons which lie over a 55Kb area at 10q11.2 (Ceccherini et al, 1993). It remains one of most important proto-oncogenes within the human genome, encoding a RTK which is a critical signaling component required for normal nervous system and kidney development, as well as spermatogenesis (Takahashi et al, 1985). RET is mainly expressed in cells of neural origin and is involved in cellular proliferation, migration and differentiation of cells (Pachnis et al, 1993). Like other RTKs, RET is made up of at least 3 functional areas (viz: extracellular, trans-membrane and intracellular regions) each appearing to have specific functions (Ceccherini et al, 1993). In terms of RET activationin the extracellular domain, the cysteine molecules appear to be key areas. A possible explanation is that genetic variation impairs the correct sequencing of the RET protein thus influencing its function (Asai et al, 1995). In addition to the usual mechanism, extracellular domain mutations (eg C620S) may affect polarity due to the unfolding of RET, which has also been shown to also result in TK activation (Carlomagno et al, 2006). The function of transmembrane domain mutations are unclear but in recent studies into the transmembrane S649L RET mutation appears to be associated with lateonset non-aggressive disease. (Colombo-Benkmann et al, 2008). Activation of RET RTK via the intracellular or TK portion of the gene activation is easier to understand. On the one hand, malfunction at critical areas probably affects molecule-molecule signaling connections with other RETrelated molecules (eg GDNF) leading to defective downstream signaling. RET receptor dimerization as well as RTK activation in the absence of the normal ligand cofactors may thus result (Carlomagno et al, 1997). On the other hand, other possible oncogenic cellular mechanisms include the autophosphorylation of RET (eg. MEN2B) as well as the modifying the sub cellular distribution of the active kinase (Santoro et al, 2002). The uncommon co-segregation of HSCR and MEN2 in the same patient is a fascinating finding as it involves both gain and loss of function in the same patient (Verdy et al, 1982; Mulligan et al, 1994; Borst et al, 1995; Blank et al, 1996; Caron et al, 1996; Peretz et al, 1997; Borrego et al, 1998; Decker and Peacock, 1998; Romeo et al, 1998; Sijmons et al, 1998; Inoue et al, 1999; Pasini et al, 2002; Dvorakova et al, 2005; Moore and Zaahl, 2008). The most commonly reported mutations associated with MEN2HSCR are C620R(65;162) and occasionally C620S and rarely C620W (Decker et al, 1998). This has led to the concept of the so-called â&#x20AC;&#x153;Janus geneâ&#x20AC;? mutation which like the Roman god of doorways can face in both directions. The association is not limited to codon 620 cysteine substitutions, however. Although it must be bourne in mind that the prevalence may differ between Paediatric surgical centers specializing in HSCR and endocrine surgical centers specializing in MTC (5% at best), a 32 35% association has been reported with codon 618 (C

618R2x >C 618S-) missense mutations (Decker and Peacock, 1998) as well as a 15-20% association with codon 609 (C 609Y mostly ) and a 4% association with 611 mutations. Genetic variation in these areas possibly resulting in the malfunction of the same or similar vital signaling pathways. The reasons why this occurs as well as the factors modifying its phenotypic expression remain areas of ongoing research. In addition to the potential tyrosine kinase activation via fusion genes, there are yet other genetic variations identified in RET malfunction (Knowles et al, 2006). The first group are those regions in the extracellular portion of the gene which control protein folding (mutations which result in a non-folded RET protein at the endoplasmic reticulum). Those occurring within the 1st cadherin-like domain in HSCR (Kjaer and Ibanez, 2003) are thought to influence its pathogenesis (Griseri, 2005). Those affecting one of the 6 cysteine radicals (viz: 609, 611, 618, 620, 630 and 634 positions) affect gene function and influence RET activity resulting in MEN2A (Carlomagno et al, 1996; Iwashita et al, 1996). The second group of RET gene variations are those within the terminal tyrosine kinase (KD) intracellular region of the gene (eg exon 16 M918/T mutations in MEN 2B (Carlomagno et al, 1996; Iwashita et al, 1996). A third area is emerging in those gene variations which affect the docking sites for downstream signaling pathways(Geneste et al, 1999). There is a further group of MEN-related genetic variations, associated with non-cysteine areas of gene which raises interesting questions as to the oncogenic mechanism. This latter group appeared to occur more frequently than expected in our own series(Moore et al, 2007) in keeping with other genetic pools (Pinna et al, 2007).

III. RET-proto-oncogene activation Oncogenic RET mutations mostly occur de novo in sporadic MTC and their effect on protein synthesis and function clearly involves a number of downstream molecular signaling pathways (Carlomagno et al, 1996). Hereditary MTC can with rare exceptions be traced back in family trees (Machens and Dralle, 2008). Genetic variations impair the RET tyrosine kinase response to tyrosine kinase activation, thus appearing to dictate downstream signaling cascade response. (Andl and Rustgi, 2005) Known downstream signaling pathways include activation of the MAPK and Ras/ERK molecular signaling cascades by a mechanism which involves grb2/mSOS recruitment (Edery et al, 1994). The multidocking intracellular portion of the RET gene appears to be both vital to tyrosine kinase function as well as downstream signaling due to the number of signaling molecules that interact there( eg. Shc, Src, FRS2, IRS1, Gab1/2, and Enigma) (Santoro et al, 1995a; Arighi et al, 1997; Lorenzo et al, 1997) Competitive Shc versus Frs2 recruitment at Tyr 1062 has been shown to mediate increased cell-survival (Lundgren et al, 2006) An alternative mechanism appears to rather signal via PI3K and Akt upregulation following recruitment of grb2/gab2 (Alberti et al, 1998). These downstream adaptor protein functions are particularly important as they play a vital

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Gene Therapy and Molecular Biology Vol 12, page 183 function in effecting the multiple diverse roles played by the RET receptor during development. The unfolding of RET due to extracellular domain mutations (eg C620S) affects polarity which may result in tyrosine kinase activation (Carlomagno et al, 2006). Mutation in the Tyrosine-kinase rich region (eg. in 95% of MEN 2B patients), on the other hand, alters the substrate specificity of RET tyrosine kinase and appears to induce a different set of downstream signaling genes from that carrying the MEN 2A mutation. This appears to function via the sic protein (Pelicci et al, 2002) which is known to induce downstream activation of the Ras/mitogen-activated protein- and P13K/Akt signaling cascades (Salvatore et al, 2001;Wong et al, 2005). It may well have bearing on the speed and aggressiveness of the oncogenic process as well as influencing the penetrance of the MEN 2 phenotype. Both the phosphorylated and nonphosphorylated forms have been shown to have a preorganized activation loop and are competent to bind ATP and substrate (Knowles et al, 2006). Recruitment at the phosphorylated Tyr 1062 intracellular site has been shown to be particularly important as it is recruited and activated by both the Ret adaptor proteins (Shc and Frs2) activated by MEN2A or by MEN2B , as well as via the rearranged Ret oncoproteins (eg Ret/ptc2) involved in papillary and other thyroid carcinomas (Arighi et al, 1997). This function does not take place in isolation, but is activated by binding to a ligand complex formed by the glial cell line-derived neurotrophic factor (GDNF) (Jing et al, 1996) and its ligand co-receptor, the GDNF-family receptor-alpha (GFR!) receptors. These receptors are GPI linked thus permitting dimerization (Baloh et al, 1997) and are known to involve lipid rafts to promote GDNF interaction (Saarma, 2001). In addition, further RET/ GDNF signaling pathways appear to be partly controlled by Heparan sulfate interactions (Wang et al, 2007). Demonstration of Tyrosine kinase activity upregulation by the RET/PTC fusion gene in papillary thyroid carcinoma (PTC) (Grieco et al, 1990) is a further known mechanism of increased RTK activity resulting in oncogenesis. In this case, RET activation which results from gene dimerization is mediated through coiled-coil motifs in the NH2 terminus of the chimeric protein(Croyle et al, 2008). Following the Chernobyl disaster (ionizing radiation)in Russia, papillary thyroid tumors occurring in children show a high prevalence of RET fusion gene rearrangements with at least 11 different gene fusions being described (the majority being a RET/PTC3 rearrangement) (Rabes and Klugbauer, 1998). Other less frequent rearrangements are also implicated in PTC and include the RET/PTC1 and some more novel rearrangements (eg RET/PTC5). Activating RET mutations are not confined to PTC but are also associated with other thyroid carcinomas (Nikiforov et al, 1997). The RET/PTC fusion gene occurs as a result of a balanced intrachromosomal inversion with fusion of the RET 3' portion to the 5' portion of various genes (eg BRAF, RET, or RAS genes of the MAPK system) to form RET/PTC and similar chimeras (Adeniran et al, 2006). As a result, the tyrosine kinase RET domain then becomes

controlled by 5' fused regulatory sequences of other genes with dimerization potential (Klugbauer and Rabes, 1999). RET receptor tyrosine kinase gene activation then occurs leading to initiation of oncogenesis. Other genes (eg EGFR) may also be involved in RET kinase activation, signaling, and the resultant growth stimulation within the final oncogenic pathway. The epidermal growth factor receptor (EGFR) has been implicated because of experimental evidence whereby the kinase inhibitor PKI166 has been shown to decrease RET/PTC kinase autophosphorylation and activation of downstream signaling cascades in thyroid cells (Croyle et al, 2008). Additional factors influencing gene penetration and the signaling pathways involved in metastasis and invasion are less clear, but it would appear that factors such as gene methylation may play an epigenetic role by altering the phenotype without affecting the genotype.

IV. Mechanisms of RET activation Abnormal disulphide homodimerization of RET is the most likely explanation of gene activation following cysteine-substitution mutations at RET codons 609, 611, 618, 620, 630 and 634 (Chappuis-Flament et al, 1998; Takahashi et al, 1999). The induction of a disulfide-linked homodimerization particularly in exon 11, (codon 634 point mutations) (Asai et al, 1995; Santoro et al, 1995b) due to genetic mutations results in oncogenesis due to the removal of a half of the intermolecular bond, thus allowing an abnormal bond with a second mutant molecule to occur, resulting in decreased RET at the plasma membrane (Arighi et al, 2004). One of the problems in the hypothesis is that it does not explain the MEN2-HSCR association and why certain commonly occurring RET variations (eg S767R and P1039L) fail to show RET malfunction (Pelet et al, 1998), whereas others (eg R231H) do (Bordeaux et al, 2000). This observation raises the role of RET ligands (eg GDNF) in the survival and differentiation of developing neurons but also raises the case for a possible second intracellular mechanism (Geneste et al, 1999) in the pathogenesis of MTC. An alternative explanation is the possible trapping of mutant RET molecules in the endoplasmic reticulum in the MEN2- HSCR association or the modifying effect of secondary inter-related pathways. In this regard, other genes (eg EGFR, VEGF) have also been shown to play a role in RET kinase activation, signaling, and the resultant growth stimulation occurring within the final oncogenic pathway. One example is the EGFR, which has been implicated because of experimental evidence whereby the kinase inhibitor PKI166 has been shown to decrease RET/PTC kinase autophosphorylation and activation of downstream signaling cascades in thyroid cells (Croyle et al, 2008). In addition, VEGF and angiogenesis are also important factors in advanced tumor biology and serum vascular endothelial growth factor receptors (sVEGF-C) levels have been shown to have a strong correlation nodal metastases in advanced PTC (Yu et al, 2008). Crosstalk between the vascular endothelial growth factor (VEGF A/VEGF2) and the GDNF/RET signaling pathways has 183

Moore: Molecular targets in medullary thyroid carcinoma also been reported (Tufro et al, 2007), suggesting a possible link in their function. Many of the tyrosine kinase inhibitors currently under study are multiple tyrosine kinase inhibitors and include VEGF and EGFR tyrosine kinase inhibition

directedâ&#x20AC;? timing of surgery. A recent long term follow-up study of 46 RET gene carriers categorized the risk to children and young adults (aged 4 - 21 years) into level 1 (low risk) and level 2 mutations (intermediate to high risk) (Frank-Raue et al, 2006). Level 1 mutations (n=11) appeared to be associated with variations in codons 790, 791, 804 and 891 and had a high incidence of cure. By way of contrast, 5 (14%) of the 35 level 2 patients (mutations of codons 618, 620, 630 and 634), had ongoing disease. On long term follow-up (mean 6.4 yrs), 2 of those with 634 mutations, developed other MEN manifestations (viz: hyperparathyroidism and bilateral Phaeochromocytoma). This latter risk would probably increase with time and long-term follow-up is necessary. It is now generally accepted that RET 609 mutations should also be added to Level 2 (Machens and Dralle, 2007b). Level 3 mutations include those at 883 and the 918 positions However, it appears to be important to separate the sporadic from familial MTC as Elisei and colleagues in 2008 observed a significantly (p<0.0001) higher representation of non-cysteine codons in the sporadic group as opposed to the familial group which were mostly cysteine-related. Furthermore, there appears to be a difference in prevalence between various population groups in terms of the frequency of RET mutations encountered. Recent increase in exon 13-15 mutations in a German series (Frank-Raue, 2007), does not appear to be necessarily reflected in certain other population groups (Dvorakova et al, 2008). In addition, a study of MTC in Sardinia has shown a 59% prevalence of the V804M mutation (against the expected 5% of other European populations) (Pinna, 2007). This demonstrates the importance of the genetic background in evaluating the RET gene in these patients. Codon-risk protocols are of considerable value but not completely watertight and the age of MTC onset does not always appear to be consistent. Recent advances into the significance of RET proto-oncogene signaling and the molecular pathways of RET signal transduction in the development of MTC, and oncogenesis have created new potential treatment modalities with exciting new possibilities for management.

V. Potential Molecular targets in MTC and thyroid tumours Understanding the biological role of the RET protooncogene in thyroid carcinoma is an important area of biomedical research which leads not only to better understanding of the pathophysiological changes involved in oncogenesis but also determines the optimal management and identifies molecular targets thus allowing the development of novel therapeutic approaches. Current understanding of RET function suggests additional molecular mechanisms and raises the case for a possible second intracellular mechanism (Geneste et al, 1999) in the pathogenesis of certain MTC carcinomas.

VII. Mutation testing prophylactic thyroidectomy

and

There appears to be fairly consistent associations between genotype and phenotype in MTC. The assessment of risk by RET gene mutation analysis and the ability to predict potential carrier states in family members with genotype-phenotype correlations as well as the evidence for an age-related progression of MTC, is becoming a reality. In general, MEN 2A mostly associated with variations in the 6 cysteine radicals in the extracellular domain (with varying degrees of risk) and 95% of MEN 2B patients are associated with a point mutation (Methionine threonine) in exon 16(M918/T) of the intracellular domain. This molecular knowledge has led to the introduction of prophylactic surgery and removal of the target organ (total thyroidectomy) in affected individuals (particularly children) (Skinner, 2003), thus preventing the onset of cancer.

VIII. Therapeutic implications of molecular RET mechanisms in Cancer A clear need for new therapeutic approaches exists due to the chemo and radio-insensitivity of the majority of these tumors, particularly when metastatic. Understanding of the molecular mechanisms involved in oncogenesis and potential molecular target sites has, in turn, led to exciting new therapeutic options by targeting of the RET tyrosine kinase action in the treatment of MEN type 2 and MTC (Ball, 2007).

B. Novel molecular targets in treatment of thyroid carcinomas Current molecularly based strategies for MTC include tyrosine kinase inhibitors, gene therapy of significant RET mutations and the promotion of cellular death (Petrangolini et al, 2006), as well as monoclonal antibodies against oncogenic proteins (Drosten and Putzer, 2003) and, nuclease-resistant factors that both recognize and inhibit RET (de Groot et al, 2006). A number of multiple kinase inhibitor drugs have shown experimental activity and are currently entering clinical trials. Of particular current interest in this regard, is the development of the RET-targeting orally administered tyrosine kinase inhibitors (eg: ZD6474; RPI-1 (Cuccuru et al, 2004) BAY 43-9006 (Carlomagno et al, 2006). These agents are mostly multi-kinase inhibitors and include effects on VEGF as well as affecting certain other kinases

A. Risk level and codon-directed surgery in MTC and FMTC Effective preventative management depends on early identification of gene carriers and prophylactic thyroidectomy prior to any clinical or biochemical abnormalities. Machens and Dralle, have stratified in 2007 the MTC risk into three categories according to the mutation-related aggressiveness, giving rise to a concept of â&#x20AC;&#x153;codon184

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which results in further angiogenesis inhibition (Petrangolini et al, 2006). Experience with ZD6484 (a specific RET, Epidermal growth factor, VEGF tyrosine kinase inhibitor ) and other multi-kinase inhibitors (eg sorafinib) are currently reporting encouraging early results (Gupta-Abramson et al, 2008) in metastatic tumors. Sorafinib appears to be particularly interesting as it inhibits a wide spectrum of kinases known to be active in thyroid tumours (including Raf kinase, VEGF, platelet-derived growth factor receptor, and RET tyrosine Kinases). This is bourne out by a reported rapid response to sorafenib/tipifarnib therapy in a patient with an exon 11 RET mutation. The agent NVPAST487 (an N,N'-diphenyl urea) has been shown to block tumor growth and Calcitonin gene expression in cell lines with RET activating mutations by inhibiting RET autophosphorylation and downstream signaling. Early results with these modalities appear encouraging with one research group reporting a statistically significant 51% tumour inhibition, a 210 % increase in apoptotic cells, a 47% loss of cellularity and a 37% decrease in micro vessel density (Petrangolini et al, 2006). Similar results are also being reported for Axitinib (AG-013736), a potent VEGF 1, 2, and 3 inhibitor, which has been experimentally shown to reduce transplanted MTC by 89% in an animal model (Johanson et al, 2007) and is active against all histologic subtypes of advanced thyroid cancer (Cohen et al, 2008). On the other hand, not all tumors respond and certain studies show that these agents may be effective in those without the usual RET mutations (Keno-Stuart et al, 2007). These observations are supported by observed differences in RET ultrastructure (Knowles et al, 2006), and raises questions as to the possibility of a secondary oncogenic mechanism in certain tumors. In addition to these TK inhibitors, an array of monoclonal antibodies has been introduced as targeted treatment in the treatment of head and neck, breast and lung cancers. They target receptors or ligands in cellular proliferation and inflammation pathways and prevent phosphorylation by blocking the ATP-binding domain. This results in inhibition of signal transduction required for upregulation of function.

IX. Conclusion Molecular targeting definitely appears to be a step forward in the management of MTC and other thyroid carcinomas. On the other hand, it would appear that more work needs to be done in this area to streamline the molecular-directed treatment and identify those cases most likely to benefit from this form of treatment.

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Pelet A, Geneste O, Edery P, Pasini A, Chappuis S, Atti T, Munnich A, Lenoir G, Lyonnet S, Billaud M (1998) Various mechanisms cause RET-mediated signaling defects in Hirschsprung's disease. J Clin Invest 101, 1415-1423. Pelicci G, Troglio F, Bodini A, Melillo RM, Pettirossi V, Coda L, De Giuseppe A, Santoro M, Pelicci PG (2002) The neuron-specific Rai ShcC adaptor protein inhibits apoptosis by coupling Ret to the phosphatidylinositol 3-kinase/Akt signaling pathway. Mol Cell Biol 22, 7351-7363. Peretz H, Luboshitsky R, Baron E, Biton A, Gershoni R, Usher S, Grynberg E, Yakobson E, Lapidot M (1997) Cys 618 Arg mutation in the RET proto-oncogene associated with familial medullary thyroid carcinoma and maternally transmitted Hirschsprung's disease suggesting a role for imprinting. Hum Mutat 10, 155-159. Petrangolini G, Cuccuru G, Lanzi C, Tortoreto M, Belluco S, Pratesi G, Cassinelli G, Zunino F (2006) Apoptotic cell death induction and angiogenesis inhibition in large established medullary thyroid carcinoma xenografts by Ret inhibitor RPI-1. Biochem Pharmacol 72, 405-414. Pinna G, Orgiana G, Riola A, Ghiani M, Lai ML, Carcassi C, Mariotti S (2007) RET Proto-Oncogene in Sardinia, V804M Is the Most Frequent Mutation and May Be Associated with FMTC/MEN-2A Phenotype. Thyroid 17, 101-104. Porter AC Vaillancourt RR (1998) Tyrosine kinase receptoractivated signal transduction pathways which lead to oncogenesis. Oncogene 17, 1343-1352. Rabes HM, Klugbauer S (1998) Molecular genetics of childhood papillary thyroid carcinomas after irradiation, high prevalence of RET rearrangement. Recent Results Cancer Res 154, 248-264. Romeo G, Ceccherini I, Celli J, Priolo M, Betsos N, Bonardi G, Seri M, Yin L, Lerone M, Jasonni V, Martucciello G (1998) Association of multiple endocrine neoplasia type 2 and Hirschsprung disease. J Intern Med 243, 515-520. Saarma M (2001) GDNF recruits the signaling crew into lipid rafts. Trends Neurosci 24, 427-429. Salvatore D, Melillo RM, Monaco C, Visconti R, Fenzi G, Vecchio G, Fusco A, Santoro M (2001) Increased in vivo phosphorylation of ret tyrosine 1062 is a potential pathogenetic mechanism of multiple endocrine neoplasia type 2B. Cancer Res 61, 1426-1431. Santoro M, Carlomagno F, Romano A, Bottaro DP, Dathan NA, Grieco M, Fusco A, Vecchio G, Matoskova B, Kraus MH, Di Fiore PP (1995a) Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science 267, 381-383. Santoro M, Grieco M, Melillo RM, Fusco A, Vecchio G (1995b) Molecular defects in thyroid carcinomas, role of the RET oncogene in thyroid neoplastic transformation. Eur J Endocrinol 133, 513-522. Santoro M, Melillo RM, Carlomagno F, Fusco A, Vecchio G (2002) Molecular mechanisms of RET activation in human cancer. Ann N Y Acad Sci 963, 116-121. Sijmons RH, Hofstra RM, Wijburg FA, Links TP, Zwierstra RP, Vermey A, Aronson DC, Tan-Sindhunata G, BrouwersSmalbraak GJ, Maas SM, Buys CH (1998) Oncological implications of RET gene mutations in Hirschsprung's disease. Gut 43, 542-547. Skinner MA (2003) Management of hereditary thyroid cancer in children. Surg Oncol 12, 101-104. Takahashi M, Iwashita T, Santoro M, Lyonnet S, Lenoir GM, Billaud M (1999) Co-segregation of MEN2 and Hirschsprung's disease, the same mutation of RET with both gain and loss-of-function? Hum Mutat 13, 331-6. Takahashi M, Ritz J, Cooper GM (1985) Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell 42, 581-588.

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Let-7, miR-125, miR-205, and miR-296 are prospective therapeutic agents in breast cancer molecular medicine Research Article

Debmalya Barh1*, Sanjeeb Parida1, Bibhu Prasad Parida1, Geetha Viswanathan2 1 2

Centre for Genomics and Applied Gene Technology, IIOAB, Nonakuri, Purba Medinipur, West Bengal -721172, India. Indian Holistic Medical Academy, EB Colony, Thanjavur, Tamil Nadu - 613006, India.

__________________________________________________________________________________ *Correspondence: Debmalya Barh, Ph.D., Centre for Genomics and Applied Gene Technology, IIOAB, Nonakuri, Purba Medinipur, West Bengal -721172, India. Email: dr.barh@gmail.com Tel: +91-944-955-0032. Key words: breast cancer, critical disease pathway, cancer, drug targets, gene therapy, key nodes, let-7, microRNA Abbreviations: Acute lymphoblastic leukemia, (AL); Breast cancer, (BC); Burkitts lymphoma; (BL); Estrogen receptor, (ER); microRNA let-7, (let-7); Loss of heterozygocity, (LOH); micro RNAs, (miRs); Replication factors, (RCFs) Received: 10 July 2008; Revised: 5 August 2008 Accepted: 22 August 2008; electronically published: September 2008

Summary Increasing evidences in recent years demonstrate that several biological processes and disease pathogenesis are regulated by micro RNAs (miRs) and restoration of normal miR activity can be new way of treating cancers. Several genetic alterations and deregulation of miRs have been reported in breast cancer. Similarly, side effects of conventional chemotherapeutic drugs are well known. In this research, using a broad bioinformatics approach we have identified critical disease pathways and drug targets in female breast cancer. Replacement therapy with let-7 is already under clinical trails for lung cancer. Here we have shown that restoration of let-7 along with miR-125 or miR-205 or miR-296 can potentially inhibit all critical disease pathways involved in breast cancer irrespective of patient specific molecular profile. Results also suggest that these miRs might be the future therapeutic agents in breast cancer molecular medicine with out side effects.

metabolism (Esau et al, 2006), miR-143 and miR-206 and in adiposity and muscle differentiation (Esau et al, 2004; Kim et al, 2006), miR-1 and miR-133 ES differentiation, mesoderm formation, and heart development and physiology (Chen et al, 2006; Zhao et al, 2007; Ivey et al, 2008), miR-208 in cardiomyocyte hypertrophy (van et al, 2007), miR-34a, miR-125b, and miR-128 in apoptosis (Lukiw and Pogue, 2007; Tarasov et al, 2007), miR-34a, miR-34b, miR-34c, miR-93, and miR-214 in aging (Kumamoto et al, 2008; Maes et al, 2008), miR-181 in Bcell progenitor determination and lineage differentiation and T-cell receptor signaling (Chen et al, 2004), and miR155 in antigen presentation (Rodriguez et al, 2007). Several reports suggest that miRs are also involved in various pathological conditions. For example, miR-203 and miR-146 in inflammatory diseases, miR-196 and miR122 in anti-viral response (Jopling et al, 2006; Sonkoly et al, 2008), miR-29a/b-1, miR-107 in Alzheimer's disease (HĂŠbert et al, 2008; Wang et al, 2008), miR-433 variation

I. Introduction A. miRs regulate biological processes and diseases microRNAs (miRs) are endogenous non-coding pool of small RNA molecules of 20-24 nucleotides in length (Ambros, 2001; Carrington and Ambros, 2003; Bartel, 2004) regulate gene expression by cleaving target mRNAs or by complementarity base pairing at 3 UTRs inhibiting translation of target mRNAs (Lai, 2002; de Moor et al, 2005; Robins and Press, 2005; Stark et al, 2005; Sun et al, 2005) and thus regulate biological processes. The total number of miRs may be more than 1% of the total protein coding genes in different species (Lai et al, 2003; Lim et al, 2003; Lim et al, 2003) and according to computational prediction around 30% of protein-coding genes may be targeted by miRs (Berezikov et al, 2005; Lewis et al, 2005). miRs have been found to involved in several biological processes. For example, miR-9 in insulin secretion (Plaisance et al, 2006), miR-122 in lipid 189

Barh et al: Let-7, miR-125, miR-205, and miR-296 as prospective therapeutic agents in breast cancer molecular medicine in Parkinson disease (Wang et al, 2008), miR-19a and miR-21 in Cowden syndrome (Pezzolesi et al, 2008), miR99a, let-7c, miR-125b-2, miR-155, and miR-802 overexpression in down syndrome (Kuhn et al, 2008), and miR-17-92 in autoimmune diseases (Xiao et al, 2008).

al, 2008), colorectal cancer (Michael et al, 2003; Akao et al, 2006; BandrĂŠs et al, 2006; Xi et al, 2006; Lanza et al, 2007; Nakagawa et al, 2007; Slaby et al, 2007; Asangani et al, 2008; Grady et al, 2008; Schetter et al, 2008), glioblastoma (Chan et al, 2005; Ciafre et al, 2005; Gillies et al, 2007; Kefas et al, 2008), hepatocellular carcinoma (Kutay et al, 2006; Murakami et al, 2006; Gramantieri et al, 2007; Meng et al, 2007; Huang et al, 2008; Jiang et al, 2008; Varnholt et al, 2008; Wang et al, 2008; Wong et al, 2008; Yang et al, 2008), lung cancer (Lewis et al, 2003; Takamizawa et al, 2004; Hayashita et al, 2005; Fabbri et al, 2007; Hurteau et al, 2007; Inamura et al, 2007; Matsubara et al, 2007; Hu et al, 2008; Ventura et al, 2008; Weiss et al, 2008), lymphomas (Metzler et al, 2004; Cimmino et al, 2005; Eis et al, 2005; He et al, 2005; Kluiver et al, 2005; Akao et al, 2007; Lawrie et al, 2007; Lum et al, 2007; Mi et al, 2007; Motsch et al, 2007; Rinaldi et al, 2007; Sampson et al, 2007; Xiao et al, 2008; Bueno et al, 2008; Faber et al, 2008; Rai et al, 2008; Lawrie et al, 2008; Navarro et al, 2008; Roehle et al, 2008), papillary thyroid carcinoma (PTC) (He et al, 2005; Weber et al, 2006; Tetzlaff et al, 2007; Visone et al, 2007; Jazdzewski et al, 2008; Nikiforova et al, 2008; Mitomo et al, 2008; Takakura et al, 2008), testicular germ cell cancer (Voorhoeve et al, 2006), and several other cancers (Table 1). High throughput miR expression data show that several miRs are differentially expressed in various cancers including breast cancer (Nam et al, 2008) (Table 2).

B. miRs and cancer 52.5% human miR genes are located at chromosomal locus those are frequently altered in human cancers (Calin et al, 2004). Increasing evidences suggest that miRs are directly involved in cancer pathogenesis and thus their expression profiles are useful for cancer diagnosis, prognosis, staging, and treatment. miRs are reported to act as oncogenes (oncomirs) (Hayashita et al, 2005; He et al, 2005; O'Donnell et al, 2005; Hammond, 2006; Cho, 2007) and tumor suppressor genes (Ambros, 2004; Bartel, 2004; Miska et al, 2004; Thomson et al, 2004; Tavazoie et al, 2008). Metastatic and angiogenic properties of miRs are also in report (Ma et al, 2007; Huang et al, 2008; Negrini and Calin, 2008; Tavazoie et al, 2008; Urbich et al, 2008). Recent studies have revealed that miRs are frequently deregulated and regulate pathological events in most common cancers. miR deregulation have been reported in breast cancer (Iorio et al, 2005; Hossain et al, 2006; Hurteau et al, 2007; Lowery et al, 2007; Ma et al, 2007; Scott et al, 2007; Sempere et al, 2007; Si et al, 2007; Yu et al, 2007; Zhu et al, 2007; Cissell et al, 2008; Frankel et al, 2008; Huang et al, 2008; Lehmann et al, 2008; Tavazoie et Table 1. miR deregulation in various cancers. Cancer Acute lymphoblastic leukemia

Upregulated miRs miR-155

miR-128a and miR-128b Anaplastic thyroid cancer Diffuse large B cell lymphoma

Downregulated miRs

References Costinean et al, 2006

miR-203 let-7b and miR-223 miR-30d, miR-125b, miR26a, and miR-30a-5p

Bueno et al, 2008 Mi et al, 2007 Visone et al, 2007

miR-155 miR-21 miR-15a miR-155, miR-210 and miR-21

B-cell chronic lymphocytic leukemia miR-150, mir-155 and mir-21

miR-15a, miR-15b, miR-161, and miR-16-2 miR-15a and miR-16-1 miR-222, miR-92a-1, miR92a-2, miR-15a, and miR-16

miR-155 miR-143 and miR-145. Burkitts lymphoma

miR-155 miR-146a and miR-155

Breast cancer

miR21

let-7a miR-143 and miR-145 miR-125b, miR-145, miR-21, and miR-155 miR-17-5p let-7a miR-126 and miR-335 miR-145, miR-205 and let-7a miR-9-1, miR-124a3, miR148, miR-152, and miR-663

miR21 miR-373 and miR-520c

Metzler et al, 2004; Eis et al, 2005 Lawrie et al, 2007 Eis et al, 2005 Lawrie et al, 2008 Calin et al, 2002 Cimmino et al, 2005 Fulci et al, 2007 Metzler et al, 2004 Akao et al, 2007 Metzler et al, 2004 Motsch et al, 2007 Sampson et al, 2007 Akao et al, 2007 Iorio et al, 2005 Hossain et al, 2006 Yu et al, 2007 Tavazoie et al, 2008 Sempere et al, 2007 Lehmann et al, 2008 Si et al, 2007 Huang et al, 2008

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Gene Therapy and Molecular Biology Vol 12, page 191 Cervical cancer

miR-127 and miR-199a miR-21

Chronic mylogenous leukemia Colorectal cancer

miR-21 miR-21 and miR-31 miR-31, miR-96, miR-135b, and miR-183. miR-15b, miR-181b, miR-191, and miR-200c miR-17-92

miR-218 miR-143 miR-203

Lee et al, 2008 Martinez et al, 2008 Lui et al, 2007 Bueno et al, 2008

miR-143 and miR-145. miR-7-3 miR-133b and miR-145.

Schetter et al, 2008 Slaby et al, 2008 Jiang et al, 2005 BandrĂŠs et al, 2006 Xi et al, 2006

miR-143 and miR-145 let-7 miR-342 Cholangiocarcinoma Follicular thyroid carcinoma

miR-21, miR-141, and miR200b miR-197 and miR-346

Glioblastoma

miR-221

Weber et al, 2006 miR-181a, miR-181b, and miR-181c

miR-21 miR221 and miR-222 miR-7 Head and neck cancer Hodgkin's lymphoma

miR-21 and miR-205 miR-155 miR-96, miR-128a, and miR128b

Insulinomas

miR-204

miR-21 miR-199a, miR-21, and miR301 miR-122, miR-100, and miR10a miR-224 miR-23a-27a

Tran et al, 2007 Kluiver et al, 2005 Navarro et al, 2008

Kutay et al, 2006

miR-122a

Gramantieri et al, 2007 Meng et al, 2007 Jiang et al, 2008

miR-198 and miR-145

Varnholt et al, 2008

miR-17-92, miR-19a, miR-20, miR-106a, and miR-106b miR-200c miR-128b let-7 let-7a-3

Mantle cell lymphoma Nasopharyngeal cancer Neuroblastoma

miR-17-92

Osteosarcoma

miR-165, miR-166, miR-17, miR-20a, and miR-21 miR-200a, miR-141, miR-200c, miR-200b, miR-21, miR-203, and miR-205 miR-214

miR-29c miR-34a

miR-199a, miR-140, miR-145 and miR-125b1

let-7a-3 Pancreatic cancer

Chan et al, 2005 Gillies et al, 2007 Kefas et al, 2008

miR-122

miR-223

Ovary cancer

Ciafre et al, 2005

Roldo et al, 2006

Hepatocellular carcinoma

Lung cancer

Lanza et al, 2007 Michael et al, 2003; Akao et al, 2006 Akao et al, 2006 Grady et al, 2008 Meng et al, 2006

miR-155, miR-21, miR-221, mirR-222, miR-301a, miR301b, miR-376a-1 and miR376a-2 miR-196a-2 miR-155

Wang et al, 2008 Huang S et al, 2008 Wong et al, 2008 Hayashita et al, 2005 Hurteau et al, 2007 Weiss et al, 2008 Takamizawa et al, 2004 Inamura K 2007 Brueckner et al, 2007 Rinaldi et al, 2007 Sengupta et al, 2008 Welch et al, 2007; Cole et al, 2008 Gao et al, 2007 Iorio et al, 2007

Yang et al, 2008 Lu et al, 2007 Lee et al, 2007

Bloomston et al, 2007 Gironella et a, 2007

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Barh et al: Let-7, miR-125, miR-205, and miR-296 as prospective therapeutic agents in breast cancer molecular medicine Prostate cancer

let-7c miR-125b-1and miR-125b-2

Testicular germ-cell tumors T-cell lymphoma Papillary thyroid carcinoma

miR-372 and miR-373 miR-363 miR-221, miR-222, and miR146 miR-221, miR-222, and miR181b

Jiang et al, 2005 Lee et al, 2005 Voorhoeve et al, 2006 Lum et al, 2007 He et al, 2005 Pallante et al, 2006

miR-138 miR-146a

Mitomo et al, 2008 Jazdzewski et al, 2008 Nikiforova et al, 2008

miR-187, miR-221, miR-222, miR-146b, miR-155, miR-224, and miR-197

Table 2. miRGator microarray analysis of deregulated miRs in breast (normal vs cancer). Upregulated miRs (63)

Downregulated miRs (86)

miR-323, miR-324-3p, miR-326, miR-328, miR-331, miR-338, miR-339, miR-340, miR-342, miR-335, miR129, miR-148a, miR-218, miR-130a, miR-199b, miR1, miR-197, miR-150, miR-23b, miR-100, miR-99a, let-7b, miR-191, miR-194, miR-204, miR-133a, miR30a-3p, miR-21, miR-154, miR-10b, miR-223, miR-28, miR-190, miR-145, and miR-134.

miR-152, miR-29a, miR-16, miR-34c, miR-26a, miR-33, let7d, miR-182*, miR-199a, miR-128b, miR-200a, miR-184, miR-185, let-7f, miR-193, miR-188, miR-130b, miR-219, miR-206, miR-216, miR-217, miR-181c, miR-138, miR-107, miR-301, miR-302a, miR-31, miR-125a, miR-106b, let-7a, miR-93, miR-30e, miR-30a-5p, miR-324-5p, miR-224, miR320, miR-137, miR-103, miR-99b, miR-15b, miR-210, miR136, miR-24, miR-203, miR-212, miR-186, miR-27a, miR147, miR-199a*, miR-101, let-7g, miR-105, miR-19b, miR30d, let-7e, miR-211, let-7c, miR-17-5p, miR-128a, miR148b, miR-149, miR-299, miR-155, miR-29b, miR-127, miR-187, miR-192, miR-23a, miR-19a, miR-26b, miR-208, miR-135a, miR-125b, miR-29c, miR-124a, miR-106a, miR126, miR-7, miR-95, miR-146, miR-27b, miR-20, miR-32, miR-183, miR-15a, miR-140, miR-143, miR-296, miR-92, miR-144, miR-215, miR-34a, miR-139, miR-34b, miR-1423p, miR-221, miR-30b, miR-141, miR-135b, miR-181a, let7i, miR-222, miR-200b, miR-96, miR-153, miR-122a, miR98, miR-142-5p, miR-9*, miR-181b, miR-18, miR-9, and miR-200c.

C. Mechanism of miRs and their targets

D. Female breast cancer biomarkers and drugs

The mechanisms of gene regulation by miR (Lai, 2002; de Moor et al, 2005; Stark et al, 2005; Sun et al, 2005; Du and Zamore, 2007; Pillai et al, 2007; Standart and Jackson, 2007), their mechanisms of growth regulation (Lewis et al, 2003; O'Donnell et al, 2005; Ma et al, 2007; Esquela-Kerscher et al, 2008; Kumar et al, 2008; Tavazoie et al, 2008), and experimentally identification and computational predictions of their target mRNAs are also under extensive study (Bentwich, 2005; Rajewsky, 2006; Doran and Strauss, 2007; Maziere and Enright, 2007; Kuhn et al, 2008). Few of such miR targets are described in (Table 3). Recent studies have also shown that restoration of key downregulated miRs (Takamizawa et al, 2004; Esquela-Kerscher et al, 2008; Grady et al, 2008) and inhibition of oncomirs (Corsten et al, 2007; Matsubara et al, 2007; Si et al, 2007; Cissell et al, 2008) in respective cancers can inhibit cancer growth and restore normal condition, which indicate the potential therapeutic applications of miRs in various cancers.

Breast cancer is one of the most common cancers with highest cancer specific death rate in women worldwide (National Cancer Instituteâ&#x20AC;&#x2122;s SEER Program database http://seer.cancer.gov/statfacts/html/breast.html, Mettlin, 1999; Miller et al, 2008). The tumor biology and epidemiology of breast cancer also varies depending on the race, geographical locations, and several other factors (Amend et al, 2006; Smigal et al, 2006; Li and Daling, 2007; Hausauer et al, 2007). Several genetic alterations and biomarkers have been found to be associated with breast cancer (Ross et al, 2003; Ross et al, 2004; Jain, 2007; McCracken et al, 2007; Levenson and Somers, 2008; Laversin et al, 2008; Marchionni et al, 2008; see breast cancer specific genetic databases also) and several groups of chemotherapeutic drugs such as selective estrogen-receptor modulators (Riggs and Hartmann, 2003; Lewis and Jordan, 2005; Ramona et al, 2007), aromatase inhibitors (Smith and Dowsett, 2003; Jonathan, 2006; Nabholtz and Gligorov, 2006; Herold and Blackwell, 2008) anthracyclines (Hennessy and Pusztai, 2005; von Minckwitz, 2007; Dean-Colomb and Esteva, 2008), and 192

Gene Therapy and Molecular Biology Vol 12, page 193 targeted therapeutics (Gerber, 2008; Higgins and Wolff, 2008; Nahleh, 2008) are commonly used in treatment of breast cancer those are also reported to evoke frequent severe side effects (Jones et al, 2006; Gianni et al, 2007; Moore, 2007; Harris, 2008; Yamada, 2008).

data, we tried to construct a universial and common critical disease pathway and drug targets, regardless to the molecular profile of specific BCs. Next we have taken the effort to target those critical components of the constructed pathway with minimum numbers of naturally occurring miRs to block the entire network, which might be a potential therapeutic strategy in breast cancer gene therapy without any side effect.

E. Objective Keeping in mind the hetrogenecity of BC and side effects of conventional therapeutics, based on available Table 3. Experimental targets of miRs in different cancers Cancers ALL and BL Diffuse large B-cell lymphoma

Deregulated miRs miR-155 miR-155

let-7 miR-17– 92 miR-127 miR-143 miR-203

Targets †BCL6 BIC PU1 †PTEN †TPM1 †DMTF1 †BCL2 †CGI-38 †BCL2 †KIT CD44 HOXD10 SOX4 and TNC CD44 AIB1 ERBB2 and ERBB3 FAM3C, ACTA2, APAF1, BTG2, FAS, CDKN1A (p21), and SESN1 TPM1 H-RAS and HMGA2 E2F1 †BCL6 †ERK5 BCR-ABL1

miR-143

ERK5

miR 145 let-7 miR-21 miR-133

IRS1 RAS and MYC PDCD4 †SRF †PTBP2 †ERG †FLJ21308 †ERK5 †Clock †CAT-1 †FLJ21308 †NFIA CCNA2, CDC34, DBF4, STK6, STK12, E2F5, CDK8, PALG12, LIN28B, DICER1, GMNN, NRAS, HMGA2, CDC2, CDC25A, CCNB1, CCNE2, CCNF, CCNJ, SKP2, CKS1B, CDC20, CDCA1, CDAC2-8, RRM1-2, CDC6, CDC45L, CTD1, ORC1L, E2F6, E2F8, CHEK1, BUB1, MAD2L1,

miR-21 B-cell chronic lymphocytic leukemia

miR-15 miR-16

Breast cancer

Cervical cancer

Chronic mylogenous leukemia Colorectal cancer

miR-222 miR-520c miR-10b miR-335 miR-373 miR-17-5p miR-125a and miR-125a miR-21

miR-145 Cholangiocarcinoma Hepatocellular cancer

miR-141 miR-122 miR-145 miR-223 let-7

193

References Saito et al, 2006 Eis et al, 2005 John et al, 2004 Meng et al, 2006 Zhu et al, 2007 Kiriakidou et al, 2004 Cimmino et al, 2005 Kiriakidou et al, 2004 Cimmino et al, 2005 Felli et al, 2005 Negrini and Calin, 2008 Ma et al, 2007 Tavazoie et al, 2008 Huang et al, 2008 Hossain et al, 2006 Scott et al, 2007 Frankel et al, 2008

Zhu et al, 2007 Yu et al, 2007 O'Donnell et al, 2005 Saito et al, 2006 Esau et al, 2004 Bueno et al, 2008; Faber et al, 2008 Akao et al, 2006; Nakagawa et al, 2007 Shi et al, 2007 Akao et al, 2006 Asangani et al, 2008 Chen et al, 2006 Boutz et al, 2007 Xiao et al, 2007 Kiriakidou et al, 2004 Esau et al, 2004 Kiriakidou et al, 2004 Chang et al, 2004 Kiriakidou et al, 2004 Fazi et al, 2005 Johnson et al, 2007

Barh et al: Let-7, miR-125, miR-205, and miR-296 as prospective therapeutic agents in breast cancer molecular medicine

Glioblastoma

Lung cancer

miR-7 miR-221, miR-222 let-7 miR-17–92

CDC23, FANCD2, BRCA2, and SOX9 EGFR and AKT p27 p57 RAS and MYC

BRCA1, Kefas et al, 2008 Gillies et al, 2007 Medina et al, 2008 Johnson et al, 2005; Kumar et al, 2008 Hayashita et al, 2005 Lewis et al, 2003 Hurteau et al, 2007 Weiss et al, 2008 Fabbri et al, 2007 Lewis et al, 2003 O'Donnell et al, 2005 Volinia et al, 2006 Meng et al, 2007 Johnson et al, 2007

Melanoma

let-7b

MYC PTEN and RB2 TCF8 EGFR DNMT3A and DNMT3B †PTEN †E2F1 †RB1 †NF2 CCNA2, CDC34, DBF4, STK6, STK12, E2F5, CDK8, PALG12, LIN28B, DICER1, GMNN, NRAS, and HMGA2 Cyclins D1, D3, A, and CDK-4

Nasopharyngeal

miR-29

†TCL1

Pekarsky et al, 2006

Neuroblastoma Osteosarcoma Ovary cancer Pancreatic cancer

miR-34a miR-20a miR-140 miR-221 miR-222 miR-196

Welch et al, 2007 Volinia et al, 2006 Tuddenham et al, 2006 Felli et al, 2005 Felli et al, 2005 Yekta et al, 2004

Testicular germ cell cancer

miR-372 miR-373

Thyroid cancer

miR-221 miR-222 miR-146

†E2F3 †TGFBR2 †HDAC4 †KIT †KIT †HOXA7, HOXC8, HOXD8, and HOXB8 LATS2 LATS2 †KIF23, GBAS, C5orf5, C2orf18, PHC2, CD24, NUPL1, MYBL1, HERPUD1, UC7L2, LMNB1, INSIG2, CDK11, MKRN1, TOPK CD83, NCB5OR, RNF149, TNFAIP1, STX11, RELA, STK4, EG1, and RPIA KIT

miR-200c miR-128b miR-29 miR-19a miR-20 miR-106a let-7a let-7

Schultz et al, 2008

cancer

Voorhoeve et al, 2006 Voorhoeve et al, 2006 Lim et al, 2005

He et al, 2005; John et al, 2004; Krek et al, 2005; Rehmsmeier et al, 2004

Highlighted miRs are upregulated and non-highlighted are downregulated in respectective cancers. † marked targets and references are taken from TarBase (Sethupathy et al, 2006) (http://www.diana.pcbi.upenn.edu). References taken from TarBase are not given in reference list.

(http://linus.nci.nih.gov/BRB-ArrayTools.html). Most frequent upregulated 15 genes and mutated 10 genes were considered for critical disease pathway construction using Osprey-Version 1.0.1 (http://biodata.mshri.on.ca/osprey/servlet/Index), Ingenuity Systems Pathways Analysis-version 5.5 (www.ingenuity.com), and Pathway Architect-version 3.0.1 (www.stratagene.com). Pathways, key nodes, and up and down stream target analysis were done following methods as described by Barh and Das, 2008. The final critical disease pathway was drawn using Cell Illustrator 3.0 (http://www.cellillustrator.com).

II. Materials and Methods A. Databases and analysis tools for identification of breast cancer critical disease pathway Using literature survey databases (Pubmed, Elsevier, Medline) and breast cancer specific three genetic alteration databases [Breast cancer genetic alterations (http://ghr.nlm.nih.gov), Tumor Gene Family of Databases (http://www.tumor-gene.org/Breast/bcgd.html), and SciMedWeb® (http://www.geocities.com/m.lacroix/ marqueurs/lismark.htm#A)] we selected several upregulated genes those were verified with microarray data from Hedenfalk and colleagues (Hedenfalk et al. 2001) for hereditary BC for their highest upregulation using BRB ArrayTools- version 3.6.0

B. Identification of cancer associated miRs PubMed was screened for literatures describing various cancer-related miRs using key words “....cancer micro RNA”.

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Gene Therapy and Molecular Biology Vol 12, page 195 Data obtained from the searches were used for the identification specific miRs and their association with related cancers. Two additional miR databases Argonaute-2 (Shahi et al, 2006) (http://www.ma.uni-heidelberg.de/apps/zmf/argonaute/disease. php) and miRGator (Nam et al, 2008) (http://genome.ewha.ac.kr/miRGator) were also used as reference for identification of cancer associated miRs.

interplay in female breast cancer too (Figure 1). All key nodes of the constructed pathway are found to be potential drug targets. Identified key nodes of the entire pathway are EGF, ESR1, ERBB2, JUN, MMP2, PCNA, AKT, VEGF, NF-!B, CCND1, and CDK4. ER signaling pathway can be disrupted by targeting CYP19A1, ESR1, NF-!B, and MYC. Identified key targets of the growth factor signaling pathway are mainly ERBB2, EGFR, JUN, AKT, !-catenin. EGF and JUN are found to be common upstream drug targets of these two pathways. RB, PCNA, E2F1, and CCND1 can be considered as critical targets in DNA repair critical path. !-catenin and CCND1 seems to be common targets for growth factor signaling and DNA repair pathways. It has been found that common down stream targets of all critical pathways are NF-!B, FOS, MYC, !-catenin, and CCND1.

C. Identification of critical disease pathway targeting miRs Five micro RNA resource and analysis databases were used for this purpose. TarBase (Sethupathy et al, 2006) (http://www.diana.pcbi.upenn.edu/tarbase.html) was used to find the experimentally supported specific miR targets corresponding to specific cancer. miRBase (Griffiths-Jones et al, 2008) (http://microrna.sanger.ac.uk/cgi-bin/targets/v5/search.pl), PicTar (Krek et al, 2005) (http://pictar.bio.nyu.edu/cgibin/PicTar_vertebrate.cgi), and TargetScan Release 4.2 (Lewis et al, 2003; Lewis et al, 2005) (http://www.targetscan.org) were used to predict miRs those can target genes involved in BC critical disease pathway. miRanda (John et al, 2004) (http://cbio.mskcc.org/cgi-bin/mirnaviewer/mirnaviewer.pl) was used for identification of common miRs those can act on several targets of our breast cancer critical pathways.

B. Up and downstream target analysis Up and downstream analysis of terminal key nodes of the critical pathway shows that one or more transcription factors, cell cycle regulators, components of replication machinery, tumor suppressor genes, and growth factors are downstream targets of several key nodes (Table-4). For example, MYC is a down stream target for all three critical pathways and CDC25A, CDK4, eIF-4E, and ERBB2 are downstream targets of MYC. Similarly, for !-catenin, downstream targets are CCND1, FOS, and MYC.

III. Results A. Breast cancer critical disease pathway and drug targets 3 critical pathways (ER signaling, mitogenic signaling, and DNA repair), similar to male breast cancer critical pathways (Barh and Das, 2008) have been found to

Figure 1. Critical disease pathways in breast cancer.

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Barh et al: Let-7, miR-125, miR-205, and miR-296 as prospective therapeutic agents in breast cancer molecular medicine Table 4. Upstream regulators and downstream targets analysis of key nodes in breast cancer critical disease pathways. Key nodes

Up stream regulators

Down stream targets

BRCA1

ESR1, FRA2, GABPA, TP53BP1, EGF, SP1 USF1, USF2, SLUG

CDKN1B, CTLP, MYC, ZBRK1, Angioprotein-1, TERT, VEGF BRCC, RAD53/CHK2, RAD51, BARD1, BRCA1, MLH1, Tubulin, SKP2, PALB2 CCND1, BRCA1 CCND1, FOS, MYC

BRCA2 CDK4 CTNNB1 (!-catenin) CYP19A1 E2F4 ESR1 FOS ERBB2/ HER2/neu JUN K-RAS MAPK9 MMP2 MYC P53 MMP9 NF-!B PCNA PGR PTEN RAD51 VEGF

MYC AKT, TNF USF1, USF2, EGF, IL6 BRCA1 FOXO3a, SIRT1, RUNX2/ AML3 EGF, AP1/JUN, ESR1, IFNG, STAT, IL22, SRC GABPA, MYC, SPI1, AR, EGF EGF, TGF, CNK, IKK, SENP1 P53, CTF, SP1, HER2 EGFR AP-2 ", P53, KRAS, HER2, ESR1 AP1/JUN, AP2, STAT, EGF, SP1, MAK, TCF4 JUN, FOS, AP1, NF1, PAX5, PTEN SDF1, MMP2, TGF, HER2 MMP9, PTEN P53, IKK-", NF! B, E2F4, P53, ESR1, AP1 ESR1, ADA3 P53, HER2 SP1, ERK, TGF, SMAD, AP1, BRCA1

TNF

ESR1, PGR, P53 PCNA, CDK1, RB, CDK2, Cyclin A, AR, P53, TGF, FOS, PGR, BRCA1, CYP1B1, FIX, MMP2, IL6 FRA1, IL2, IL6, IL8, ET1, MMP1, FLG, P53 PTEN, KRAS, CCND1, ESR1, MMP2, MMP9, MYC MSH2, ESR1 MMP2, MMP9, CCND1 JUN, RARA MMP9, TSP1 CDC25A, CDK4, eIF-4E, ERBB2, TERT PCNA, MSH2, K-RAS, PPM1D TSP1, TSP2, NF-! B PCNA, FOS, MYC POL-D and- E, DNA Ligase-1, RFCs, MSH3, GTBP, RARA CSF, IGFBP NF-!B BRCC, ATM, BRCA2, GTBP, P53, BRCA1 VEGF-A, VEGF-165, VEGF-145 MYC, MMP2, MMP9

PCNA which is either upregulated or mutated in breast cancer have been found to be regulated by P53, IKK-", NF!B, E2F4, P53, ESR1, and AP1/JUN from its upstream and PCNA regulates its downstream targets mainly replication machinery key molecules such as POL-D andE, DNA Ligase, and RCFs etc. All key nodes and their upstream and down stream targets can be considered as drug targets depending on the molecular profile of the specific case of the cancer.

data not shown). Out of 150 identified targets including key nodes of the critical disease pathways (Table 4), a total of 73.3% key molecules are found to be targeted by let-7, miR-125, miR-205, and miR-296 (Tables 3 and 5) where they respectively covers 63%, 21.8%, 20.9% and 6.4% key targets. Experimental data show that let-7 targets oncogenes such as RAS and MYC and also inhibits cell cycle machinery by targeting several cell cycle regulators mainly CDC25A, CDK4, CDK6, E2F5-6, HMG2, several cyclins, check point regulators, and DNA polymerases (Johnson et al, 2005; Johnson et al, 2007) (Table 3). Predicted targets for let-7 show that it can also inhibit CCND1-2, CYP19A1, ESR2, FGF11, FGFR, IGF1 and IGFR1, IL6 and other ILs, MAPKKs, MMP8, DNA polymerases, and TGFBR (Table 5). Interestingly, the data also suggest that let-7 might play a role in targeting DNA damage response and repair genes such as BRCA1, BRCA2, MLH3, and RAD51C (Tables 3 and 5). Using miR and a precictions for common miRs for several targets, additional let-7 targets have been identified as ESR1, MMP2, MAPK4-6, RB1, TP53, and GRB2 (Table 6).

C. Let-7 targets ER and mitogenic signaling pathways and also blocks cell cycle Next we tried to identify miRs those can potentially target key nodes and their upstream regulators and downstream targets of the critical pathway network. miRs were identified from miR prediction databases (mirBase, PicTar, TargetScan, and miRanda), experimentally identified target database (TarBase), and form extensive Pubmed literature search. Several miRs have been found to target multiple key nodes along with their upstream regulators and down stream targets (Table 3) (predicted

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Gene Therapy and Molecular Biology Vol 12, page 197 Table 5. Predicted targets form Target Scan, Pic Tar, and Mir Base. Target Gene AKT3 BCL2 BCL6 BRCA1 CCND1 CCND2 CDC25A CDC34 CDK11 CDK4 COL4A2 CSF1 CYP19A1 DLC1 E2F1 E2F3 E2F5

Gene Name v-akt murine thymoma viral oncogene homolog 3 B-cell CLL/lymphoma 2 B-cell lymphoma 6 protein Breast cancer 1, early onset Cyclin D1 Cyclin D2 Cell division cycle 25 homolog A Cell division cycle 34 Cyclin-dependent kinase 11 Cell division protein kinase 4 Collagen, type IV, alpha 2 Macrophage colony-stimulating factor 1 Cytochrome P450, family 19, subfamily A, polypeptide 1 Deleted in liver cancer 1 E2F transcription factor 1 E2F transcription factor 3 E2F transcription factor 5

Target Scan miR-125a miR-205 miR-205

E2F6 EIF4A3 EIF4E1B ERBB2 ERBB3 ERBB4

E2F transcription factor 6 Eukaryotic translation initiation factor 4A isoform 3 Eukaryotic translation initiation factor 4E-1B Receptor tyrosine-protein kinase erbB-2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 v-erb-a erythroblastic leukemia viral oncogene homolog 4

let-7c

ESR2 ESRRA FGF11 FGF4 FGF5 FGFR2 FGFR4 HGF HRAS IGF1 IGF1R IGF2 IL10 IL13 IL16 IL2 IL5 IL6 IL6ST IL8 MAP2K2 MAP2K7 MAP3K10 MAP3K3 MAP4K3 MAP4K4 MAPK14 MAPK3 MAPK6 MAPK9 MLH3 MMP11 MMP26 MMP8 MSH2 MYC NFIB

Estrogen receptor beta Estrogen-related receptor alpha Fibroblast growth factor 11 Fibroblast growth factor 4 precursor Fibroblast growth factor 5 Fibroblast growth factor receptor 2 Fibroblast growth factor receptor 4 Hepatocyte growth factor GTPase HRas precursor Insulin-like growth factor IA precursor Insulin-like growth factor 1 receptor Insulin-like growth factor II precursor Interleukin 10 Interleukin 13 Interleukin 16 Interleukin 2 Interleukin-5 precursor Interleukin 6 (interferon, beta 2) Interleukin-6 receptor subunit beta precursor Interleukin 8 Mitogen-activated protein kinase kinase 2 Mitogen-activated protein kinase kinase 7 Mitogen-activated protein kinase kinase kinase 10 Mitogen-activated protein kinase kinase kinase 3 Mitogen-activated protein kinase kinase kinase kinase 3 Mitogen-activated protein kinase kinase kinase kinase 4 Mitogen-activated protein kinase 14 Mitogen-activated protein kinase 3 Mitogen-activated protein kinase 6 Mitogen-activated protein kinase 9 DNA mismatch repair protein Mlh3 Matrix metalloproteinase 11 Matrix metalloproteinase-26 Matrix metalloproteinase-8 DNA mismatch repair protein Msh2 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) Nuclear factor I/B

NRAS

Neuroblastoma RAS viral (v-ras) oncogene homolog

197

let-7b let-7f let-7d let-7

Pic Tar

Mir Base

miR-205 miR-205 let-7a let-7a let-7a let-7a miR-125a, miR-205 miR-205

let-7

let-7a

let-7f

let-7a let-7a miR-205 miR-125a let-7a miR-205 let-7a

miR-125b

miR-205 miR-125a

miR-205 miR-205 miR-205 miR-205 miR-125b miR-205

miR-205 miR-125a miR-205

let-7a miR-98

miR-125a let-7a

miR-125a

miR-125a miR-125a

miR-205

let-7a-b let-7b miR-205 let-7b let-7b miR-125b let-7a let-7a miR-125a

let-7a-b miR-125b let-7a-b miR-205

let-7d let-7b let-7a let-7b miR-125a miR-98 let-7a

miR-125a miR-125a let-7a let-7a-b let-7a miR-125a miR-125b

let-7b mir-205

miR-205 miR-125a

let-7i miR-125a, miR-205 let-7f

let-7a-b miR-125b miR-205 let-7b miR-205 let-7a-b

Barh et al: Let-7, miR-125, miR-205, and miR-296 as prospective therapeutic agents in breast cancer molecular medicine POLA2 POLI POLL POLQ POLR2D POLR2G POLR3D POLR3G RAD51C S100A1 SP1 SP4 TGFBR1

DNA polymerase subunit alpha B DNA polymerase iota DNA polymerase lambda DNA polymerase theta DNA-directed RNA polymerase II 16 kDa DNA directed RNA polymerase II polypeptide G Polymerase (RNA) III (DNA directed) polypeptide D, 44kDa DNA-directed RNA polymerase III subunit G DNA repair protein RAD51 homolog 3 Protein S100-A1 Sp1 transcription factor Sp4 transcription factor Transforming growth factor, beta receptor I

TGFBRAP1 TNF VEGFA VEGFB

TGF beta receptor associated protein -1 Tumor necrosis factor precursor Vascular endothelial growth factor A Vascular endothelial growth factor B

growth

let-7d let-7a let-7a miR-205 miR-125a miR-205 let-7g, miR-125b

let-7a

miR-205

Thus in respect to our breast cancer critical disease pathway, let-7 might potentially inhibit both the estrogen receptor and mitogenic signaling pathways by directly targeting CYP19A1, ESR1, TGFB, RAS, SKP2, MMP2, ILs, and MYC (Table 7). In addition to these two pathways, the current evidences suggest that let-7 also can induce cell cycle arrest and growth inhibition by directly targeting several cell cycle regulators (Johnson et al, 2007). It is also found that let-7 can inhibit ITGB3 and angiogenin thus inhibits angiogenesis, cell migration, and metastasis.

D. miR-125 blocks signaling and cell cycle

let-7a let-7a-b let-7a-b let-7b let-7a-b let-7b

E. miR-205 and miR-296 targets multiple key nodes of the BC critical disease pathway From prediction databases it has been found that miR-296 might inhibit multiple key components of all three critical pathways. Identified potential targets of miR296 are ERBB2, ESR1, MMP2, JUN, CCND1, CCND3, and TGFB1 (Table 6). Similarly, miR-205 have been found to potentially target BCL2, BRCA1, CDK11, CDK4, E2F1, E2F5, EIF4A3, EIF4E1B, ERBB3, ERBB4, FGF4, HRAS, IL5, MAPK9, NFIB, S100A1, SP4 and VEGFA (Table 5), MMP2, KRAS, E2F6 and FGF1 (miRanda precictions). miRanda common target preciction also shows that EGFR signaling can potentially be disrupted by miR-27, miR-34, and miR-214 (Table 6).

factor

The potential second miR for precisely targeting growth receptor signaling was identified as miR-125 which can block the pathway by targeting numbers of key molecules, growth factors, and cell cycle regulators. Identified targets of miR-125 are AKT, ERBB2-4, FGF, FGFR, IGF, MAPKs, MMP11, NFIB, SP1, TGFBR1, TNF, VEGF, CDK11, E2F3, ESRRA (Tables 3 and 5). miRanda precictions give additional targets for miR-125 as CTNNB1/ !-catenin, FOS, CDC25A, and IL6R (Table 6). Thus miR-125 can potentially inhibit ERBB2 signaling by inhibiting key nodes (ERBB2, AKT, !-catenin, growth factor signaling molecules) along with key transcription factors (FOS, NFIB, E2F3) and cell cycle regulators (CDC25A and CDK11) (Table 7).

IV. Discussion Population based studies show that the genetic makeup of BC pathogenesis varies from person to person and also varies depending on particular group of population, race, and socioeconomic factors (Gloeckler et al, 2003; Chlebowski et al, 2005; Fejerman and Ziv, 2008). Apart from several genetic alterations, a considerable numbers of miRs have been found deregulated in breast cancer (Iorio et al, 2005; Hossain et al, 2006; Sempere et al, 2007; Si et al, 2007; Yu et al, 2007; Huang et al, 2008; Lehmann et al, 2008).

Table 6. miRanda precictions for common miR targets. miRs let-7 miR-27 miR-34 miR-125 miR-205 miR-214 miR-296

miR-205 miR-125b miR-205 miR-125b

Targets THBS1, CDC25A, POLD3, MMP2, IGF1, E2F5/6, MAPK6, ESR1, CYP19A1, MYC, CCND3, MUC4, MAPK4, STAT2, TP53, RB1, GRB2 JUN, EGFR MYC, EGFR, KRAS CTNNB1, VEGF, FOS, CDC25A, TNF, IL6R, E2F2, MMP11, BCL2 MMP2, KRAS, E2F6, FGF1, CTNNB1, NFKB1, EGFR ERBB2; ESR1, MMP2, JUN, CCND1, CCND3, TGFB1, NOTCH3

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Gene Therapy and Molecular Biology Vol 12, page 199 Several recent studies have shown that miRs are promising agents for cancer therapy. miR-17-92 cluster which is involved in lung development (Ventura et al, 2008) is highly upregulated and induces MYC in aggressive smallcell lung cancer (Hayashita et al, 2005). Induced inhibition of miR-17-5p and miR-20a induces apoptosis in lung cancer cells overexpressing miR-17-92 cluster (Matsubara et al, 2007). miR-128b LOH is frequent in NSCLC and restoration of miR-128b is reported to inhibit tumor growth by inhibiting EGFR (Weiss et al, 2008). Similarly, let-7 is downregulated in lung cancer and overexpression or restoration of let-7 represses lung cancer growth (Takamizawa et al, 2004; Fabbri et al, 2007; Inamura et al, 2007; Esquela-Kerscher et al, 2008, Kumar et al, 2008) by inhibiting RAS and MYC (Johnson et al, 2007). In colorectal cancer miR-143 and miR-145 are downregulated (Michael et al, 2003) and overexpression miRNA 143 and miR145 inhibit colon cancer growth by suppressing respectively ERK5 (Akao et al, 2006) and IRS1 (Shi et al, 2007). let-7 which is also downregulated in colorectal cancer induces growth inhibition by lowering RAS and c-MYC expression when overexpressed (Akao et al, 2006). Reconstitution of miR-342 reported to induce apoptosis in colorectal cancer (Grady et al, 2008). Similarly, miR-126 and miR-335 those are lost in primary breast cancers, reduces tumour growth, proliferation, and metastasis when overexpressed (Tavazoie et al, 2008). Inhibition of oncomir miR-21 is reported to suppress MCF-7 cell growth (Si et al, 2007; Cissell et al, 2008). Several other studies show that inhibition of miR-21 inhibits growth of glioblastoma (Chan et al; 2005; Corsten et al, 2007) and hepatocellular carcinoma (Meng et al, 2007). Treatment with miR-7 reduces invasiveness of

glioblastoma by suppression EGFR and AKT (Kefas et al, 2008). miR-221 and 222 are also reported to be ideal therapeutic agent for glioblastoma (Medina et al, 2008). miR-203 is lost in CML and ALL and that the epigenetic silencing of miR-203 enhances ABL1 and BCR-ABL1 oncogene expression (Bueno et al, 2008). Restoration of miR-203 expression has been reported to reduce ABL1 and BCR-ABL1 levels and inhibit cell proliferation in CML (Faber et al, 2008). Restoration of several other deregulated miRs in different cancers (Tables 1 and 2) and their targets (Tables 3, 5, 6 and 7) may also prove beneficial for cancer therapy. In our current study we demonstrated that, growth factor receptor (EGFR) signaling, estrogen receptor signaling, and DNA repair pathways interplay in breast cancer. EGF, CYP19A1, ESR1, ERBB2, JUN, MMP2, PCNA, AKT, VEGF, NF-!B, Cyclin-D, and CDK4 are found to be key nodes of the entire network those are potentially be good drug targets. The downstream key effecter molecules those are also needed to be targeted are identified as components of replication machinery, DNA polymerases, cell cycle regulators, and transcription factors such as FOS and MYC. let-7 is downregulated in breast cancer and induction of let-7 suppress cancer growth by inhibiting H-RAS and HMGA2 expression (Sempere et al, 2007; Yu et al, 2007). In our study we have found that let-7completely inhibit ER signaling and partially represses growth factor signaling pathway by targeting key components of these pathways. let-7 also have been found to bring cell cycle arrest and inhibition of angiogenesis by targeting several cell cycle and angiogenic regulators and transcription factors.

Figure 2. Targets of ler-7, miR-125, and miR-205 in breast cancer critical disease pathway.

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miR-125

miR-205 miR-296

Targets RAS, MYC, CDC25A, CDK4, CDK6, E2F5-6, HMG2, DNA-Polymerases, CCND1-2, CYP19A1, ESR1, ESR2, FGF11, FGFR, IGF1 and IGFR1, IL6 and other ILs, MMP2, MMP8, TGFBR, MAPK4-6, RB1, TP53, GRB2, TGFB, SKP2, ITGB3, and angiogenin AKT, ERBB2-4, FGF, FGFR, IGF, MAPKs, MMP11, NFIB, SP1, TGFBR1, TNF, VEGF, CDK11, E2F3, ESRRA, CTNNB1/ !-catenin, FOS, CDC25A, and IL6R BCL2, CDK11, CDK4, E2F1, E2F5, E2F6, EIF4A3, EIF4E1B, ERBB3, ERBB4, FGF4, HRAS, IL5, MAPK9, NFIB, S100A1, SP4, VEGFA, MMP2, KRAS, and FGF1 ERBB2, ESR1, MMP2, JUN, CCND1, CCND3, and TGFB1

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Reduced expression of miR-125b correlates with metastasis in breast cancer (Iorio et al, 2005) and the overexpression of either miR-125a or miR-125b inhibit breast cancer growth by inhibiting ERBB2 and ERBB3 at both the transcript and protein level (Scott et al, 2007). Apart from these targets our analysis shows that miR-125 can target multiple key molecules (ERBB2, AKT, !catenin, FOS, NFIB, E2F3, CDC25A, and CDK11) of the entire network of our breast cancer critical pathways. Similarly, miR-205 is downregulated in breast cancer (Sempere et al, 2007) and we have shown that restoration of miR-205 might inhibit growth factor receptor signaling pathway by repressing ERBB 3-4, FGF, HRAS, MAPK9, NFIB, VEGFA, MMP2, KRAS, CDK4, and E2F1. Another predicted miR i.e., miR-296 have been also found potential to disrupt EGFR signaling by targeting ERBB2, ESR1, MMP2, JUN, CCND1, CCND3, and TGFB1. Thus appropriate combination of these microRNAs can potentially cover all targets (Figure 2, Table 7) to inhibit tumor growth and to restore normalcy in breast cancer.

V. Conclusion In conclusion, our results suggest that let-7 replacement therapy might be effective in estrogen and ERBB2 positive breast cancers. Treatment with either miR-125 or miR-205 or miR-296 has best possibility to be effective in ER negative breast cancers overexpressing ERBB2. A combination of let-7 along with any one of the above mentioned three miRs might be an efficient future molecular medicine irrespective of the type of breast cancer. Being miR a natural agent, miR replacement therapy will not evoke any side effect those are occasionally found in chemotherapy.

Acknowledgements We are thankful to all Bioinformatics software and database providers, whose tools and data were used in this research. We highly appreciate their public, academic, limited trial and free licensing options.

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From left to right: Debmalya Barh, Sanjeeb Parida, Bibhu Prasad Parida and Geetha Viswanathan

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Gene Therapy and Molecular Biology Vol 12, page 207 Gene Ther Mol Biol Vol 12, 207-218, 2008

Suppression of Primary and Disseminated Murine Tumor Growth with eIF5A1 Gene Therapy Research Article

Songmu Jin, Catherine A. Taylor, Zhongda Liu, Zhong Sun, Bin Ye, John E.Thompson* Department of Biology, University of Waterloo, Waterloo, ON, Canada, N2L 3G1

__________________________________________________________________________________ *Correspondence: John E.Thompson, Dept. of Biology, University of Waterloo, 200 University Ave. W., Waterloo, ON, Canada, N2L 3G1; Tel: (519) 888-4465; Fax: (519) 746-0614; e-mail: jet@uwaterloo.ca Key words: eukaryotic translation initiation factor 5A (eIF5A), gene therapy, Adenovirus, DOTAP, melanoma, lung cancer, apoptosis Abbreviations: Adenoviral vectors, (Adenovirus 5 serotype, E1 and E3-deleted) expressing eIF5A1, (Ad-eIF5A1); Adenoviral vectors, (Adenovirus 5 serotype, E1 and E3-deleted) expressing eIF5A2, (Ad-eIF5A2); adenovirus vector expressing the bacterial lacZ gene, (Ad-LacZ); deoxyhypusine hydroxylase, (DOHH); deoxyhypusine synthase, (DHS); Eukaryotic translation initiation factor 5A, (eIF5A); hemagluttin, (HA); Hematoxylin and eosin, (H&E); multiplicities of infection, (MOI) Received: 27 June 2008; Revised: 21 July 2008 Accepted: 24 July 2008; electronically published: September 2008

Summary Eukaryotic translation initiation factor (eIF5A) is the only known protein that is post-translationally modified to contain hypusine. The purpose of this study was to establish whether eIF5A1 gene delivery might be an effective therapy against primary and disseminated tumors. The effects of adenoviral-mediated eIF5A1 gene transfer on tumor growth and animal survival were examined using a syngeneic murine melanoma (B16-F0) model and a human lung xenograft (A549) model. Significant suppression was observed in both primary melanoma (p < 0.001; B16-F0) and lung tumor (p < 0.001; A549) growth following intra-tumoral injections of Ad-eIF5A1. Increased incidence of apoptosis was evident in melanoma tumors following Ad-eIF5A1 treatment. Gene transfer of the second member of the eIF5A family, eIF5A2, also gave rise to significant delays in growth of primary melanoma tumors. Animal survival experiments revealed prolonged survival [median survival time: 25 days (treated), 7 days (control) for B16-F0; and 54 days (treated), 24 days (control) for A549]. Systemic administration of DOTAP:pCpGeIF5A1 complexes into C57BL/6 mice suppressed tumor growth (p < 0.05) in a B16-F10 model of experimental disseminated metastases. Our findings suggest that eIF5A1 may be an important target in the development of treatments for primary and disseminated cancers.

function of eIF5A remains elusive, numerous functions have been proposed, including roles in protein translation (Kang and Hershey, 1994; Zanelli et al, 2006), mRNA transport (Kang and Hershey, 1994; Hanauske-Abel et al, 1995; Liu et al, 1997; Xu et al, 2004), and mRNA stability (Zuk and Jacobson, 1998; Schrader et al, 2006). EIF5A has also been implicated in the regulation of cell proliferation (Schnier et al, 1991; Kang and Hershey, 1994) and apoptosis (Li et al, 2004; Taylor et al, 2007). There are two isoforms of eIF5A in the human genome, eIF5A1, which is abundant in all tissues and cancer cell lines, and eIF5A2, for which expression appears to be restricted to testis, parts of the brain, and certain cancers, such as colon cancer (Jenkins et al, 2001). Over-expression of eIF5A1 has been found to induce apoptosis in lung (Li et al, 2004) and colon cancer cell

I. Introduction Despite advances in cancer treatment, statistics show that cancer deaths are still on the rise and that the 5-year survival rates for both lung cancer (Sรถrenson et al, 2001) and melanoma (Balch et al, 2001) remain low. Thus there is a growing need for new treatments. Eukaryotic translation initiation factor 5A (eIF5A) is the only protein found in nature that contains the amino acid, hypusine. The formation of hypusine occurs posttranslationally on a conserved lysine residue in a two-step process. The first step is the transfer of a butylamine group from spermidine and is catalyzed by deoxyhypusine synthase (DHS) (Wolff et al, 1990). A second hydroxylation reaction, catalyzed by deoxyhypusine hydroxylase (DOHH), results in the formation of the mature hypusine-containing eIF5A. Although the precise 207

Jin et al: Suppression of tumor growth with eIF5A1 gene therapy seven-week-old female BALB/c nude mice. All mice were obtained from Charles River Canada (Saint Constant, Quebec).

lines (Taylor et al, 2007), but no studies have yet determined whether eIF5A1 gene therapy provides therapeutic benefit in in vivo cancer models. In this study, we address this question by looking at survival of melanoma and lung tumor-bearing mice treated with an adenovirus expressing eIF5A1. The ability of eIF5A1 to treat disseminated tumors was also examined in an experimental metastasis lung cancer model.

E. B16-F0 subcutaneous tumor model and therapy B16-F0 tumors were established by subcutaneous injection of 500,000 cells into the right flank of C57BL/6 mice. When tumor size reached an average diameter of 5 mm the mice were randomized into three groups of nine to ten mice each and received the following treatments: (a) PBS/10% glycerol, (b) AdLacZ, or (c) Ad-eIF5A1. Adenovirus (1 x 109 pfu diluted in 100 microliters of PBS/10% glycerol) was injected intra-tumorally at multiple sites every other day (for a total of 3 injections/week) until the animals were sacrificed. Tumors were measured every day with calipers, and the mice were sacrificed when tumor size reached 10 % of body weight (tumor diameter exceeding 17 mm). The average weight of the mice at the initiation of treatment was 20 grams. Tumor volume was calculated using the equation: tumor volume (mm 3) = L * W2 * 0.52. For TUNEL, H&E, and immunohistochemical analysis, mice bearing B16-F0 subcutaneous tumors were treated as above except that three mice per group were sacrificed 0, 3, and 6 days after the initiation of treatment. The tumors were cut in half, and one segment was frozen at - 80°C while the other was immediately fixed in 4 % formaldehyde/PBS, embedded in paraffin, and sectioned (10 mm).

II. Material and Methods A. Cell lines B16-F0 (CRL-6322) and B16-F10 (CRL-6475) murine melanoma cell lines were purchased from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Media containing 10 % fetal bovine serum. A549 lung carcinoma cells were obtained from Anita Antes (University of Medicine and Dentistry, New Jersey) and were cultured in RPMI 1640 supplemented with 1 mM sodium pyruvate and 10 % fetal bovine serum. Cell cultures were maintained at 37 °C in a humidified environment containing 5 % CO 2.

B. Construction of Adenoviral Vectors Adenoviral vectors (Adenovirus 5 serotype, E1 and E3deleted) expressing eIF5A1 (Ad-eIF5A1) or eIF5A2 (AdeIF5A2) were generated using the AdMax! Hi-IQ system (Microbix Biosystems Inc., Toronto, Canada). The creation and propagation of Ad-eIF5A1 is described elsewhere (Taylor et al, 2007). Ad-eIF5A2 was constructed by subcloning a PCRamplified cDNA of eIF5A2 into the SmaI site of the adenovirus shuttle vector pDC516(io) using primers having the following sequences: forward 5'ATCAAGCTTGCCCACCATGGCAGACG-3 ; and reverse 5'AACGAATTCCATGCCTGATGTTTCCG-3. An adenovirus vector expressing the bacterial lacZ gene (Ad-LacZ) was purchased from Qbiogene (California, USA) and used as a reporter gene and negative control. Pure, high-titer adenovirus stocks were prepared using the ViraBind! Adenovirus Purification Mega Kit (Cell Biolabs, San Diego, CA, USA). The viral stocks were titered by plaque assay on 293-IQ cells (Microbix Biosystems Inc., Toronto, Ontario). Viral stocks were diluted with PBS/10% glycerol for injection into mice.

F. B16-F10 lung tumor model and therapy B16-F10 lung tumors were established by tail vein injection of 50,000 cells into C57BL/6 mice. The mice were randomized into three groups of five mice each and received the following treatments: (a) PBS, (b) pCpG-LacZ:DOTAP, and (c) pCpG-eIF5A1:DOTAP. Plasmid DNA was delivered to the lung by tail vein injection of DOTAP:DNA complexes. Immediately prior to injection, fifty micrograms of plasmid DNA was diluted in 100 microliters of PBS and mixed with 80 microliters (80 mg) of DOTAP (Roche Applied Science) diluted to 100 microliters in PBS. The complexes were incubated for 15 minutes at room temperature, and 200 microliters of complexed DNA was injected via tail vein into each mouse. Complexed DNA was injected on days 7, 14, and 21 after tumor seeding. Animals were sacrificed as soon as they showed signs of distress such as lethargy, ruffled fur, or difficulty breathing. Lungs were removed, weighed, and photographed.

C. Construction of pCpG-eIF5A1 vector An expression vector lacking immunostimulatory CpG dinucleotides and expressing LacZ (pCpG-LacZ) was purchased from Invivogen (San Diego, California, USA). In order to create a CpG-free expression vector expressing a hemagluttin (HA) epitope-tagged eIF5A1, pCpG-LacZ was digested with NcoI and NheI, and the vector backbone was isolated. The pCpG vector backbone was ligated to an eIF5A1 cDNA fragment generated by PCR using a pHM6 vector (Roche Molecular Biochemicals) containing eIF5A1 cDNA as a template. The following primers were used to generate the PCR fragment for subcloning: forward 5'-GCTCCATGGCAGATGATTTGGACTTCG-3'; and reverse 5'CGCGCTAGCCAGTTATTTTGCCATCGCC-3'. The plasmids were propagated in E. coli GT115 and purified using the QIAGEN EndoFree Plasmid Giga kit.

G. A549 subcutaneous tumor model and therapy Twenty-five BALB/c nude mice were injected subcutaneously with 1 million A549 cells on the right flank and randomized into the following three treatment groups (a) PBS/10% glycerol, (b) Ad-LacZ, or (c) Ad-eIF5A1. Treatment was initiated when the tumors reached approximately 4 mm in diameter. Mice received intra-tumoral injections of 1 x 109 pfu of adenovirus three times weekly (days 1, 3, 5, 8, 10, 12, 15, 17, 19, ect...). Tumors were measured three times per week, and mice were sacrificed when tumor size reached 10 % of body weight (tumor diameter exceeding 17 mm).

H. Immunohistochemistry and TUNEL

D. Animal Experimentation

In order to detect expression of LacZ, tumor sections were deparaffinized in xylene and rehydrated. A beta-galactosidase antibody (ab616; Abcam, Cambridge, MA) was used at a dilution of 1:2000 in combination with a FITC-conjugated goat antirabbit antibody (Sigma). Apoptotic cells in tumor sections were detected using the DeadEnd! Fluorometric TUNEL system (Promega) according to the manufacturer’s instructions. The

Animal experiments were conducted in accordance with the guidelines set out by the University of Waterloo Animal Care Committee (Waterloo, Ontario, Canada). All animal experiments using murine melanoma cell lines B16-F0 and B16-F10 were performed using five- to seven-week-old female C57BL/6NCRL mice. A549 xenograft experiments were performed using five- to

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were examined for DNA fragmentation using TUNEL. A significant number of TUNEL-positive cells were observed 3 days (Figure 1) and 6 days (supplemental data Figure 2) after intra-tumoral injection with Ad-eIF5A1 but not after injection with Ad-LacZ or PBS. A significant number of apoptotic cells were also observed in AdeIF5A1-treated tumors sections from mice that were sacrificed on day 6 (supplementary data Figure 2). Sections derived from tumors treated with Ad-eIF5A1 for 6 days were also found to have many regions of decreased cell density compared to Ad-LacZ controls when examined by H&E staining (Figure 1, supplementary data Figure 3), indicating considerable cell loss due to apoptosis and/or necrosis. The therapeutic effects of delivering the eIF5A1 gene to subcutaneous murine tumors were also evaluated in the syngeneic B16-F0 model. Injections of Ad-LacZ or AdeIF5A1 were given intra-tumorally three times per week until sacrifice. Treatment with Ad-eIF5A1 over a course of several weeks resulted in a substantial increase in survival compared to control mice that received injections of PBS or Ad-LacZ (Figure 2A). Mice that received injections of

I. Hematoxylin and Eosin Staining Hematoxylin and eosin (H&E) staining was used to observe tumor ultrastructure. B16-F0 tumor sections were prepared as described above. Sections were deparaffinized, rehydrated, and stained in Mayer’s hematoxylin solution (15 minutes; Sigma). The sections were washed with water and stained with aqueous Eosin Y (30-60 minutes; Sigma). Following staining, the sections were dehydrated, mounted with resinous mounting media and photographed using light microscopy.

K. Western blotting A549 cells were infected with adenovirus constructs at increasing multiplicities of infection (MOI). Forty-eight hours after infection, the cells were lysed [2 % SDS, 62.5 mM Tris-HCl (pH 7.4), 10 % glycerol]. The protein was fractionated by SDSPAGE and western blotted using antibodies against eIF5A (BD Transduction Laboratories; 1:20,000), eIF5A2 (Novus Biologicals; 1:2000), p53 (Cell Signalling; 1:1000) or "-actin (Oncogene; 1:20,000).

L. Annexin/PI A549 cells were infected with Ad-LacZ, Ad-eIF5A1, or Ad-eIF5A2 at an MOI of 80. Four hours later fresh media was added and the cells were incubated for seventy-two hours with media changes every 24 hours. Cells were labeled using the Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen) according to the manufacturer’s instructions. The cells were sorted by flow cytometry (Becton Dickinson FACSVantage SE with a 488 nm argon laser) and the data were analyzed using WinMDI 2.8. The percentage of cells in early apoptosis (Ann+/PI-) was combined with the percentage of the cell population in late apoptosis (Ann+/PI+) to give the total percentage of cells in apoptosis.

M. Statistical analyses Student’s t-test was used for statistical analysis. Significance was deemed to be a confidence level above 95 % (p < 0.05).

III. Results A. Intra-tumoral injection of Ad-eIF5A1 induces apoptosis and prolongs survival in a murine melanoma model To determine whether eIF5A1 gene therapy could have therapeutic value in in vivo cancer models, we used adenovirus-mediated gene delivery in a murine melanoma model. B16-F0 cells were injected subcutaneously into the right flank of C57BL/6 mice. Tumors of 5 mm in diameter were given intra-tumoral injections of either Ad-LacZ or Ad-eIF5A1 every other day. Three mice per group were sacrificed on days 0, 3, and 6, and the tumors were paraffin-embedded for further analysis. Expression of "galactosidase was observed after 3 days (supplemental data Figure 1) and 6 days (Figure 1, supplemental data Figure 1) in tumor sections from mice that had received intra-tumoral injections of Ad-LacZ, indicating efficient gene transfer. In order to determine whether intra-tumoral injections of Ad-eIF5A1 resulted in the induction of apoptosis in tumors, paraffin-embedded tumor sections

Figure 1. Subcutaneous B16-F0 tumors exhibit increased apoptosis following intra-tumoral injection of Ad-eIF5A1. C57BL/6 mice were injected subcutaneously on the flank with B16-F0 cells. Intra-tumoral injections of PBS, Ad-LacZ, or AdeIF5A1 were given on days 0, 2, and 4. Tumors were harvested on days 0, 3, and 6. Paraffin-embedded tumor sections were examined for LacZ expression (day 6) using an antibody against "-galactosidase. Apoptotic cells in tumor sections were labelled by TUNEL (day 3) and tumor structure was observed by H&E staining (day 6). Data shown are representative of results obtained from three different mice. All photographs were taken at 400x magnification.

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Jin et al: Suppression of tumor growth with eIF5A1 gene therapy potential oncogene (Guan et al, 2001, 2004). Although eIF5A1 and eIF5A2 share 82% homology at the amino acid level, it is not known whether they have conserved functions. In order to determine whether eIF5A2 may also have anti-cancer properties, we examined the effect of AdeIF5A2 intra-tumoral injections on tumor growth in the B16-F0 subcutaneous melanoma model. Ad-eIF5A2treated mice also exhibited a significant delay in tumor growth compared to control mice, although it was not as great as that seen in mice that received Ad-eIF5A1 (Figure 3). The median survival for mice that received injections of PBS was 8 days while the median survival for mice injected with Ad-eIF5A1 and Ad-eIF5A2 was 16 and 14 days, respectively (data not shown). Thus eIF5A2 and eIF5A1 gene delivery have similar anti-cancer properties.

C. Systemic administration of eIF5A1 plasmid DNA reduces tumor burden in a murine experimental metastasis model The ability of eIF5A1 to control disseminated tumors was also examined using a metastatic lung cancer model. Lung tumors were established in C57BL/6 mice by tail vein injection of the highly metastatic melanoma cell line, B16-F10. Although viral delivery of genes can be very efficient, repeated dosing, particularly intravenously, can give rise to an immune response targeted against the viral vector, thereby limiting the effectiveness of subsequent doses (Bessis et al, 2004). Non-viral gene therapy can also elicit immune responses that inhibit expression from later doses (Tan et al, 1999). Consequently, in order to get the highest possible transgene expression in this lung tumor model, an expression plasmid (pCpG) that has been modified to remove all CpG dinucleotides was administered. CpG-reduced plasmids have been shown to decrease toxicity and increase the duration of transgene expression in vivo (Yew et al, 2000, 2002; Hodges et al, 2004). Gene delivery to the lung was accomplished by tail vein injection of plasmid DNA complexed with DOTAP, a combination that has been used previously to deliver genes to the lung (Li and Huang, 1997; Bragonzi et al, 1999, 2000). Plasmid DNA complexed with DOTAP was injected by tail vein once per week (days 7, 14, and 21 after injection of B16-F10 cells). A small, but statistically insignificant, decrease in lung weight was observed in mice treated with pCpG-LacZ compared to mice that only received injections of PBS (Figure 4A) and could be due to residual immune stimulation from the DOTAP:DNA complex. Injections with pCpG-eIF5A1 resulted in a 59 % decrease in average lung weight compared to mice that received pCpG-LacZ (Figure 4A) indicating a significant reduction in tumor burden. The lungs of mice treated with eIF5A1 plasmid DNA were noticeably smaller and had considerably less metastatic tumor growth than control groups (Figure 4B). Thus eIF5A1 treatment may also be feasible as a therapy for disseminated tumors.

Supplemental Figure 1. Subcutaneous B16-F0 tumors express "-galactosidase following intra-tumoral injection of AdLacZ. C57BL/6 mice were injected subcutaneously on the flank with B16-F0 cells. Intra-tumoral injections of Ad-LacZ were given on days 0, 2, and 4. Tumors were harvested on days 0, 3, and 6. Paraffin-embedded tumor sections were examined for "galactosidase expression by immunohistochemistry using an antibody against "-galactosidase. Images of the same field of view under white light are also shown. Data shown are representative of results obtained from three different mice. All photographs were taken at 400x magnification.

PBS or Ad-LacZ had a median survival of 7 days following the initiation of treatment, whereas mice that received injections of Ad-eIF5A1 had a median survival of 25 days (Figure 2A). There was also a significant delay in tumor growth in mice that received intra-tumoral injections of Ad-eIF5A1 (Figure 2B). Regression of tumors was observed in 5 out of 10 (50%) mice in the AdeIF5A1 treatment group, and in one mouse the tumor completely regressed and did not return for the duration of the study. Additionally, no decrease in body weight or activity level was observed in any of the treatment groups indicating that the treatments were well tolerated.

B. Intra-tumoral injection of Ad-eIF5A2 delays growth of tumors in a murine melanoma model A second isoform of eIF5A, eIF5A2, has been localized to a chromosomal region that is frequently amplified in ovarian cancer and has been identified as a

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Figure 2. Mice bearing B16-F0 subcutaneous tumors exhibit prolonged survival following intra-tumoral injection of Ad-eIF5A1. C57BL/6 mice were injected subcutaneously on the flank with B16-F0 cells. Intra-tumoral injections of PBS (buffer), Ad-LacZ, or AdeIF5A1 were given three times per week until sacrifice. Survival of mice (A) and mean tumor volumes + SE of surviving mice (B) are shown. Supplemental Figure 2: Subcutaneous B16-F0 tumors exhibit increased apoptosis following intra-tumoral injection of Ad-eIF5A1. C57BL/6 mice were injected subcutaneously on the flank with B16-F0 cells. Intra-tumoral injections of PBS, Ad-LacZ or AdeIF5A1 were given on days 0, 2, and 4. Tumors were harvested on days 0, 3, and 6. Apoptotic cells in paraffin-embedded tumor sections were labelled by TUNEL. Data shown are representative of results obtained from three different mice. All photographs were taken at 400x magnification.

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Figure 3: Mice bearing B16-F0 subcutaneous tumors exhibit delayed tumor growth following intra-tumoral injections of either Ad-eIF5A1 or Ad-eIF5A2. C57BL/6 mice were injected subcutaneously on the flank with B16-F0 cells. Intra-tumoral injections of PBS, Ad-eIF5A1 or Ad-eIF5A2 were given three times per week until sacrifice. The graph depicts mean tumor volume + SE for each treatment group (** p<0.01, n = 9; *** p <0.001, n = 8).

Supplemental Figure 3: H&E staining of B16-F0 tumor sections following intra-tumoral injection of Ad-eIF5A1. C57BL/6 mice were injected subcutaneously on the flank with B16-F0 cells. Intra-tumoral injections of PBS, Ad-LacZ, or AdeIF5A1 were given on days 0, 2, and 4. Tumors were harvested on days 0, 3, and 6. Paraffin-embedded tumor sections were examined by H&E staining. Data shown are representative of results obtained from three different mice. All photographs were taken at 400x magnification

(Figure 5A), although the upregulation of p53 was not as strong as that seen in response to Ad-eIF5A1 infection. These data suggest that eIF5A1 and eIF5A2 overexpression may activate similar apoptotic pathways. A significant increase in apoptosis was observed in A549 cells infected with either Ad-eIF5A1 or Ad-eIF5A2 (Figure 5B, 5C). It will be interesting to determine in future experiments the extent to which p53 up-regulation may contribute to apoptosis resulting from eIF5A1 and eIF5A2 over-expression in cell lines, such as A549, in which p53 is not mutated. Subcutaneous A549 tumors were established in SCID mice, and treatment was initiated when the tumors reached 4 mm in diameter. Mice were then divided into

D. Intra-tumoral injection of Ad-eIF5A1 prolongs survival in a murine lung cancer model In order to determine whether use of eIF5A1 to delay tumor growth is feasible in cancer models other than melanoma, the ability of Ad-eIF5A1 and Ad-eIF5A2 to infect and kill A549 cells was confirmed in vitro (Figure 5). A dose-dependent increase in p53 expression was observed in A549 cells infected with increasing amounts of Ad-eIF5A1 (Figure 5A). Over-expression of eIF5A1 has been correlated with increased p53 expression in lung cancer cells (Li et al, 2004) and that result is confirmed in this study. An increase in p53 expression was also observed in response to increasing expression of eIF5A2

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Figure 4: Mice bearing B16-F10 lung tumors exhibit reduced tumor burden following systemic treatment with pCpGeIF5A1:DOTAP complexes. B16-F10 cells were injected into the tail vein of C57BL/6 mice. Fifty micrograms of plasmid DNA was complexed with DOTAP and injected via the tail vein on days 7, 14, and 21 following tumor seeding. (A) Mean lung weights + SE for each treatment group (* p<0.05; n = 5) and (B) photographs of lungs for each treatment group.

three treatment groups and received injections of PBS, AdLacZ, or Ad-eIF5A1 three times weekly until sacrifice. As with the melanoma tumor models, Ad-eIF5A1 treatment provided a significant survival advantage in challenged mice (Figure 6A). The median survival of mice receiving injections of PBS or Ad-LacZ was 24 and 28 days, respectively. A549-bearing mice that were treated with Ad-eIF5A1 had a median survival of 54 days, and tumor growth was considerably delayed (p<0.001; Figure 6B). These results indicate that eIF5A1 has potent anti-tumor activity against both murine and human tumors.

expressing p53 has been approved for use in malignant cancers in China (Peng, 2005). The major finding of the present study is that either local or systemic delivery of the eIF5A1 gene suppresses tumor growth in vivo and that this therapy does not result in any apparent toxic side effects. Although the precise function of eIF5A1 has yet to be clearly defined, it has been proposed that hypusinemodified eIF5A1 is necessary for cell growth based on the finding that depletion of hypusine-containing eIF5A1 by genetic or biochemical means induces cell cycle arrest (Park et al, 1993, 1994). A growing body of evidence indicates that eIF5A1 may also be an important component of the apoptotic process. EIF5A1 was recently identified as a gene target of p53 and demonstrated p53dependent up-regulation in response to mitomycin C, a DNA damaging agent (Rahman-Roblick et al, 2007)

IV. Discussion Gene therapy has shown promise as a therapeutic strategy in the treatment of cancer. Numerous clinical trials using adenovirus to deliver therapeutic genes to tumors have been performed, and an adenovirus

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Figure 5: Ad-eIF5A1 and Ad-eIF5A2 infection up-regulate p53 and induce apoptosis in A549 cells. (A) A549 cells were infected with increasing multiplicity of infection (MOI) of adenovirus expressing either eIF5A1 or eIF5A2, and cell lysate was harvested fortyeight hours later. Western blot analysis of cell lysate using antibodies against eIF5A1, eIF5A2, p53 or "-actin is shown. Densitometry analysis of p53 and actin expression levels was performed using TotalLab TL100 v2006 software and the results expressed as a ratio of p53: actin. (B) A549 cells were infected with Ad-LacZ, Ad-eIF5A1, or Ad-eIF5A2 (MOI 80). Seventy-two hours later, the cells were labelled with Annexin/PI to identify apoptotic cells and analyzed by FACS. Data are means + SE (* p<0.05; *** p<0.0001; n = 3). (C) Dot-plots of Annexin-FITC (x-axis) and PI (y-axis) labelling following adenovirus infection (without pifithrin) and Annexin/PI labelling as in B.

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Figure 6. Mice bearing A549 subcutaneous tumors exhibit prolonged survival following intra-tumoral injection of Ad-eIF5A1. C17-SCID mice were injected subcutaneously on the flank with A549 cells. Intra-tumoral injections of PBS, Ad-LacZ, or Ad-eIF5A1 were given three times per week until sacrifice. A) Survival of mice and (B) mean tumor volumes + SE are shown (*** p < 0.001; n = 10).

Studies with cell lines have indicated that up-regulation of eIF5A1 stimulates the expression of p53 and induces both p53-dependent and p53-independent apoptosis (Li et al, 2004; Taylor et al, 2007), while suppression of eIF5A1 using an siRNA protected lamina cribrosa cells from TNFa-mediated apoptosis (Taylor et al, 2004). The present study confirmed these findings by demonstrating induction of apoptosis as well as a dose-dependent increase in p53 expression in A549 cells infected with Ad-eIF5A1. In addition, A549 cells exhibited a similar increase in apoptosis and p53 expression in response to eIF5A2 overexpression, indicating that eIF5A1 and eIF5A2 may activate similar pathways. EIF5A1 and eIF5A2 share 82 % amino acid identity, including the minimum domain and lysine residue

required for the hypusine modification. While eIF5A1 is abundant in all tissues, expression of eIF5A2 is weak except in testis and parts of the brain, although it has been found to be over-expressed in colon and ovarian cancer. A growing body of evidence has linked eIF5A2 overexpression in cancers to tumor growth and progression. EIF5A2 has been mapped to 3q26, a chromosomal region that is frequently amplified in human ovarian cancer (Guan et al, 2001; Clement et al, 2003) and colorectal carcinomas (Xie et al, 2008). Over-expression of eIF5A2 in a liver cell line was found to increase colony formation and tumor growth in nude mice (Guan et al, 2004), while over-expression of eIF5A2 in colorectal carcinomas was positively correlated to tumor stage and tumor cell proliferation (Xie et al, 2008). Over-expresson of eIF5A2 215

Jin et al: Suppression of tumor growth with eIF5A1 gene therapy has also been linked to advanced stages of ovarian cancer (Guan et al, 2004), and to a higher risk of lymph node metastasis in human gastric cancer (Marchet et al, 2007). Higher eIF5A1 expression has been correlated to K-ras mutations and shorter survival in lung cancer patients (Chen et al, 2003) and was identified as a marker of aberrant proliferation in neoplasia of the vulva (Cracchiolo et al, 2004), suggesting that it may contribute to tumor progression as well. In the present study both eIF5A1 and eIF5A2 gene delivery significantly inhibited melanoma tumor growth in vivo. While the anti-cancer effect of eIF5A1 and eIF5A2 over-expression may appear contradictory to their suspected roles in oncogenesis, we believe this apparent contradiction can be explained by distinct functions of the unhypusinated and hypusinated forms of eIF5A. The oncogenic activity of eIF5A can be attributed to the hypusinated form of the protein (Cracchiolo et al, 2004), particularly since DHS has also been observed to be upregulated in cancers (Ramaswamy et al, 2003; Clement et al, 2006) and inhibition of DHS inhibited the growth of melanoma tumors in mice (Jasiulionis et al, 2007). Furthermore, the in vitro formation of hypusine is responsive to the addition of serum and is greatly increased in Ras transformed NIH3T3 cells (Chen and Chen, 1997). Although the hypusination status of eIF5A1 and eIF5A2 that accumulate in vivo following adenovirus treatment were not identified in this study, it is likely to be the unhypusinated forms due to limiting amounts of DHS and DOHH (Clement et al, 2006). Indeed, we previously demonstrated that adenovirus-mediated over-expression of eIF5A1 in colon cancer cells resulted in the accumulation of the unmodified rather than hypusinated form of the protein and that this accumulation resulted in the induction of apoptosis (Taylor et al, 2007). Over-expression of both DHS and DOHH in the presence of eIF5A1 precursor has been found necessary to obtain an accumulation of hypusinated exogenous eIF5A1 in mammalian cells (Park et al, 2006). Thus, it is not surprising that the data presented here, which results from over-expression of exogenous eIF5A that is likely very inefficiently hypusinated, should be very different from findings resulting from studies looking at over-expression of the endogenous hypusinated protein. In contrast to the proliferative function of the hypusinated form of the protein, previous studies have indicated that the unhypusinated form of eIF5A is involved in apoptosis (Jin et al, 2003). Adenovirus mediated expression of an eIF5A mutant that is incapable of being hypusinated, induced apoptosis in colon cancer cells (Taylor et al, 2007). Furthermore, the use of inhibitors of DHS results in an accumulation of unmodified eIF5A and the induction of apoptosis (Tome et al, 1997; Caraglia et al, 2003; Jin et al, 2003). Since DHS is highly expressed in certain cancer cell lines (Clement et al, 2006) and has been identified as a marker for metastatic disease (Ramaswamy et al, 2003), these studies suggest the possibility that DHS overexpression in cancer cells not only enhances growth of tumors by increasing levels of hypusinated eIF5A but also enhances survival by preventing the accumulation of the unhypusinated, apoptosis-inducing form of eIF5A1.

In this study we demonstrate for the first time the anti-tumor effects of eIF5A1 and eIF5A2 in syngeneic and xenograft cancer models. Treatment of both melanoma and lung subcutaneous tumors with intra-tumoral injections of Ad-eIF5A1 resulted in delayed tumor growth and a significant survival benefit. In the murine melanoma subcutaneous model, regression of tumors was observed in 50% of the Ad-eIF5A1-treated mice, indicating that eIF5A1 treatment may be effective against established tumors. Evidence of nuclear fragmentation and cell loss in situ indicated that apoptotic cell death accounts, at least in part, for the significant inhibition of tumor cell growth observed in the murine melanoma model. The reduction in tumor growth following treatment with Ad-eIF5A1 was more pronounced for murine B16 tumors than for human A549 tumors. This difference could reflect involvement of the immune system in eIF5A-mediated reduction in tumor growth since the A549 tumors were formed in immunocompromised mice. However, the possibility that the difference is attributable to the slower growth of A549 tumors is not ruled out. Indeed, the longer Ad-eIF5A1 treatment required for A549 tumor-bearing mice, 66 days compared with only 37 days for B16 tumor-bearing mice, means that there was a higher prospect of antibody production against the adenovirus in the A549 tumorbearing mice. This would result in more rapid clearing of Ad-eIF5A1 in the A549 tumor-bearing mice and a less pronounced reduction in tumor growth in comparison with B16 tumors. The systemic therapeutic effects of eIF5A1 in the treatment of disseminated tumors were also examined in an experimental metastatic lung cancer model. Intravenous administration of cationic liposomes complexed with bacterially derived plasmid DNA elicits pro-inflammatory cytokine production resulting in toxicity (Tousignant et al, 2000; Zhao et al, 2004) and ensuing loss of transgene expression (Tan et al, 1999; Yew et al, 1999). Bacterial DNA differs from mammalian DNA in that CpG dinucleotides are more frequent and remain unmethylated, resulting in elicitation of a pro-inflammatory response (Krieg et al, 1995). Methylation of bacterial plasmids or removal of their CpG motifs results in a reduced inflammatory response (Yew et al, 2000; Reyes-Sandoval and Ertl, 2004) and higher levels of transgene expression (Tan et al, 1999; Yew et al, 2002; Hodges et al, 2004). The immune response elicited by bacterially derived plasmid DNA limits the effectiveness of repeated doses (Song et al, 1997; Li et al, 1999) and thus removal of CpG motifs permits more effective redosing (Tan et al, 1999). Therefore, in order to maximize transgene expression, a CpG-free expression plasmid complexed with DOTAP was used to deliver eIF5A1 to lung tumors in mice. Treatment with pCpG-eIF5A1 resulted in a 60 % reduction in lung weight compared to pCpG-LacZ vector control indicating that systemic delivery of eIF5A1 has a significant anti-tumor effect on experimental lung metastasis. The present study demonstrates that the proapoptotic effects of eIF5A1 extend to tumors in mice and suggests that up-regulated expression of this protein could have therapeutic value in the treatment of both primary

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Gene Therapy and Molecular Biology Vol 12, page 217 Frequently Amplified Region at 3q26 in Ovarian Cancer. Cancer Res 61, 3806-3809. Hanauske-Abel HM, Slowinska B, Zagulska S, Wilson RC, Staiano-Coico L, Hanauske AR, McCaffrey T, Szabo P (1995) Detection of a sub-set of polysomal mRNAs associated with modulation of hypusine formation at the G1S boundary. Proposal of a role for eIF-5A in onset of DNA replication. FEBS Lett 366, 92-98. Hodges BL, Taylor KM, Joseph MF, Bourgeois SA and Scheule RK (2004) Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides. Mol Ther 10, 269-278. Jasiulionis MG, Luchessi AD, Moreira AG, Souza PP, Suenaga AP, Correa M, Costa CA, Curi R, Costa-Neto CM (2007) Inhibition of eukaryotic translation initiation factor 5A (eIF5A) hypusination impairs melanoma growth. Cell Biochem Funct 25, 109-114. Jenkins ZA, Haag PG and Johansson HE (2001) Human eIF5A2 on chromosome 3q25- q27 is a phylogenetically conserved vertebrate variant of eukaryotic translation initiation factor 5A with tissue-specific expression. Genomics 71, 101-109. Jin BF, He K, Wang HX, Wang J, Zhou T, Lan Y, Hu MR, Wei KH, Yang SC, Shen BF, Zhang XM (2003) Proteomic analysis of ubiquitin-proteasome effects: insight into the function of eukaryotic initiation factor 5A. Oncogene 22, 4819-4830. Kang, HA and Hershey, JW (1994) Effect of initiation factor eIF5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J Biol Chem 269, 3934-3940. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546-549. Li AL, Li HY, Jin BF, Ye QN, Zhou T, Yu XD, Pan X, Man JH, He K, Yu M, Hu MR, Wang J, Yang SC, Shen BF, Zhang XM (2004) A novel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis. J Biol Chem 279, 49251-49258. Li S and Huang L (1997) In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes. Gene Ther 4, 891-900. Li S, Wu SP, Whitmore M, Loeffert EJ, Wang L, Watkins SC, Pitt BR, Huang L (1999) Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors. Am J Physiol 276, L796 -L804. Liu YP, Nemeroff M, Yan YP and Chen KY (1997) Interaction of eukaryotic initiation factor 5A with the human immunodeficiency virus type 1 Rev response element RNA and U6 snRNA requires deoxyhypusine or hypusine modification. Biol Signals 6, 166-174. Marchet A, Mocellin S, Belluco C, Ambrosi A, DeMarchi F, Mammano E, Digito M, Leon A, D'Arrigo A, Lise M, Nitti D (2007) Gene expression profile of primary gastric cancer: towards the prediction of lymph node status. Ann Surg Oncol 14, 1058-1064. Park JH, Aravind L, Wolff EC, Kaevel J, Kim YS and Park MH (2006) Molecular cloning, expression, and structural prediction of deoxyhypusine hydroxylase: a HEAT-repeatcontaining metalloenzyme. Proc Natl Acad Sci U S A 103, 51-56. Park MH, Wolff EC and Folk JE (1993) Is hypusine essential for eukaryotic cell proliferation? Trends Biochem Sci 18, 475479. Park MH, Wolff EC, Lee YB and Folk JE (1994) Antiproliferative effects of inhibitors of deoxyhypusine synthase: Inhibition of growth of Chinese hamster ovary cells by guanyl diamines. J Biol Chem 269, 27827-27832.

and disseminated cancers.

Acknowledgements We are grateful to Anita Antes of the University of Medicine and Dentistry, New Jersey, for generously providing cell lines. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and by a research contract from Senesco Technologies Inc. Some of the experimental observations detailed in this manuscript have formed, in part, the basis for patent applications filed by Senesco Technologies Inc.

References Balch CM, Soong SJ, Gershenwald JE, Thompson JF, Reintgen DS, Cascinelli N, Urist M, McMasters KM, Ross MI, Kirkwood JM, Atkins MB, Thompson JA, Coit DG, Byrd D, Desmond R, Zhang Y, Liu PY, Lyman GH, Morabito A (2001) Prognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer melanoma staging system. J Clin Oncol 19, 36223634. Bessis N, GarciaCozar FJ and Boissier MC (2004) Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther 11, S10-S17. Bragonzi A, Boletta A, Biffi A, Muggia A, Sersale G, Cheng SH, Bordignon C, Assael BM, Conese M (1999) Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs. Gene Ther 6, 1995-2004. Bragonzi A, Dina G, Villa A, Calori G, Biffi A, Bordignon C, Assael BM, Conese M (2000) Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs. Gene Ther 7, 1753-1760. Caraglia M, Marra M, Giuberti G, D'Alessandro AM, Baldi A, Tassone P, Venuta S, Tagliaferri P, Abbruzzese A (2003) The eukaryotic initiation factor 5A is involved in the regulation of proliferation and apoptosis induced by interferon-alpha and EGF in human cancer cells. J Biochem 133, 757-765. Chen G, Gharib TG, Thomas DG, Huang CC, Misek DE, Kuick RD, Giordano TJ, Iannettoni MD, Orringer MB, Hanash SM, Beer DG (2003) Proteomic analysis of eIF-5A in lung adenocarcinomas. Proteomics 3, 496-504. Chen ZP and Chen KY (1997) Marked elevation of hypusine formation activity on eukaryotic initiation factor 5A in vHA-RAS transformed mouse NIH3T3 cells. Cancer Lett 272, 15865-15871. Clement PM, Henderson CA, Jenkins ZA, Smit-McBride Z, Wolff EC, Hershey JW, Park MH, Johansson HE (2003) Identification and characterization of eukaryotic initiation factor 5A-2. Eur J Biochem 270, 4254-4263. Clement PM, Johansson HE, Wolff EC and Park MH (2006) Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells. FEBS J 273, 1102-1114. Cracchiolo BM, Heller DS, Clement DMJ, Wolff EC, Park MH and Hanauske-Abel HH (2004) Eukaryotic initiation factor 5A-1 (eIF5A-1) as a diagnostic marker for aberrant proliferation in intraepithelial neoplasia of the vulva. Gynecol Oncol 94, 217-222. Guan XY, Fung JM, Ma NF, Lau SH, Tai LS, Xie D, Zhang Y, Hu L, Wu QL, Fang Y, Sham JS (2004) Oncogenic role of eIF-5A2 in the development of ovarian cancer. Cancer Res 64, 4197-4200. Guan XY, Sham, JS, Tang TC, Fang Y, Huo KK and Yang JM (2001) Isolation of a Novel Candidate Oncogene within a

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Jin et al: Suppression of tumor growth with eIF5A1 gene therapy Peng Z (2005) Current Status of Gendicine in China: Recombinant Human Ad-p53 Agent for Treatment of Cancers. Hum Gene Ther 16, 1013-1024. Rahman-Roblick R, Roblick UJ, Hellman U, Conrotto P, Liu T, Becker S, Hirschberg D, Jรถrnvall H, Auer G, Wiman KG (2007) p53 targets identified by protein expression profiling. Proc Natl Acad Sci U S A 104, 5401-5406. Ramaswamy S, Ross KN, Lander ES and Golub TR (2003) A molecular signature of metastasis in primary solid tumors. Nat Genet 33, 49-54. Reyes-Sandoval A and Ertl HC (2004) CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses. Mol Ther 9, 249-261. Schnier J, Schwelberger HG, Smit-McBride Z, Kang HA and Hershey JW (1991) Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiaie. Mol Cell Biol 11, 31053114. Schrader R, Young C, Kozian D, Hoffman R and Lottspeich F (2006) Temperature-sensitive eIF5A mutant accumulates transcripts targeted to the nonsense-mediated decay pathway. J Biol Chem 281, 35336-35346. Song YK, Liu F, Chu SY and Liu DX (1997) Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Hum Gene Ther 8, 1585-1594. Sรถrenson S, Glimelius B, Nygren P, SBU-group, Swedish Council of Technology Assessment in Health Care (2001) A systematic overview of chemotherapy effects in non-small cell lung cancer. Acta Oncol 40, 327-339. Tan Y, Li S, Pitt BR and Huang L (1999) The Inhibitory Role of CpG Immunostimulatory Motifs in Cationic Lipid VectorMediated Transgene Expression in Vivo. Hum Gene Ther 10, 2153-2161. Taylor CA, Senchyna M, Flanagan J, Joyce EM, Cliche DO, Boone AN, Culp-Stewart S, Thompson JE (2004) Role of eIF5A in TNF-alpha-mediated apoptosis of lamina cribrosa cells. Invest Ophthalmol Vis Sci 45, 3568-3576. Taylor CA, Sun Z, Cliche DO, Ming H, Eshaque B, Jin S, Hopkins MT, Thai B, Thompson JE (2007) Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor alpha signalling. Exp Cell Res 313, 437-449. Tome M, Fiser S, Payne C and Gerner E (1997) Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis. Biochem J 328, 847-854. Tousignant JD, Gates AL, Ingram LA, Johnson CL, Nietupski JB, Cheng SH, Eastman SJ, Scheule RK (2000) Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice. Hum Gene Ther 11, 2493-2513. Wolff EC, Park MH and Folk JE (1990) Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD. J Biol Chem 265, 4793-4799.

Xie D, Ma NF, Pan ZZ, Wu HX, Liu YD, Wu GQ, Kung HF, Guan XY (2008) Overexpression of EIF-5A2 is associated with metastasis of human colorectal carcinoma. Hum Pathol 39, 80-86. Xu A, Jao DL and Chen KY (2004) Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity copurification and differential display. Biochem J 384, 585590. Yew NS, Zhao H, Przybylska M, Wu IH, Tousignant JD, Scheule RK, Cheng SH (2002) CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo. Mol Ther 5, 731-738. Yew NS, Zhao H, Wu IH, Song A, Tousignant JD, Przybylska M, Cheng SH (2000) Reduced Inflammatory Response to Plasmid DNA Vectors by Elimination and Inhibition of Immunostimulatory CpG Motifs. Mol Ther 1, 255-262. Yew NS, Wang KX, Przybylska M, Bagley RG, Stedman M, Marshall J, Scheule RK, Cheng SH (1999) Contribution of Plasmid DNA to Inflammation in the Lung after Administration of Cationic Lipid:pDNA Complexes. Hum Gene Ther 10, 223-234. Zanelli CF, Maragno AL, Gregio AP, Komili S, Pandolfi JR, Mestriner CA, Lustri WR, Valentini SR (2006) eIF5A binds to the translational machinery components and affects translation in yeast. Biochem Biophys Res Commun 348, 1358-1366. Zhao H, Hemmi H, Akira S, Cheng SH, Sheule RK and Yew NS (2004) Contribution of toll-like receptor 9 signaling to the acute inflammatory response to nonviral vectors. Mol Ther 9, 241-248. Zuk D and Jacobson A (1998) A single amino acid substitution in yeast eIF-5A results in mRNA stabilization. EMBO J 17, 2914-2925.

John E.Thompson

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Gene Therapy and Molecular Biology Vol 12, page 219 Gene Ther Mol Biol Vol 12, 219-238, 2008

Hexavalent chromium exposure, genomic instability and lung cancer Review Article

Ana M. Urbano1,2,3, Carlos F.D. Rodrigues1,2, Maria Carmen Alpoim1,2,4,* 1

Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Coimbra, Portugal Centro de Investigação em Meio Ambiente, Genética e Oncobiologia (CIMAGO), Faculdade de Medicina, Universidade de Coimbra, Coimbra, Portugal 3 Unidade I&D Química-Física Molecular, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Coimbra, Portugal 4 Centro de Neurociências e Biologia Celular, Coimbra, Portugal 2

__________________________________________________________________________________ *Correspondence: Maria Carmen Alpoim, Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Apartado 3126, Coimbra, Portugal; Tel: + 351239853603; Fax: + 351239853607; e-mail: mcalpoim@iav.uc.pt Key words: Hexavalent chromium, lung cancer, genomic instability, microsatellite instability Abbreviations: Ascorbate, (Asc); ataxia telangiectasia mutated kinase, (ATM); benzo[!]pyrene, (B[!]P); base excision repair system (BER); chromium, (Cr); DNA-protein crosslinks (DPCs); hexavalent chromium [Cr(VI)]; Falconi anemia, (FA); homologous recombination, (HR); International Agency for Research on Cancer, (IARC); interstrand cross-links, (ICLs); major mismatch repair, (MMR); non-small cell lung cancer (NSCLC); Occupational Safety and Health Administration (OSHA); permissible exposure limit (PEL); reactive oxygen species, (ROS); US Environmental Protection Agency, (USEPA); World Health Organization (WHO) Received: 28 July 2008; Revised: 25 August 2008 Accepted: 29 August 2008; electronically published: October 2008

Summary Worldwide, several million workers experience occupational exposure to different hexavalent chromium [Cr(VI)] compounds (chromates) which have long been recognized as human respiratory tract carcinogens through chronic exposure. Although the majority of lung cancers were found among Cr(VI)-exposed workers who smoked, smoking does not affect chromium accumulation in the lung and chromate exposure was clearly established as an independent risk factor for lung cancer. Compatible with the smoking-unrelated origin of the majority of chromate malignancies are the findings that the molecular features of chromate- and smoking-associated cancers are very different and that the location of chromate lung tumors, i.e. the bronchial bifurcations, corresponds to the sites of chromium accumulation in ex-chromate workers. More recent studies also revealed that environmental exposure to particulate Cr(VI) compounds is increasing due to chromium-containing dusts generated from industrial waste disposal, portland cement, concrete pavement, milling, demolition, cigarette smoke and fuel combustion. Experimentally, it has been demonstrated that Cr(VI) compounds can neoplastically transform cells in culture. They are also genotoxic and can induce a wide spectrum of DNA damage, gene mutations, sister chromatid exchanges and chromosomal aberrations. Albeit extensive information and studies on Cr(VI)-induced effects, the mechanisms mediating Cr(VI)-induced toxicity and carcinogenicity are still poorly understood. This happens mostly due to the use, in a vast majority of the studies, of inadequate model systems and exposure regimens, as well as to the restricted access to lung tumor tissues from Cr(VI)-exposed workers, critical for the identification of consistent cellular and molecular changes. Consequently, additional studies using adequate model systems and exposure regimens mimicking occupational exposure conditions will have to be performed in order to understand the signalling mechanisms involved in the cellular response to Cr(VI), as well as the role of genomic instability and of specific DNA repair pathways in the development of this pathology. Knowledge of these pertinent issues will be a step forward for the discovery of biomarkers of malignant transformation that might lead to new treatment approaches or new ways to prevent this particular subtype of lung cancer. This review will discuss several aspects underlying Cr(VI)-induced carcinogenicity. However, particular emphasis will be given to the recently identified roles of Cr(VI)-induced DNA lesions, particularly single- and double-strand breaks, on genomic instability, one of the hallmarks of lung cancer.

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Urbano et al: Hexavalent Chromium Exposure, Genomic Instability and Lung Cancer Cr(III) and chiefly Cr(VI) (International Agency for Research on Cancer, 1990). Cr(III) is a very stable oxidation state for chromium and no adverse effects were observed in workers exposed to Cr(III) oxide and chromic sulfide for up to 25 years (National Library of Medicine, 1995). Although Hathaway and collaborators (Hathaway et al, 1991) and epidemiological studies have found no clear association between the exposure to Cr(III) compounds and the risk of cancer (International Agency for Research on Cancer, 1990; Agency for Toxic Substances and Disease Registry, 1993; Gibb et al, 2000; Mancuso, 1997) Mancuso alleged that lung cancer death rates increase by gradient level of exposure to insoluble Cr(III). In contrast, chronic exposure to Cr(VI)-containing compounds, which is primarily produced by anthropogenic sources, poses serious risks and Cr(VI) has been considered carcinogenic for more than a century (Långard, 1990). Cr(III) forms very large, low-spin octahedral coordination complexes and chelates which are unable to easily pass through the cell membrane (Cohen et al, 1993). This behavior may explain, at least in part, the lack of mutagenicity of Cr(III) using internationally standardized CHO cell assays (Slesinski et al, 2005), and the lack of toxic effects in vivo following a 3-month chronic exposure of rats and mice to Cr(III) piccolinate (up to 50,000 ppm) (Rhodes et al, 2005). Yet, as there is evidence of Cr(III) cellular uptake (Coogan et al, 1991; Gao et al, 1993; Sipowicz et al, 1997) and of transgenerational carcinogenicity in the offspring of male mice exposed intraperitoneally to CrCl3 (Yu et al, 1999), a National Toxicology Program 2-year bioassay on Cr(III) supplementation is ongoing in the USA in order to elucidate its potentially harmful effects (Vincent, 2004). As to Cr(VI), it readily enters cells, where it induces genotoxic, clastogenic and cytotoxic damage (Singh et al, 1998; De Flora, 2000; O’Brien et al, 2003). In fact, at physiological pH Cr(VI) exists as the oxyanion chromate (CrO42"), whose tetrahedral structure, resembling sulfate and phosphate oxyanions, facilitates its transport and intracellular accumulation (Alcedo and Wetterhahn, 1990). This may explain the greater relative absorption and toxicity of Cr(VI) versus Cr(III) (Costa, 1997). Moreover, lung tissue readily retains Cr(VI) (Ishikawa et al, 1994a,b, Kondo et al, 2003), causing upper respiratory tract and pulmonary carcinogenesis, as well as noncancerous tissue erosion, respiratory distress, fibroproliferative disease (Glaser et al, 1985; Adachi et al, 1986, 1987; Bright et al, 1997; Singh et al, 1998) and airway hypersensitivity (Bright et al, 1997). Autopsies of chromate-exposed workers revealed that high levels of particulate (insoluble) chromates accumulate and persist in the workers’ lungs for as long as 20 years after exposure (Ishikawa et al, 1994a,b; Kondo et al, 2003). Although both Cr(III) and Cr(VI) compounds induced dose-dependent anchorage independence (a characteristic often associated with malignant transformation) in cultured diploid human fibroblasts, several experiments revealed that Cr(VI) compounds are approximately 1,000-fold more cytotoxic and mutagenic than Cr(III) compounds (Biedemann and Landolph, 1987,

I. Introduction Certain Cr(VI) compounds are carcinogenic to humans, potent inducers of tumors in experimental animals (International Agency for Research on Cancer, 1990; Landolph, 1990; Långard, 1990; Agency for Toxic Substances and Disease Registry, 1993) and can transform cells in culture (Leonard, 1988; Patierno et al, 1988; Elias et al, 1989; Xie et al, 2007). They are also genotoxic and can induce a wide spectrum of DNA damage, gene mutations, sister chromatid exchanges (O’Brien et al, 2003) and chromosomal aberrations (Manning et al, 1994; Wise et al, 2002, 2003, 2004a,b, 2006; Xie et al, 2004). Although there is vast information on Cr(VI)induced effects, it has been difficult to draw consistent conclusions as to the mechanisms mediating Cr(VI)induced toxicity and carcinogenicity, since the large majority of information was obtained using inadequate model systems and/or exposure regimens. In particular, crucial information for the design of new treatment approaches and/or new ways to avoid this particular subtype of lung cancer, such as biomarkers of malignant transformation for early detection of Cr(VI)-induced cancers and those events that are important to lung cancer progression, is yet missing. It must be acknowledged that access to lung tumor tissue from Cr(VI)-exposed workers is restricted, hindering the identification of consistent cellular and molecular changes. Moreover, drawing conclusions from the study of lung tumor tissues is not a straightforward process, since many workers are also smokers. The limited work done to this day (fewer than 10 tumors in a couple of studies) indicates that the model of clonogenic expansion of mutated cells may not apply to Cr(VI)-induced tumors, as common oncogenes such as Ras appear unaffected (Ewis et al, 2001). In terms of cell systems, primary epithelial lung cells should be used. However, this type of study requires prolonged exposure to sub-toxic Cr(VI) doses, a requirement that is not compatible with the very short lifespan of primary cultures. Hence the option for immortalized nontumorigenic cell lines.

II. Chromium carcinogenicity: background information Chromium (Cr) is a transition metal element whose environmental behavior and biological activity depend mostly on its oxidation state. Cr(0), Cr(III) and Cr(VI) are the oxidation states of Cr most commonly found in the workplace and environment. Cr(0) is found in alloys with other metals, such as nickel (Ni), iron (Fe) and cobalt (Co), being largely used in stainless steel production. Although metallic Cr is chemically and biologically inert, exposure to Cr(0)-containing dusts may cause nonspecific irritation in the respiratory tract (Zhitkovich, 2005). Moreover, even though Cr(0) is stable to oxidation by atmospheric oxygen under environmental conditions, when present in prostheses can undergo slow oxidation, thus releasing higher oxidative Cr compounds, particularly Cr(III) and Cr(VI) (Case et al, 1995; Merritt and Brown, 1995). Also, high temperature processes such as welding or exposure to corrosive chemicals lead to the formation of

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Gene Therapy and Molecular Biology Vol 12, page 221 1990; De Flora et al, 1990; Slesinski et al, 2005). Thus, based on epidemiologic studies and on in vivo and in vitro carcinogenicity studies, the US Environmental Protection Agency (USEPA) and the International Agency for Research on Cancer (IARC) classified Cr(VI) as a human carcinogen of Group I (USEPA, 1992) and Group A (International Agency for Research on Cancer, 1990), respectively. In contrast, Cr(III) is considered an essential nutrient required for normal carbohydrate and lipid metabolism (Morris et al, 1992; Anderson, 2000; Ding and Shi, 2002). Occupational exposure to different Cr(VI) compounds affects several million workers worldwide (International Agency for Research on Cancer, 1990; Agency for Toxic Substances and Disease Registry 1993). Moreover, non-occupational exposure to particulate Cr(VI) compounds is increasing due to chromiumcontaining dusts generated from industrial waste disposal, portland cement, concrete pavement, milling, demolition, cigarette smoke and fuel combustion (Freeman et al, 1997; Singh et al, 1998; O’Brien et al, 2003). The situation is even more complex, as very recent in vitro data revealed that cells bearing deficiencies in DNA repair genes may have a higher Cr(VI) uptake capacity when compared to the parent wild type cells (Grlickova-Duzevika et al, 2006; Camrye et al, 2007; Saverya et al, 2007; Stackpole et al, 2007). These findings are consistent with previous reports on the existence of functional polymorphisms in DNA repair genes which correlate with increased chromosome damage in Cr(VI)-exposed workers (Lei et al, 2002; Mateuca et al, 2005). Not all Cr(VI) compounds are equally effective as carcinogens. Pathology data of chromate lung cancers indicated that particulate Cr(VI) compounds exhibit the highest carcinogenicity (Ishikawa et al, 1994a,b; Kondo et al, 2003). This is in agreement with what was found in laboratory animals, where the Cr(VI) compounds that induced tumors were the slightly soluble to highly insoluble particulate forms of sintered calcium chromate, zinc chromate and lead chromate administered in their non solubilised particulate forms (Leonard and Lauwerys, 1980; Levy and Vanitt, 1986; Levy et al, 1986; International Agency for Research on Cancer, 1990). Also, in vitro studies have demonstrated the possibility of inducing malignant transformation in different cell lines by using insoluble (particulate) Cr(VI) compounds (Patierno et al, 1988; Xie et al, 2007, 2008). Hence, it is generally accepted that Cr(VI) is specifically a respiratory tract carcinogen when particulate forms are inhaled at relatively high doses for long periods of time (De Flora et al, 1990; De Flora, 2000; O’Brien et al, 2003). It must be acknowledged, however, that there are reports in the literature of epidemiological studies revealing that human exposure to soluble chromates also significantly increases the risk of lung cancer (Mancuso, 1997; Sorahan et al, 1998; Gibbs et al, 2000; Park et al, 2004). Therefore, taking into consideration all the conflicting results, it may be wise to consider all Cr(VI) compounds as potentially carcinogenic. The negative results obtained in in vivo carcinogenesis experiments with soluble chromates were

ascribed to the vast extracellular capacity of mammals to reduce Cr(VI) (De Flora et al, 1997). On the other hand, low solubility Cr(VI) nanoparticles adhere to lung airway epithelial cells and slowly release high concentrations of Cr(VI) at the cell surface. A detailed study using human lung fibroblasts indicated that the genotoxicity of Cr(VI) particulates is due to the slow chronic extracellular dissolution of the solid particles and the consequent release of the soluble Cr(VI) oxyanion that, once inside the cells, induces DNA damage (Xie et al, 2004). This study also suggested that the difference in the carcinogenicity between particulate and soluble Cr(VI) compounds is not a consequence of genotoxic effects from the cation, although an indirect role for the cation, such as allowing Cr-damaged cells to escape cell death, cannot be excluded. In fact, in another study it was demonstrated that following exposure of normal human lung small airway epithelial cells to lead chromate particles (1-4 µm size), lead associated with DNA (Singh et al, 1999). It is also possible that a relationship exists between the degree of carcinogenicity of a given compound and the time it persists at sites of exposure (Xie et al, 2004). Data indicate that Cr(VI)-induced tumors possibly start off in bronchial cells at the site of bifurcations where Cr(VI) particles are most likely to impact and persist (Ishikawa et al, 1994a,b), although no tumors were observed when the in vivo implantation model of lung carcinogenesis and insoluble salts of lead and barium chromates were used (Levy et al, 1986). In 2000, based on epidemiological and risk assessment studies, the Occupational Safety and Health Administration (OSHA) established a permissible exposure limit (PEL) to Cr(VI) (as CrO3) in air in the work place of 100 #g/m3 (Lurie and Wolfe, 2002). However, the publication of consistent reports on the induction of lung cancer in humans upon exposure to 100 #g/m3 Cr(VI) and less (Långard, 1990; Gibb et al, 2000) urged a reevaluation of the lifetime risk of dying from lung cancer for this exposure limit. An estimated lifetime risk of 25% was obtained by Park and collaborators (Park et al, 2004), which was comparable to previous estimates by the USEPA, California EPA and OSHA using different occupational data (Park et al, 2004). Therefore, early in 2006 OSHA lowered the PEL for Cr(VI) to 5 #g/m3 (Occupational Safety and Health Administration, Department of Labor, 2006).

III. The specificities of Cr(VI)-induced lung cancer Lung cancer is the leading cause of cancer death worldwide and non-small cell lung cancer (NSCLC) accounts for nearly 80% of the disease (American Cancer Society, 2001). In terms of cell morphology, adenocarcinoma and squamous cell carcinoma are the most common types of NSCLC (Travis et al, 1996). Whereas squamous cell carcinoma is closely associated with tobacco smoking habits, the etiology of the adenocarcinoma remains unclear (Bennett et al, 1999; Hainaut and Pfeifer, 2001). The clinical courses of the two types of tumors are similar but whereas adenocarcinomas are characterized by their peripheral location in the lung 221

Urbano et al: Hexavalent Chromium Exposure, Genomic Instability and Lung Cancer and often have activating mutations in the K-ras oncogene (Gazdar et al, 1994; Graziano et al, 1999), squamous cell carcinomas are usually centrally located and carry p53 gene mutations more frequently (Therrien et al, 1999; Niklinska et al, 2001). However, it must be said that even though the incidence of p53 mutations is much lower in lung adenocarcinomas (Niklinska et al, 2001), expression of p53-dependent genes such as p21waf1/CIP1 and 14-3-3! is very often decreased (Waldman et al, 1996; Chan et al, 1999; Nacht et al, 2001), thus suggesting that inactivation of genes in the p53-pathway may also play an important role in lung adenocarcinomas. Squamous cell carcinoma is the most frequent type of lung cancer among Cr(VI)-exposed workers (Ishikawa et al, 1994a; Kondo et al, 2003). Interestingly, unlike the other types of lung squamous cell carcinomas, the majority of Cr-related cancers exhibit microsatellite instability, aberrant methylation of p16INK4a and a low incidence of p53 mutations (Kondo et al, 1997, 2006; Takahashi et al, 2005). Moreover, when observed, the pattern of p53 mutations differs from that of common squamous cell lung cancers (Kondo et al, 1997). Although the majority of chromate workers that developed lung cancers were also smokers (Ishikawa et al, 1994a,b; Gibb et al, 2000; Kondo et al, 2003), the very different molecular features of these two types of cancer and the fact that the location of chromate lung tumors corresponds to the sites of chromium accumulation point to a smoking-unrelated origin of the common chromate malignancies. Therefore, chromate exposure is now clearly established as an independent risk factor for lung cancer (Gibb et al, 2000). Whether Cr(VI) is a selective cancer agent is a subject of intense dispute and controversy. Indeed, Cr(VI) is considered a selective carcinogen to the lung and sinonasal cavity by the World Health Organization (WHO) (World Health Organization, 1988) and by the IARC, but several authors claim that, in addition to lung cancer, Cr(VI)-exposed workers also developed other types of cancers, such as stomach, kidney and bladder cancers (Royle, 1975; Franchini et al, 1983; L책ngard et al, 1990; Moulin et al, 1990; Becker et al, 1991; Kusiak et al, 1993; Costa, 1997; Gibb et al, 2000). More recently, in a large epidemiological study, Cr(VI) exposure was associated with prostate cancer in nonwhites (Park et al, 2004). In a review published in 2000 (De Flora, 2000), De Flora dismissed most reports of Cr(VI)-induced cancers at sites other than the lower respiratory tract and the sinonasal cavity as inconsistent and referred to a review article (Cohen et al, 1993) and to an "authoritative article on carcinogenesis" (Hayes, 1997) dealing with more recent data where the conclusions of the WHO and the IARC were reiterated. The increase in mental, psychoneurotic and personality disorders among all race groups, reported by Park and collaborators (Park et al, 2004), in association with the findings that Cr(VI) exposure results in chromium accumulation in the central nervous system of rodents, raises the possibility that Cr(VI) is also neurotoxic (Bagchi et al, 1997; Travacio et al, 2001; Ueno et al, 2001;

Attenburrow et al, 2002; Wesseling et al, 2002) and causes brain cancer in exposed workers (Wesseling et al, 2002). On the other hand, although it is well known that dermal contact with chromium compounds is usually associated with allergic responses, characterized by eczema and contact dermatitis (US Department of Health and Human Services, 1993), no significant increase in skin cancer was reported among Cr(VI) exposed workers (L책ngard et al, 1990; Becker et al, 1991). Nevertheless, a recent report revealed that, in mice, chronic exposure to Cr(VI)-contaminated drinking water in combination with solar ultraviolet light induced skin tumors (Costa and Klein, 2006).

IV. Cr(VI) metabolism Many studies demonstrated, in both rodent and human cells, that Cr(VI) oxyanions are quickly actively transported across cell membranes and build up massively inside the cells, reaching levels 10-20 times over those outside the cell within 3 h (Wise and Little, 2002; Wise et al, 2004b, 2006; Xie et al, 2005, 2007; Grlickova-Duzevik et al, 2006; Holmes et al, 2006; Savery et al, 2007; Stackpole et al, 2007). Inside the cells, low molecular weight thiols (glutathione and cysteine) and ascorbate (Asc), believed to be the major intracellular reducers of Cr(VI), rapidly reduce it to the more stable Cr(III) form (Suzuki and Fukuda, 1990; Standeven and Wetterhahn, 1991, 1992; Quievryn et al, 2001). In the case of massive exposures to Cr(VI), hemoglobin (Fernandes et al, 2000), hydrogen peroxide (Kawanishi et al, 1986) and flavoenzymes (Banks and Cooke, 1986; Mikalsen et al, 1989; Shi and Dalal, 1989), as well as the mitochondrial electron transport complexes (Ryberg and Alexander, 1990), may also contribute to Cr(VI) reduction. The intracellular levels of some reducing agents influence Cr(VI) uptake and its consequent intracellular accumulation (Cohen et al, 1993), as well as the ability of the cells to repair DNA-induced damage, as revealed in very recent in vitro studies (Grlickova-Duzevika et al, 2006; Camrye et al, 2007; Saverya et al, 2007; Stackpole et al, 2007). Although the final product of Cr(VI) reduction is always Cr(III), the concentration ratio of reducers to Cr(VI) and the nature of the chief reducer will determine the reduction rate, as well the nature of intermediates and other final products (Stearns and Wetterhahn, 1994; Lay and Levina, 1998; Zhitkovich, 2005; Salnikow and Zhitkovich, 2008). For instance, reduction of Cr(VI) by cysteine is a relatively slow and sequential process that produces transient Cr(V) and Cr(IV) species, whereas reduction by the two electron donor Asc, at physiological concentrations, is fast and generates Cr(IV) as the main Cr intermediate (Levina and Lay, 2005; Zhitkovich, 2005) (Figure 1). In rat lung extracts, Asc is the chief reducer of Cr(VI), accounting for at least 80% of Cr(VI) metabolism (Standeven and Wetterhahn, 1991, 1992). This observation can be rationalised in terms of the rate of Cr(VI) reduction by this agent (the highest among all biological reducing agents) (Standeven and Wetterhahn, 1991, 1992; Zhitkovich, 2005) and by the fact that Asc concentration is 222

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Figure 1. In vivo and in vitro Cr(VI) metabolism and the biological effects of the formation of ternary Cr(III) complexes [L = Asc; Cys (cysteine); GSH (glutathione)]. Adapted from Zhitkovich, 2005.

very high in the target tissues of Cr(VI) toxicity, such as the lung (Slade et al, 1985), which exhibits Asc:Cr(VI) ratios higher than one. A similar finding was reported in extracts of rat liver and kidney (Standeven and Wetterhahn, 1991, 1992). Therefore, taking into account that Asc does not directly generate Cr(V) under physiological conditions (Stearns and Wetterhahn, 1994; Lay and Levina, 1998), it is predictable that only a rapid inflow of massive Cr(VI) doses, resulting in a severe drop in Asc levels, could cause a significant Cr(V) production (Reynolds et al, 2007). The causes for the reported increase on Cr(V) formation under Asc deficiency may include a shift to a thiol-dependent metabolism and/or increased stability of Cr(IV) intermediates capable of generating Cr(V) via secondary reactions (Zhitkovich, 2005). At this point, it is important to mention that, in sharp contrast to what is observed in vivo in lung cells, cultured cells maintained in standard growth media are severely Asc deficient, even when cultivated in its presence, which results in a distorted metabolism and in potentially abnormal responses to Cr(VI) (Reynolds and Zhitkovich, 2007). Thus, results obtained using cellular systems whose Asc levels were not restored to physiological values must be interpreted with care.

are genotoxic. The genotoxicity of Cr(VI) has been ascribed to the reactive intermediates Cr(V) and Cr(IV), to carbon/sulfur and oxygen radicals, as well as to Cr(III), all of them produced during Cr(VI) intracellular reduction (Oâ&#x20AC;&#x2122;Brien et al, 2003; Xie et al, 2005; Zhitkovich, 2005; Wise et al, 2006). The unique spectrum of nuclear matrix associated DNA damage (Xu et al, 1994), induced by some or all of these reaction products, particularly Cr(III), includes DNA single- and double-strand breaks, DNAprotein cross-links, Cr(III)-DNA binary and ternary complexes, and DNA-Cr(III)-DNA interstrand cross-links (Oâ&#x20AC;&#x2122;Brien et al, 2003; Ha et al, 2004; Zhitkovich, 2005).

A. Cr(III)-DNA complexes Due to its chemical properties, Cr(III) tends to form coordinated complexes with its intracellular reducers, glutathione, cysteine and Asc, which react with DNA giving rise to ternary complexes (Zhitkovich et al, 1995, 1996b; Quievryn et al, 2002). These ternary complexes represent the main forms of Cr-DNA adducts in mammalian cells. They are all premutagenic (Voitkun et al, 1998; Quievryn et al, 2002; Quievryn et al, 2003), and it has been suggested that the mutagenic forms are probably Cr(III) chelates involving a phosphate group and the N7 position of guanines (Figure 2) (Zhitkovich et al, 2001), although nucleotide level mapping of major ternary Cr(III)-DNA adducts detected no apparent base specificity in Cr(III)-DNA binding (Voitkun et al, 1998).

V. Cr(VI)-induced genetic lesions Although Cr(VI) itself does not react with isolated DNA under physiological conditions, Cr(VI) compounds

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Figure 2. Proposed structure for L–Cr(III)–DNA ternary complexes. Adapted from Zhitkovich, 2005.

Back in the nineties, Zhitkovich and collaborators showed, in an in vitro study, that cysteine-Cr(III)-DNA and glutathione-Cr(III)-DNA cross-links were the most abundant Cr(III)-DNA ternary complexes (Zhitkovich et al, 1995). As to Asc-Cr(III)-DNA cross-links, which appear to be the most mutagenic of all Cr-DNA adducts (Quievryn et al, 2003; Zhitkovich, 2005), their detection in cultured cells required restoration of physiological levels of Asc (Quievryn et al, 2002). This finding may, at least in part, explain the lower mutagenicity observed under low Asc concentrations as compared to that observed with physiological concentrations (Quievryn et al, 2006). Interestingly, although results from shuttle-vector experiments have shown that these ternary adducts inhibit replication in human cells (Quievryn et al, 2003), they demonstrated very little, if any, blocking potential in acellular systems that used purified polymerases, suggesting that the inhibitory action observed in vivo results from an indirect effect, rather than from direct interference of these adducts with replicative polymerases. Finally, these ternary complexes represent a major form of Cr(VI)-induced toxicity, as human cells unable to remove these lesions upon inactivation of nucleotide excision repair (NER) became much more sensitive to apoptosis and clonogenic lethality (Reynolds et al, 2004). Although less frequent, Cr(III)-DNA binary complex formation was also observed (Zhitkovich, 2005). Apparently, the binary complexes can be generated directly by reaction of Cr(III) with DNA or indirectly through binding of reactive Cr(IV) or Cr(V) species to DNA (Zhitkovich, 2005). Contrary to Cr-DNA ternary adducts, these binary adducts are only weakly mutagenic (Voitkun et al, 1998; Quievryn et al, 2003). DNA-Cr(III)-DNA interstrand cross-links (ICL) could only be detected during in vitro reduction of Cr(VI) by Asc (Bridgewater et al, 1994b; O’Brien et al, 2001; O’Brien et al, 2002) or cysteine (Zhitkovich et al, 2000), but not by glutathione (O’Brien et al, 2001). Also, their formation was revealed to be highly dependent on the ratio of reducer to Cr(VI), and the most extensive DNA crosslinking was always observed under conditions of limited reducer concentrations. Taking into account that the

formation of bifunctional complexes involving sterically hindered molecules, such as DNA, is an unlikely event, it was proposed that their formation would involved oligomeric Cr(III) complexes with each DNA strand bound to a different Cr(III) atom (Zhitkovich, 2005). Considering Cr(V)-DNA complexes, it has already been mentioned that Cr(V) formation is believed to occur only under very specific conditions. In any case, Cr(V) complexes exhibited little or no direct binding to DNA in vitro (Molyneux and Davies 1995; Levina et al, 2001) and were not required for the formation of Cr-DNA adducts (Quievryn et al, 2002, 2003; Reynolds et al, 2007; Reynolds and Zhitkovich, 2007). Moreover, in vitro studies on the formation of mutagenic damage showed that increased Cr(V) formation did not translate into higher levels of mutagenic damage (Quievryn et al, 2006). Also, Cr(V) was reported a weak mutagen in mammalian cells that rely on thiols to reduce Cr(VI) (Cohen et al, 1993). Altogether, these observations argue against an important role of Cr(V) in Cr(VI)-induced carcinogenicity. Cr(VI)-induced DNA lesions in mammalian cells also include the formation of cross-links between proteins and DNA (DPCs) (Zhitkovich, 1996a). Cr-DPCs are stable, ternary DNA adducts and constitute a significant class of Cr-related genetic lesions that may represent a major obstacle for the replication and transcription processes (Fornace et al, 1981; Manning et al, 1994; Wei et al, 2004; Schnekenburger et al, 2007). In both the liver and kidneys of injected rats, Cr-DPCs have been reported to extensively develop between DNA and non-histone proteins (Cupo and Wetterhahn, 1985a). Some of the proteins cross-linked to DNA were shown to be nuclear lamins, actin and nuclear matrix proteins (Miller and Costa, 1988; Miller et al, 1991). However, as much as 50% of the cross-linking did not involve a Cr atom but, instead, appeared to be catalyzed by oxidative mechanisms (Mattagajasingh and Misra, 1996). Although it was claimed that this type of lesion represents only a very small fraction of the initially formed DNA adducts in cultured cells, about 0.1% according to Zhitkovich (Zhitkovich, 2005), it is used as a biomarker of genetic damage in Cr-exposed human populations (Costa et al,

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the addition of catalase and iron chelators or the increase of glutathione levels inhibited Cr(VI)-mediated singlestrand break formation (Messer et al, 2006). However, cDNA microarray analysis of normal human lung cells treated with toxicologically relevant concentrations of Cr(VI) found no clear evidence for the involvement of reactive oxygen species (ROS) (O’Brien et al, 2003). A point that further complicates the elucidation of the relationship between Cr(VI) reduction and ROS is the strong oxidizing potential of high-valent Cr(V) (Zhitkovich, 2005). Therefore, the relative contribution of oxidative mechanisms involving ROS to the genotoxicity and mutagenicity of Cr(VI) is still a subject of controversy (O’Brien et al, 2003). Although a variety of observations, such as an increased frequency of chromosomal breaks (Sen et al, 1987; Wise et al, 1992) and micronuclei (Witt et al, 2000), did suggest the induction of double-strand breaks upon Cr(VI) exposure, it was only recently, following significant work by the groups of Patierno and of Zhitkovich, that stronger evidence was obtained through the use of indirect immunofluorescence for $-H2AX as a biochemical marker for DNA double-strand breaks (Ha et al, 2004; Peterson-Roth et al, 2005 ; Reynolds et al, 2007). H2AX is a protein that is specifically phosphorylated ($H2AX) and forms foci at the sites of double-strand breaks (Rogakou et al, 1999; Celeste et al, 2002).

B. Strand breaks In vivo, the production of single-strand breaks, either directly as a consequence of Cr-DNA interactions and/or of oxygen/carbon radical generation (Figure 3) or indirectly as a result of replication past/repair of Crlesions, is a commonly reported lesion in the livers and kidneys of mice intraperitoneally exposed to Cr(VI) (Ueno et al, 2001). The presence of this type of lesion is a common indicator of a more generalized oxidative insult on DNA, which can include other forms of damage to the DNA backbone, as well as the formation of oxidized bases leading to abasic sites (O’Brien et al, 2003; Messer et al, 2006). Given the fact that, in vitro, the conditions for the formation of abasic sites were reported to be very similar to those required for the formation of single-strand breaks, it appears that oxidant species such as hydroxyl radicals generated by a "Fenton-like" reaction between the reactive intermediate species [Cr(V)/Cr(IV)] and H2O2 (Figure 4) may be involved in both types of DNA lesions (Shi and Dalal, 1990; Casadevall and Kortenkamp, 1995; Tsou and Yang, 1996; Shi et al, 2004; Messer et al, 2006). In support of this hypothesis, a recent study, with normal lung bronchial epithelial cells and CHO cells, revealed that

Figure 3. Possible mechanisms of Cr(VI)-induced single-strand breaks formation [GSH = glutathione].

Figure 4. Generation of hidroxyl radical by a Fenton-like mechanism.

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Urbano et al: Hexavalent Chromium Exposure, Genomic Instability and Lung Cancer When colon HCT116+ch3 cells were exposed to Cr(VI), there was an extensive accumulation of $-H2AX foci, indicating the formation of double-strand breaks (Peterson-Roth et al, 2005). The formation of these highly toxic lesions was followed by a rapid activation of apoptotic processes. The fact that a major increase in $H2AX focus-containing cells occurred at 6 h post exposure suggested that these lesions were not directly caused by Cr(VI), but were rather the result of damaged DNA processing. Also, the majority of $-H2AXcontaining cells were positive for cyclin B1, a protein that is highly expressed in G2 phase (Sherwood et al, 1994; Hwang et al, 1995). These results point to the possibility that passage of these cells through S phase is a requisite for double-strand breaks formation. This suggestion is supported by recent reports that state that Cr(VI)-induced double-strand breaks occurred in cycling human fibroblasts, but not in growth arrested ones (Ha et al, 2004). Interestingly, Cr(VI) exposure of HCT116 cells deficient for MLH1 (MLH1â&#x20AC;&#x201C;/â&#x20AC;&#x201C;), one of the major mismatch repair (MMR) proteins, yielded much lower focus formation than in MLH1+/+ cells (Peterson-Roth et al, 2005), which strongly supports the hypothesis that doublestrand breaks are secondary lesions, probably resulting from abnormal processing of Cr-DNA damage by the MMR system. Data obtained in this study suggest the involvement of Cr-DNA adducts. Indeed, MMR complexes can specifically bind to the abundant Cr-DNA adducts found in Cr(VI)-exposed cells, which can lead to a strong replication blockage, resulting in frequent stalling of replication complexes and, as a consequence, in a high probability of complete arrest and collapse of replication forks. In turn, these collapsed replication forks will ultimately result in the formation of double-strand breaks (Sogo et al, 2002; Courcelle et al, 2003). The G2 specificity of double-strand breaks formation is in agreement with this hypothesis, as it may reflect the requirement of Cr-damaged cells to pass through the Sphase so that the MMR system activates aberrant processing. Considering all these data, Reynolds and collaborators (Reynolds et al, 2007) proposed the model that highly mutagenic adducts such as Asc-Cr-DNA crosslinks induce mismatches during the replication of damaged DNA and that these compound lesions (mismatches at the sites of Cr-Adducts) lead to abnormal MMR, ultimately leading to the formation of double-strand breaks. Curiously, although the same mechanism for the induction of double-strand breaks could also operate for Asc-deficient cells, extensive double-strand breaks formation was not observed. This was possibly related to the fact that thiol-stimulated DNA damage is weakly mutagenic, inducing low levels of base mispairing and mild activation of MMR (Quievryn et al, 2006). Finally, other mechanisms of double-strand breaks formation cannot, at this stage, be excluded. For instance, it is possible that the transition of cells with single-strand breaks from S to G2 could inactivate fork viability mechanisms resulting in collapsed forks and double-strand breaks formation (Reynolds et al, 2007). This proposal is reinforced by the studies of Kuzminov (Kuzminov, 2001)

in replicating chromosomes, and by the reports of Merrill and Holm on the generation of double-strand breaks following the accumulation of single-strand breaks in response to hydroxyurea in mec-srf mutated Saccharomyces cerevisiae (Merrill and Holm, 1999).

VI. Consequences of Cr(VI)-induced genetic damage A. Genomic instability: the role of strand breaks and DNA repair systems Although genomic instability is a hallmark of cancers in general, Cr-related lung cancers can be distinguished from the other types of lung squamous cell carcinomas in that they exhibit a high incidence of microsatellite instability. Cr(VI) is also known to induce clastogenic effects such as sister chromatid exchanges, chromosomal aberrations and genetic deletions in several cell lines, including human lung fibroblasts and bronchial epithelial cells exposed to Cr(VI) (Sen and Costa, 1986; Wise et al, 1994, 2002, 2006; Seoane et al, 2002; Grlickova-Duzevik et al, 2006; Holmes et al, 2006). Although the exact mechanisms underlying Cr(VI)-induced genomic instability are still largely unknown, the damage that takes place in nuclear matrix-associated DNA, particularly single- and double-strand breaks, is bound to play an important part in this effect. DNA damage normally triggers an arrest in either S or G2 phases of the cell cycle in order to allow cells to repair their genomic damage. However, when this arrest is very prolonged, it usually uncouples cell cycle progression from centrosome duplication, thus generating cells with aberrant centrosome numbers (Xie et al, 2005; Doxsey et al, 2005; Brito and Rieder, 2006, Ganem et al, 2007). These cells are potentially harmful because they can undergo mitotic catastrophe, engendering potentially malignant aneuploid progeny (Doxsey et al, 2005; Brito and Rieder, 2006, Ganem et al, 2007) (Figure 5). Quite recently, it was reported that the Falconi anemia (FA) pathway may be involved in Cr(VI)-induced chromosome damage. Both FA and homologous recombination (HR) pathways are known to be involved in DNA interstrand cross-links repair (Savery et al, 2007). Hence, it is not surprising that FA A cells, cells deficient in FANCA gene, were described as hypersensitive to Cr(VI)-induced interstrand cross-links formation (Vilcheck et al, 2002). Some of the Cr(VI)-induced clastogenic effects, such as sister chromatid exchanges, chromosomal aberrations and genetic deletions, may be explained by increased levels of single-strand breaks (Levis and Majone, 1979; Bianchi et al, 1980; Sugiyama et al, 1993; Thompson and West, 2000; Caldecott, 2003; Grlickova-Duzevik et al, 2006) and, indeed, the involvement of this type of lesion is supported by the observation that XRCC1, a major component of the DNA base excision repair (BER) system that also plays a major role in facilitating the repair of single-strand breaks in mammalian cells (Christie et al, 1984; Thompson and West, 2000; Caldecott, 2003), was reported to prevent Cr(VI)-induced chromosome structural

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Gene Therapy and Molecular Biology Vol 12, page 227 modifications in CHO cells (Grlickova-Duzevik et al, 2006). It was also described that DNA damage (Takada et al, 2003) and/or defects on a number of key genes and on NER repair system, a repair system that in conjunction with BER is mostly involved in single-strand breaks repair, will possibly play important roles on the generation of genomic instability (Xu et al, 1999; Yamaguchi-Iwai et al, 1999; Griffin et al, 2000; Brooks et al, 2008). Of all the various forms of DNA damage induced by Cr(VI) exposure, double-strand breaks are probably the most dangerous, as it is now believed that inappropriate repair of double strand-breaks by the nonhomologous endjoining repair system (NHEJ), the most important DNA repair system in higher eukaryotes (Karran, 2000; Lewis and Resnick, 2000) (Figure 6), and/or mutations in many of the factors involved in their detection and repair can lead to an increased predisposition to cancer (Jackson et al, 2002). Indeed, a causal link between the formation of double-strand breaks and the induction of mutations and chromosomal translocations with tumorigenic potential is now supported by experimental data (Jackson et al, 2002). Actually, in response to a Cr(VI) insult MMR activation generates repair-generated gaps (double-strand breaks) that will dictate the cell fate. Accumulation of unrepaired double-strand breaks leads usually to apoptosis, while its abnormal processing by the NHEJ will generate chromosome rearrangements and thus genomic instability (Peterson-Roth et al, 2005; Reynolds et al, 2007; Reynolds and Zhitkovich, 2007). Cr(VI)-induced genomic instability can also result from prolonged arrest at either S or G2

phases of the cycle as a consequence of the long-lasting repair of double-strand breaks (Figure 5). At this point, it is important to stress that doublestrand breaks are not only strong inducers of mutations and major threats to genomic integrity, they are also potent inducers of cell death (Rich et al, 2000). This is in line with the finding of Peterson-Roth and collaborators (Peterson-Roth et al, 2005) of a much higher Cr(VI) cytotoxicity in MMR-proficient cells than in their MMRdeficient counterparts, due to a lower apoptotic stimulus in response to Cr-DNA damage of the latter. Considering that the levels of double-strand breaks are also much lower in MMR-deficient cells, it seems likely that this type of DNA lesion is strongly involved in Cr(VI)-induced cytotoxicity. One important consequence of this tolerance to Cr(VI) by MMR-deficient cells is that chronic exposure to toxic doses of Cr(VI) may result in the selective outgrowth of these cells (Peterson-Roth et al, 2005; Zhitkovich et al, 2005), which is actually observed in Cr(VI)-induced lung cancers (Hirose et al, 2002; Takahashi et al, 2005). As MMR-deficient cells exhibit very high rates of spontaneous mutagenesis, this model could also explain the high incidence of microsatellite instability in these cancers (Kondo et al, 1997; Hirose et al, 2002; Takahashi et al, 2005). Although the NHEJ repair system is crucial for DNA double-strand breaks repair (Karran, 2000; Lewis and Resnick, 2000), it was recently reported that this pathway was not involved on particulate Cr(VI)-induced chromosome instability (Camrye et al, 2007).

Figure 5. Schematic representation of the cellular responses to Cr(VI)-induced DNA damage. The occurrence of double- and singlestrand breaks may be repaired by the activation of cell cycle checkpoints and DNA damage repair systems, whereas cells with extensive damage may be eliminated by the induction of cell death. Impairment of these systems can lead to genomic instabilities including chromosomal aberrations.

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Figure 6. Schematic representation of the main NHEJ repair system components and mechanism.

Nevertheless, it has been claimed that Cr(VI)-induced premutagenic adducts, such as Asc-Cr(III)-DNA, can promote a wide range of chromosomal abnormalities as a result of the error-prone repair of double-strand breaks through the activation of this repair system (Karran, 2000; Lewis and Resnick, 2000; Salnikow and Zhitkovich, 2008). A possible explanation for the conflicting results relies on the fact that the results of Camrye and collaborators (Camrye et al, 2007) were obtained using CHO and CHO-derived cell lines and a non-physiological Asc concentration which, as already mentioned, is a mandatory condition to favor oxidative damage and single-strand breaks formation rather than double-strand breaks formation (Reynolds et al, 2007; Reynolds and Zhitkovich, 2007; Salnikow and Zhitkovich, 2008). A call of attention for the important role played by double-strand breaks on particulate Cr(VI)-induced chromosome instability was the fact that deficiency in the DNA HR repair proteins, specifically the RAD51 paralogs XRCC3 and RAD51C, increased Cr(VI)-induced chromosomal damage and caused a dramatic shift in the spectrum of chromosomal aberrations (Stackpole et al, 2007) (Figure 5). As already mentioned, Cr(VI)-induced DNA double-strand breaks are preferentially formed after the S-phase of the cell cycle, precisely when HR repair is particularly active (Van den Bosch et al, 2002; Ferreira and Cooper, 2004; Ha et al, 2004). The kinetochorenegative micronuclei detected in human lung epithelial cells following Cr(VI) exposure is one of the many

Cr(VI)-induced clastogenic effects that can be explained through double-strand break formation (Reynolds et al, 2007). The role of the BER and HR DNA repair systems in dealing with Cr(VI)-associated lesions is consistent with recent data enlightening the relationship between functional polymorphisms in XRCC1 and XRCC3 and increased levels of chromosome damage and risk of lung cancer in workers exposed to hard-metal dusts (Lei et al, 2002; Mateuca et al, 2005). In this context, it is important to mention that many lines of evidence revealed that knowledge of the mechanisms by which specific DNA single- and double-strand breaks are generated and of the DNA repair pathways involved can provide important indications as to the genomic instability outcome. Which DNA repair system(s) is activated depends on whether double-strand breaks are generated in the lagging or in the leading strand, is a consequence of replication fork collapse or the reversal. The understanding of how cells decide which DNA repair system(s) they will use to repair double-strands breaks will strongly contribute to the knowledge of the different mechanisms and factors governing genomic instability (Aguilera and G贸mezGonz谩lez, 2008).

B. Effects on cytokinesis Even though it is commonly accepted that Cr(VI) is a respiratory tract carcinogen when particulate forms are inhaled, there is a large body of information on the

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Gene Therapy and Molecular Biology Vol 12, page 229 clastogenic effects of both water-soluble and insoluble Cr(VI) compounds (Sen and Costa 1986; Wise et al, 1994, 2002, 2006; Seoane et al, 2002; O’Brien et al, 2003; Glaviano et al, 2005; Holmes et al, 2006;). However, the exposure consequences are different, since particulate Cr(VI) was reported to induce an increase in both the number of metaphases with too few chromosomes (hypodiploidy) and the number of metaphases with twice the number of chromosomes (tetraploidy) in human lung fibroblasts (Holmes et al, 2006), whereas soluble Cr(VI) [sodium dichromate] induced only hypodiploidy (Seoane et al, 2002). These different clastogenic outcomes appear to be dependent not only on the solubility characteristics of the Cr(VI) compound but also on the cell line and exposure regimens used. In the case of particulate Cr(VI), exposures to very low and low to moderate concentrations lead, in human lung fibroblasts, to a long-term increase in tetraploid cells, while short-term exposures to low and moderate concentrations induced an increase in hypodiploid cells (Holmes et al, 2006; Xie et al, 2007). In addition, short-term exposures to very low concentrations did not appear to have any effect on centrosome amplification. In contrast, low to moderate doses induced, both in human lung fibroblasts and human bronchial epithelial cells, centrosome amplification (Holmes et al, 2006; Xie et al, 2007). These findings lead to the hypothesis that, in the aforementioned cell lines, centrosome amplification was inducing hypodiploidy, whereas tetraploidy was induced by a different mechanism possibly by effects on the spindle assembly checkpoint (Holmes et al, 2006). It is important to note that hypodiploidy was reported to occur before tetraploidy in human lung cells exposed to low to moderate particulate Cr(VI) concentrations (Holmes et al, 2006). In contrast, in cells exposed to soluble Cr(VI), tetraploidy was the first outcome of centrosome amplification (Güerci et al, 2000; Seoane et al, 2002). Thus, there are apparently different mechanisms for Cr(VI)-induced hypodiploidy and tetraploidy (Holmes et al, 2006) which cannot be only ascribed to the solubility and the exposure regimen (concentration and exposure time) to the Cr(VI) compound. The cell line used appears to be the key feature. In fact, the same exposure regime to particulate Cr(VI) induced only hypodiploidy in hTERTimmortalised human lung epithelial cells, while in human lung fibroblasts tetraploid cells exceeded by far the number of hypodiploid cells (Holmes et al, 2006). It is possible that these dissimilar results may be explained by Cr(VI) effects on hTERT expression, since recent studies revealed that although hTERT protected cells against many features of Cr(VI)-induced genomic instability, it allowed Cr(VI) to induce tetraploidy rather than aneuploidy (Glaviano et al, 2005). Aberrant mitotic figures (lagging metaphase, cmetaphase and ball metaphase, as well as lagging and disorganized anaphase and mitotic catastrophe) (Holmes et al, 2006) may explain Cr(VI) effects on chromosome instability, particularly the tetraploid phenotype, one of the hallmarks of lung cancer (Masuda and Takahashi, 2002). Both soluble and particulate Cr(VI) compounds were also reported to induce structural chromosome

modifications (chromatid lesions, isochromatid lesions, dicentric chromosomes and centromere spreading) in both human lung epithelial cells and human lung fibroblasts (Manning et al, 1994; Wise et al, 2002, 2003, 2004b, 2006; Xie et al, 2004; Xie et al, 2007). All these findings are in line with the epidemiological evidence suggesting that chromosomal abnormalities and genomic instability may be involved in the induction of human lung cancer by Cr(VI) (Kondo et al, 1997; Hirose et al, 2002; Takahashi et al, 2005). Therefore, determining how Cr(VI) causes genomic instability will be a significant step forward in the prevention of Cr(VI)-induced cancers and events that are important to lung cancer progression in general and, ultimately, in the design of new treatment approaches.

C. Genomic instability: the success of proliferating pathways and the failure of apoptotic pathways Although undoubtedly relevant, the establishment of a whole spectrum of Cr(VI)-induced DNA lesions cannot, by itself, explain Cr(VI)-induced toxicity and carcinogenicity and a lot more effort will have to be put into the elucidation of the signalling pathways mediating the cellular responses to Cr(VI) exposure. In fact, it must be acknowledge that, in terms of signalling pathways, many of the results obtained to this day are probably seriously compromised by the use of inadequate systems and/or of exposure regimens that are not representative of any toxicologically relevant human exposures. In particular, which signalling pathways are activated and their role on genomic instability and consequently in lung cancer onset and progression needs to be established. In vitro studies revealed that extensive Cr-DNA interactions play a critical role in DNA polymerase arrest (O’Brien et al, 2002) and on the processivity of both prokaryotic and mammalian DNA and RNA polymerases (Bridgewater et al, 1994a,b, 1998; Xu et al, 1996; O’Brien et al, 2001, 2002), leading generally to apoptosis. Instead, low levels of DNA damage can induce the activation and/or the inactivation of signalling pathways which may contribute to Cr(VI)-induced genomic instability because they allow cells with unrepaired DNA damage to progress through the cell cycle. One such a signalling pathway is mediated by the ataxia telangiectasia mutated kinase (ATM), a serine/threonine kinase that belongs to a family of large proteins that contain the phosphatidylinositol 3-kinaserelated domain (Beamish et al, 1996). This protein is a key element in multiple biochemical pathways linking, through phosphorylation of various substrates, the recognition and repair of chromatin structure lesions (ICLs and doublestrand breaks) to downstream cellular processes, such as activation of cell cycle checkpoints, DNA repair, apoptosis and also cell proliferation (Kastan and Lim, 2000) (Figure 7). As revealed by in vitro studies, in normal human dermal fibroblasts and human bronchial cells, Cr(VI)induced double-strand breaks formation activates ATM which was shown to be a major signal initiator for Cr(VI)induced apoptosis that also contributes to cell survival by facilitating recovery/escape from terminal growth arrest 229

Urbano et al: Hexavalent Chromium Exposure, Genomic Instability and Lung Cancer (Ha et al, 2003; Xie et al, 2005) (Figure 7). Moreover, in normal dermal fibroblasts, the physiological targets of ATM, i.e. p53 Ser15 and Chk2 Thr68, were also activated by phosphorylation following Cr(VI) exposure (Ha et al, 2003) (Figure 7). However, the signalling pathways mediated through ATM in response to Cr(VI) insult were shown to depend on the intensity of the insult. Thus, low Cr(VI) concentrations induced the phosphorylation of "H2AX (Ha et al, 2004; Xie et al, 2005; Wakeman and Xu, 2006) and of 53BP1, the ATM substrates reported to lead to foci formation over large chromatin domains surrounding the break (Rogaku et al, 1999; Celeste et al, 2002). Instead, high Cr(VI) concentrations were reported to induce ATM-Rad3-related (ATR) activation (Ha et al, 2004; Xie et al, 2005; Wakeman and Xu, 2006) (Figure 7). The chromosome breaking and centromere-negative micronuclei observed, in lung epithelial cells, following Cr(VI)-induced DNA double-strand breaks formation and subsequent phosphorylation of "-H2AX and of 53BP1 (Ha

et al, 2003; Reynolds et al, 2007) confirms the involvement of ATM-dependent signalling pathways in Cr(VI)-induced in vitro events (Figure 7). Additionally, the finding that, in lung epithelial cells exposed to Cr(VI), there is a direct connection between the MMR proteins (MSH2 or MLH1), "-H2AX phosphorylation and micronuclei formation (Reynolds et al, 2007) may shed some light on the mechanisms through which MMR deficiency promotes expansion of cells with pre-malignant lesions and genomic instability. As to the ATM substrate 53BP1, it is known to be required for p53 accumulation, G2-M checkpoint arrest and the intra-S-phase checkpoint (Wang et al, 2002). Therefore, it is not surprising that several reports state that p53 is involved in apoptotic cell death following widespread DNA damage caused by Cr-DNA adducts formation (Cupo and Wetterhahn, 1985b; Ye et al, 1999; Charlisle et al, 2000a,b; Chuang et al, 2000; Costa et al, 2002; Bagchi et al, 2001; Wang and Shi, 2001).

Figure 7. Schematic representation of the ATM-53BP1-dependent signalling pathway. The pathway is triggered by DNA double-strand breaks following genotoxic events such as Cr(VI). Phosphorylation of histone "-H2AX by ATM occurs at or near the DNA doublestranded break site and is required for phosphorylation of 53BP1 by ATM and localization of 53BP1 into nuclear foci. In turn, 53BP1 function is important for coupling ATM to several of its downstream targets, including p53. In the case of the Chk2 kinase, the coupling mechanism to ATM seems to be largely independent of 53BP1 and may involve the BRCT family of proteins. Adapted from Wang et al, 2002.

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Gene Therapy and Molecular Biology Vol 12, page 231 However, some authors reported that, in lung epithelial cells, both p53-dependent and p53-independent pathways were simultaneously involved (Ye et al, 1999; Wang and Shi, 2001). Curiously, a very recent and more detailed study revealed that, in normal lung epithelial cells exposed to a occupational relevant Cr(VI) concentration, elevated levels of p53 and increased p53 Ser-15 phosphorylation did not result in its transactivation (Reynolds and Zhitkovich, 2007). This result suggests that the products of Cr(VI) metabolism disrupt the interactions of transcription factors with transcriptional co-regulators, silencing the apoptotic p53-dependent pathway. This interpretation is supported by studies demonstrating that expression of proapoptotic gene targets requires the participation of other tightly regulated proteins, as well as the presence of additional posttranslational modifications at Lys residues of p53 (Flores et al, 2002; Knights et al, 2003, 2006; Feng et al, 2005; Flores et al, 2005). These findings lead the group of Zhitkovich to propose that the loss of apoptotic response to Cr(VI)-induced damage is more likely due to inactivation of the MMR repair system than to p53 inactivation (Peterson-Roth et al, 2005; Reynolds et al, 2007; Reynolds and Zhitkovich, 2007). Overall, the in vitro findings that Cr(VI) toxicity is MMR dependent but p53-independent may explain why chromate cancers exhibit high frequency of MMR deficiency and low incidence of p53 mutations (Kondo et al, 1997; Hirose et al, 2002; Takahashi et al, 2005). The disruption of transcriptional activator-coactivator complexes (Hamilton and Wetterhahn, 1989; Alcedo et al, 1994; Manning et al, 1994; Shumilla et al, 1999) may account for the failure of Cr(VI) to block the expression of metal-inducible genes without affecting the expression of housekeeping genes (Hamilton and Wetterhahn, 1989; Wetterhahn and Hamilton, 1989; Wetterhahn et al, 1989; Alcedo et al, 1994; McCaffrey et al, 1994). These observations have led some authors to hypothesize that the chromatin structure of inducible promoters, perhaps by virtue of being more open, may offer a better target for the formation of Cr-DNA adducts and Cr-DNA cross-links than the more closed chromatin of constitutive promoters (Manning et al, 1992). In fact, as already mentioned, Cr(VI)-induced DPCs were reported preferentially in nuclear matrix DNA (Xu et al, 1994; Manning et al, 1994), where many replication, repair and transcription proteins associate, suggesting that formation of cross-links between DNA and these proteins may be responsible for effectively blocking their function. Thus, the selective expansion of pre-malignant cells due to inactivation of pro-apoptotic pathways may result from Cr(VI) persistent repression of regulatory pathways that affect the function of transcriptional co-regulators. In fact, it has been documented that the reductive metabolism of Cr(VI) to Cr(V) and Cr(IV) induces an increase of the binding activity of NF#B to DNA, but did not cause a concomitant increase in the expression of NF#Bdependent genes (Ye et al, 1995; Chen et al, 1997; Kaltreider et al, 1999). A possible explanation is that intermediate oxidation states of chromium block the binding of the NF#B p65 subunit to CBP/p300, a transcriptional co-activator with intrinsic histone

acetyltransferase activity, whose association with p65 was described as essential for NF#B-enhanced transcriptional activity (Shumilla et al, 1999). Also, the inhibitory effects of Cr(VI) on the expression of Cyp1a1 and several other genes induced by benzo[!]pyrene (B[!]P) were found to depend on the presence of promoter-proximal sequences and not on the cis-acting enhancer sequences that bind the aryl hydrocarbon receptor-aryl hydrocarbon receptor nuclear translocator (Wei et al, 2004). The authors demonstrated that exposure to Cr(VI) prevented the B[$]P-dependent release of histone deacetylase (HDAC-1) from Cyp1a1 chromatin, maintaining a state of histone deacetylation and transcriptional repression and preventing the recruitment of p300. Thus, the increased mutagenicity, due to the slow repair of B[$]P adducts-DNA adducts, was recently proposed to result from a decrease in the NER system activity (Salnikow and Zhitkovich, 2008). Interestingly, in vivo evidence revealed that Cr(VI) appeared to cause a selective increase in the number of B[!]P-DNA adducts at the mutational â&#x20AC;&#x153;hotspotsâ&#x20AC;? of p53 in smoking-induced lung cancer: codons 248, 273 and 282 (Feng et al, 2003). Cr(VI) has also been found to modify the transactivation potential of MTF-1 without affecting basal or inducible binding to metal-response elements (Majumder et al, 2003). Consequently, it appears that the molecular mechanism underlying Cr(VI) inhibition of inducible but not constitutive gene expression is likely to involve interactions of transcription factors with transcriptional co-regulators and chromatin remodelling factors, more so than the binding of the factors themselves to their cognate recognition sites. Therefore, the reaction of the products of the reductive metabolism of Cr(VI) with other biomolecules besides DNA may also contribute to, or occur in addition to, genotoxic damage (Banks and Cooke, 1986; Sugiyama et al, 1986; Mikalsen et al, 1989; Shi and Dalal, 1989; Ryberg and Alexander, 1990; Salnikow et al, 1992; Shi et al, 1992, 1994), and to the activation of signalling pathways which may inhibit apoptosis. Thus, the loss of apoptotic signalling in response to Cr(VI) insults promotes accumulation of damaged cells and, thereafter, genomic instability.

VII. Conclusions Despite the remarkable work of many researchers and all the data and knowledge acquired using simple models the understanding of the metabolism of Cr in complex biological systems remains incomplete. The acquisition of additional basic knowledge using more appropriate model systems and exposure regimens that mimic, as much as possible, occupational human exposures will help to ascertain new facets of Cr(VI) toxicity and, most importantly, the potential role that each event plays in the induction of human respiratory cancer by Cr(VI) compounds exposure.

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Gene Therapy and Molecular Biology Vol 12, page 239 Gene Ther Mol Biol Vol 12, 239-246, 2008

Screening of coding region of metastasis suppressor genes KISS1 and KAI-1 for germ line mutations in breast cancer patients Research Article

Fraz Arshad Malik1,2,*, Mahmood Akhter Kayani1, Hina Iqbal1, Wen G Jiang2, Rafshan Sadiq3 1

Cancer Genetics Lab, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan University Department of Surgery, Cardiff University School of Medicine, Cardiff, Wales, United Kingdom 3 Punjab Institute of Nuclear Medicine, PINUM, Faisalabad, Pakistan 2

__________________________________________________________________________________ *Correspondence: Dr Fraz Malik, Department of Surgery, Cardiff University School of Medicine, Cardiff, Wales, CF14 4XN, UK; Tel: 44 29 2074 2895; Fax 44 29 2074 2896; e-mail: Fraz_cemb@yahoo.com Key words: sporadic breast cancer, KISS1, KAI1, Single strand Conformational polymorphism, SSCP Abbreviations: Combined Military Hospital of Rawalpindi, (CMH); Metastasis suppressor genes, (MSGs); Nuclear Medicine Oncology and Radiotherapy Institute, (NORI); Punjab Institute of Nuclear Medicine, (PINUM); Single Strand Conformational Polymorphism, (SSCP) Received: 15 July 2008; Revised: 21 August 2008 Accepted: 5 September 2008; electronically published: October 2008

Summary Breast cancer is one of the most common female cancers worldwide. Abnormalities of genetic or epigenetic factors are mainly responsible for the development and progression of mammary tumours. In patients with breast cancer, metastasis is the leading cause of death. In recent years, a group of genes has been identified as metastasis suppressor genes (MSGs), which are involved in the suppression of the growth of secondary tumours. Down regulations of MSG expression have been frequently observed in advanced tumours. The present study was designed to screen two of the most frequently down-regulated MSGs (KAI1 and KISS1) for germ line mutations in sporadic breast cancer cases of the Pakistani population. 170 cases of unilateral breast cancer patients, who had no prior history of breast cancer and no other disease in general in their families with age ranging from 35-75yrs, were included in this study. Mutational analysis for the entire coding region of KAI1 and KISS1 (including 10 exons and 3 exons, respectively) was carried out by using the Single Strand Conformational Polymorphism (SSCP) technique. No germ line mutation was observed on the entire coding region in the samples from patients with breast cancer in the Pakistani population. Splice site variants on these genes were also absent in breast cancer patients. Involvement of germ line mutations for these MSGs is thus considered to be an event that occurs less frequently in breast cancer patients of Pakistani population. Conserved coding regions of both MSGs indirectly enlighten the involvement of transacting factor on DNA sequence as major contributor in the progression and aggression of tumours rather than any high risk associated mutation itself. A detailed analysis of regulatory mechanism is required to explore the genetic basis of down regulation of these MSGs for a better understanding of breast cancer progression.

altered expression of several genes. In recent years a group of genes has been identified and characterized as suppressors of metastasis; termed as metastasis suppressor genes (MSGs) (Steeg et al, 1998). These genes suppress metastasis at certain steps of metastatic cascade without affecting the primary tumour growth. In several types of cancer, frequent down regulation of proteins encoded by these genes have been found in conjunction with clinical progression (Stafford et al, 2008). The two most promising genes in this family of MSGs: KAI1 (Ichikawa et al, 1992)

I. Introduction Metastasis is the leading cause of cancer related death in patients with solid tumours. Metastasis is not a random process and its progression requires contribution of genetic and epigenetic factors in cancer. Metastasis is usually characterized by a chain of events that begins with the dislodging of cancer cells from the primary site, crossing the basement membrane, surviving in circulation and finally its adherence to the secondary site for proliferation. The whole cascade of events is controlled by 239

Malik et al: Lack of KiSS-1 and KAI-1 mutation in breast cancer and KISS1 (Lee et al, 1996) are found to have a tumour suppressor role for different types of cancer. Expressional regulation of these genes in relation to various clinical parameters during cancer progression have been extensively investigated in prostate (Jackson et al, 2003), breast (Stark et al, 2005, Huang et al, 2005), lung (Wang et al, 2005), ovarian (Houle et al, 2002), ocular (Martin et al, 2008), gastric (Tsutsumi et al, 2005; Guan-Zhen et al, 2007), pancreatic (Sho et al, 1998), oesophageal (Ikeguchi M et al, 2004; Farhadieh et al, 2004), bladder (Sanchez et al, 2003; Jackson e al, 2007) and cervical (Schindi et al, 2000) cancers. The KAI1 gene, also termed as prostate metastasis marker, encodes a glycoprotein with 267 amino acids. KAI1 protein has a transmembrane location (White et al, 1998). KISS1 protein has 138 amino acids, with a cytoplasmic location (Kevin et al, 2006). The mechanism by which KAI1 exerts metastasis suppression has shown to be induced by their interaction with different molecules, integrins and epidermal growth factor receptor EGFR, for example (Odintsova et al, 2000). Forced expression of these proteins results in decreased invasion and adhesion in breast cancer cell lines. 4-10 % of breast cancer cases are due to germ line mutations. So far KISS1 and KAI1 have not been screened for germ line mutations in mammary tumours. The only available report for KAI-1 mutation is a study on oesophageal squamous cell carcinoma, in which node metastasis was found to be correlated with KAI1 expression along with mutational screening of KAI1 (Miyazaki et al, 2000). An altered splice variant of KAI1 has also been identified and is known as KITENIN. The carboxy terminal portion of this variant is unable to induce suppression of tumour invasion due to lack of exon 7 coding amino acids (Lee et al, 2004). This suggests the importance of screening KAI1 for any germ line mutations to distinguish whether there is germ line involvement for regulation of this protein. KISS1, termed as metastin or kisspeptin (KP54), is known to have spliced variations and play a role in cancer progression (Rouille et al, 1995). However, germ line variants of the coding region of KISS1 has not been explored in relation to breast cancer. The present study was designed with an intention to screen the above mentioned genes for germ line mutations. We have recently reported that KISS-1 expression in human breast cancer is aberrant. In the study breast tumours were found to have high levels KISS-1 transcript (but not KiSS-1 receptor), compared with normal mammary tissues. Furthermore, our study demonstrated that highly expressed KISS-1 transcript was linked to nodal metastasis and long term survival (Martin et al, 2005). The study has further shown that over-expression of KISS-1 in breast cancer cell lines rendered the cells more motile and invasive. In a recent study, we have shown that the pattern of KAI-1 expression in human breast cancer cells is inversely linked to the invasiveness of the cells, in that over-expression of KAI-1 reduced the in vitro invasion and knocking down of KAI-1 in breast cancer cells resulted in cells becoming more invasive (Malik et al, 2008). The aim of this study was to explore these MSGs genome for any deletion, insertion or frame shift mutations

in breast cancer patients. The prime objective of this study was to assess the presence of any germ line mutations on these genes and also to estimate the penetrance of those mutated alleles, if any, in the breast cancer patients. Allelic variations were assessed to estimate whether there was any correlation to breast cancer patients. Splice sites for each exon of the respective genes were screened for any complete or partial loss of genomic portion.

II. Materials and Methods All reagents used in the following experiment were purchased from Sigma Chemical Ltd. (Dorset, UK) unless stated otherwise. Primers were synthesized from Invitrogen (Paisley, UK) and BigDye®Terminator used for sequencing was purchased was purchased from ABI (Applied Biosystems, USA). 170 patients at different stages of breast cancer were included in this study. Blood samples were collected with prior approval from the Ethical Committees of the participating oncology institutes and hospitals of Pakistan. Oncologists at the Punjab Institute of Nuclear Medicine (PINUM), Allied Hospital of Faisalabad, Nuclear Medicine Oncology and Radiotherapy Institute (NORI) and Combined Military Hospital of Rawalpindi (CMH) helped in identifying and communicating with the respective patients. Blood samples of 200 healthy normal females of similar age group were also included in this study as controls. Samples from these normal women were used as a positive control and also to observe any polymorphism on the respective genes. Blood sampling was done after stringent initial screening (no family history of breast cancer, age of onset of breast cancer, no other family prevailing disorders, no earlier sampling from any other group for any study). The clinical and histological details of the tumours are given in Table 1. Incidence of breast cancer within the male population was not found to be very frequent and adequate enough to justify the penetration, so only female patients were selected for this study.

A. Sample collection and storage Blood samples from each case were collected in blood vaccutainer with EDTA as an anticoagulant. For storage, transportation and preservation, recommended guidelines were followed (Anderson et al, 1998). 200 blood samples from normal females exonerated from any disorder were also collected from respective ethnic groups of Punjabi, Pathan, Sindhi and Balochi so that a mutation or polymorphism of the respective genes could be differentiated.

B. DNA isolation and dilution Genome isolation was carried out following the recommended protocol (Köchl et al, 2005) with minor modifications of ethanol precipitation. DNA isolated was first confirmed by agarose gel electrophoreses, than quantified by using a spectrophotometer for the polymerase chain reaction. DNA samples were stored at -20C°, while dilutions were kept at 4C° for further usage.

C. Amplification and Mutation Screening Sequences for the coding region of KAI1 and KISS1 available on NCBI with gene IDs as 3732 and 3814, respectively, were included in this study. Primers were designed using the Primer-3 software and intron/ exon junctions were also included in this study for a better identification of splice variants. Primer sequences for respective genes KISS1 (3 exons) and KAI1 (10 exons) involved in this study are given in the Table 2 and Table 3 respectively. After optimization, amplification conditions set for each exon were as 95C° for 4min; 95C°for 30s, 55C° for 30s, 72C° for 1min for 30cycles with a final extension of 72C° for 45

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Gene Therapy and Molecular Biology Vol 12, page 241 min. Amplified products were then run on 2% agarose gels to confirm chances of non-specificity and the yield of amplified products. For mutation detection, SSCP technique (Lallas et al, 1997) with few modifications was used and samples were screened for any mobility shift in their banding pattern. This change in mobility shift, either predicting any frame shift

alterations or base substitution in the specified region, was confirmed by running normal controls along with the samples from the patients. To check and confirm findings, DNA sequencing of the suspected samples was carried out using BigDye速Terminator Reaction kit (ABI, USA). Bio Edit software was use to compare normal and suspected samples.

Table 1. Clinical stages and age group of breast cancer patients Type of Breast Cancer IDC

ILC

Stages of cancer (number of samples) Stage 1 (35) Stage 2 (82) Stage 3 (12) Stage 4 (8) Stage1 (12) Stage 2 (15) Stage 3 (6)

Age ranges among patients 23 ! to " 45

26 ! to" 53

IDC: Invasive Ductal carcinoma, ILC: Invasive Lobular Carcinoma

Table 2. Primer sequences for KISS1 genomic sequence EXONS EXON1 KISS1

EXON2 EXON3

F R F R F R

PRIMER Sequences(5'-to-3') TCTTCAGGAGGGTCTGAGGAG GGCTGGTAAACAGGAAAGATCA CTCTACCAGGAGCCTCCAAAG TAGATTTCCACCAAATGCAATG AGGTCAAGGAAGGAAAAGAAGG GCACTGACCTTAATGACACCAG

Product sizes 371 395 675

Table 3. Primer Sequences for KAI1 genomic sequence EXONS EXON1 EXON2 EXON3 EXON4 KAI1

EXON5 EXON6 EXON7 EXON8 EXON9 EXON 10

F R F R F R F R F R F R F R F R F R F R

PRIMER Sequences(5'-to- 3')

Product sizes

GTTGGGGTACGGCCATAGTG CCTGTCACTAGTTCCGAGGAAAG GGGAGCCTGGATTTAAAGTGA AATGCTGTAGGAGCCAGAGAAG ACAGGGTTAGTACCCACCTCCT CTCAGTCCCTACCCACCAAAT GACTTGGGTTCCAGGGACAG AAAAGCAACAGGCATTGAAG CAATCCTGAGAAGCCTTACGAA ATGCTCTCCACCCGATGTTAC CTGCCCATTTCCTCTCATCC CCCCATTTGATCACAAGGAATA GGGGGAGGCAGTTTAAGTAGG CAGATGCCAGTCCCAGACAT GTGGCATATCTCAGTCTCTGTCC GCTCTGCCTGTTCCTTGAATAA CAGCAATTCCTTCCTGCATTTA CACCTCCCCAAGGAGATCAG TCAAGTTGAGGATCCACTTAATC GCCTTATCTAACGCCCTTCC

458

241

381 458 261 492 381 278 478 373 875

Malik et al: Lack of KiSS-1 and KAI-1 mutation in breast cancer Germ line mutations of both KAI1 and KISS1 are less frequent in sporadic breast cancer patients. Although genetic instability is a well established fact in breast cancer cases, the coding regions of KAI1 and KISS1 genes probably contribute little to the genetic instability, as no partial or complete loss of any coding region has been observed. Coding regions of these genes are conserved among the patients with breast cancer and the controls. Splice site variations of KAI1 and KISS1 are less common in sporadic breast cancer patients of Pakistani population. Almost complete absence of any germ line mutations for these MSGs indirectly indicates the involvement of some other regulatory mechanisms responsible for downregulation of the genes. Thus, metastasis suppression aberration can be attributed more towards these regulatory factors rather than the impaired coding genome. Contribution of germ line mutation of these MSGs is less likely responsible for their down-regulation in cancer.

III. Results After extensive screening for the whole coding region of both KISS1 and KAI1, we were unable to find any mobility shift as mentioned in Figures 1 and 2 for KAI1 and KISS1 genes respectively. Blood samples from normal females with no previous history of any type of cancer were included as positive controls. Suspected samples after sequencing were also found to be negative for frame shift deletions or insertions. Splice sites for coding sequences of both genes were screened by using the same set of primers employed for amplifying the respective exons as they also included an intronic portion. Primer binding sites on KISS1 and KAI1 genes were shown in Figure 3 and 4 respectively. No partial or complete loss of any exonic portion for both genes had been observed in the respective breast cancer samples. No splice site variations were observed either on KISS1 or KAI1 genomic sequence with respect to the control group. The finding that there was almost no germ line mutation in these genes in sporadic breast cancer patients highlights the following features:

IV. Discussion Breast cancer is a polygenic disease, in which coordinated aberrations in expression of several genes are responsible for the disease spread. Genomic instability is a hallmark feature of breast cancer progression and is induced by a number of factors out of which germ line or

Figure 2. SSCP Gel Stained with Ethidium Bromide for KISS1. All exons of KISS1 show banding pattern among normal and mammary tumour patients on 8% non-denaturing polyacrylamide gel.

Figure 1. SSCP Gel Stained with ethidium bromide for KAI1. All exons of KAI1 show normal banding pattern among normal and mammary tumour patients on 8% non-denaturing polyacrylamide gel.

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Gene Therapy and Molecular Biology Vol 12, page 243 AGCCTGTATCAAAGTCACCAAATACTTTCTCTTCCTCTTCAACTTGATCTTCTTTg taagtatgtcccatgccgtcctgaccacctccgaaaagattctccttccaggggca tcccagcctgagcctttccagagccgaccgggccggggattggggggatttggtgg gtagggactgaggacgaactatctccaacgga

Primers binding sites for KISS1 Genomic Sequence Exon1: tcccgcctcggagggctcgggtggggcaggaggagaggctgcctccagtcacagag cccggaggcagaggcctcccaaggtgccatgcTCTTCAGGAGGGTCTGAGGAGgag ggaggggcggcagagtgggcctgtgcttggagacgtcaccccgggctttataaaag ggatgtgatcagGGAGCTGGGGAGAACTCTTGAGACCGGGAGCCCAGCTGCCCACC CTCTGGACATTCACCCAGCCAGGTGGTCTCGTCACCTCAGAGGCTCCGCCAGACTC CTGCCCAGGCCAGGACTGAGGCAAGgtaggcacactgcagtgtccacccctgggaa gggggtctgccctgacctggggatgctctggaggggagcagggaatccgttctaag acggacagatgcatccaggcaggcaaaggcaggggagggagacactgatctttcct gtttaccagcctggccagattatcctgggatg

Exon4: gaagggtggatgtggtgccctctgtggccccctgccctgcccaccctGACTTGGGT TCCAGGGACAGctggcagggggagggctatctgctcttggctccccattaactgct cccttctccttccagATCCTGGGCGCAGTGATCCTGGGCTTCGGGGTGTGGATCCT GGCCGACAAGAGCAGTTTCATCTCTGTCCTGCgtaaggacccctcagcttccccag acccaggcccactgaagaggggagggccagggcgcagatacagctgctcccataag tgcttcggcttcaatgcctgttgcttttcccgctgacctcagggatgtggcgaggt cctgctgcccaccactgcccatgg

Exon2:

Exon5:

tccccagtcactcctatatatggcatctcaccccacctttctcaaacattcctctc aagcaaccctgagattcagaatcctggccattctctgacccaagcagtcctgcccc ttccaccaaattgggtctttccttttttcctaactttactgtcctcacctcctcca ggaagccctccctgatgaccctcacctagcatgaattcagctttaactacactgtg taaagagtctcaatccCatttcactgatatatttgtttagactcatccCTCTACCA GGAGCCTCCAAAGgcaagaacttgcccctctcttggaggactgtcccttttgcact gggctctgccctcagcacccagcccagatcctgtgcctgacctagtctttgttccc tctctctgtctcagCCTCAAGGCACTTCTAGGACCTGCCTCTTCTCACCAAGATGA ACTCACTGGTTTCTTGGCAGCTACTGCTTTTCCTCTGTGCCACCCACTTTGGGGAG CCATTAGAAAAGGTGGCCTCTGTGGGGAATTCTAGACCCACAGgtatgtatcctct ggggaaaggagtgggagggagcaagtgggttgttgcaaaatgagctttcccgtatt ttccatctagtcgactggtgtgagtttaacattgcatttggtggaaatctaagact ggcacacatgcaactcattgagggggtcctgttaaccctgcaagtgacggatgcct tgcctctgtgatggcctcagtccgcccacctaggttccagggcttattaacc

agtgaaatgtagtctattCAATCCTGAGAAGCCTTACGAAgtaaaaaataaagccg ggaaggggcttaggctggatgagggtgtggattgcaattttactgggggagggaca caagtggggatgggggtgacctggttccctgttgggcagtgagaagccagcagggg aatgcagctgaccccaacaccctggctccaaagaggggatccggaagaagacctgg acggttaggccaggtgtttatacctgctgtgcccactgattttgtacttcttcttc cccctagAAACCTCCTCCAGCTCGCTTAGGATGGGGGCCTATGTCTTCATCGGCGT GGGGGCAGTCACTATGCTCATGGGCTTCCTGGGCTGCATCGGCGCCGTCAACGAGG TCCGCTGCCTGCTGGGGCTGgtgagtacggatccctccgcagctgcctgcccattt cctctcatccagccgagtgcagcctgaccgcggcgctggccgtaacatcgggtgga gagcatctctcggggcacgcaggctgggt

Exon6:

ggatccctccgcagctgcCTGCCCATTTCCTCTCATCCagccgagtgcagcctgac cgcggcgctggccgtaacatcgggtggagagcatctctcggggcacgcaggctggg tgcacctggtcggggaccctcagctgactttgtgcctgctctgtgtccccagTACT TTGCTTTCCTGCTCCTGATCCTCATTGCCCAGGTGACGGCCGGGGCCCTCTTCTAC TTCAACATGGGCAAGGtaagcccctctctccctccctcttcactgggctggaccaa ccatgggggtgattgactgagtgtgggggatggacaaggaacccccccagttgtca cagacagatccagtaggtgtcagggacggcctcctggacactattccttgtgatca aatgggggagcgttagaagagaaggcgg

Exon3: aagagggaggcgcctagggcctaatcatcccagctaaggtgatcgtgctggttcgc atggcactgagctccgagcttccaaaAGGTCAAGGAAGGAAAAGAAGGataggatg ggatgacaggaggtgttgcaaagccatctttcccggaccccggcctcatctttctg tgccctctgtcctagGCCAGCAGCTAGAATCCCTGGGCCTCCTGGCCCCCGGGGAG CAGAGCCTGCCGTGCACCGAGAGGAAGCCAGCTGCTACTGCCAGGCTGAGCCGTCG GGGGACCTCGCTGTCCCCGCCCCCCGAGAGCTCCGGGAGCCCCCAGCAGCCGGGCC TGTCCGCCCCCCACAGCCGCCAGATCCCCGCACCCCAGGGCGCGGTGCTGGTGCAG CGGGAGAAGGACCTGCCGAACTACAACTGGAACTCCTTCGGCCTGCGCTTCGGCAA GCGGGAGGCGGCACCAGGGAACCACGGCAGAAGCGCTGGGCGGGGCTGAGGGCGCA GGTGCGGGGCAGTGAACTTCAGACCCCAAAGGAGTCAGAGCATGCGGGGCGGGGGC GGGGGGCGGGGACGTAGGGCTAAGGGAGGGGGCGCTGGAGCTTCCAACCCGAGGCA ATAAAAGAAATGTTGCGTAACTCactgacgtgtggtctgcggctcatttttgcaag cagctctgatgaggctgttccccttccatcctcaatggatggttagctgatttcaa gaggcctctggtgtcattaaggtcagtgcttctcacactaggtgtaatgaaggaac aggcggttagttctccaatccattgcagattggtacttaaaaaaaaaaattaaaat

Exon7:

ggcaggagtggttggcactgcctgcatcgggcaccctgggagcgccaggaaagtgg ctggagccatgagcgtgtcccagggttgctgaggggaacgGGGGGAGGCAGTTTAA GTAGGggtgaccacaggtgggcacgggtttcaggaaatctgaccctgacctttgtc ctcccccctgcaGCTGAAGCAGGAGATGGGCGGCATCGTGACTGAGCTCATTCGAG ACTACAACAGCAGTCGCGAGGACAGCCTGCAGGATGCCTGGGACTACGTGCAGGCT CAGgtgaggtggggcggggctgcaggaggctctctggcctgggtgtccctgcattt ggggctctgtgcacccacatgtctgggactggcatctgcagtgctcgtgtgtgcct gacagtttgtaggagagtgtgcttctat

Exon8: ttcggctgggaccaggggcctggaaGTGGCATATCTCAGTCTCTGTCCtggggagg tcctccctctgccaggagggcagcctgcctagggtgagccgtgagcacaagcagtc tgtcccctgccttgcccagGTGAAGTGCTGCGGCTGGGTCAGCTTCTACAACTGGA CAGACAACGCTGAGCTCATGAATCGCCCTGAGGTCACCTACCCCTGTTCCTGCGAA GTCAAGGGGGAAGAGGACAACAGCCTTTCTGTGAGGAAGGGCTTCTGCGAGGCCCC CGGCAACAGGACCCAGAGTGGCAACCACCCTGAGGACTGGCCTGTGTACCAGGAGg tgtgcggggggctgcggatcgggggcggggctccgagggcgttgggggccatctgg gctactgctcagcaattccttcctgcatttagttccttcccttaattcatctgtca tttgtaccttcatctacttctttgttaatgtatttattcaaggaacaggcagagcc ctctgtaggggcttcc

Figure 3. Primer binding sites on KISS1 Genomic Sequence. Exon sequences are in upper case over orange background and introns in plain text. Forward primers are marked in bold underlined upper case over a grey background. Reverse primers are in bold underlined lower case over a grey background.

Exon9: cgagggcgttgggggccatctgggctactgctCAGCAATTCCTTCCTGCATTTAgt tccttcccttaattcatctgtcatttgtaccttcatctacttctttgttaatgtat ttattcaaggaacaggcagagccctctgtaggggcttccgggctgggactgggggg ctctcggtggttctgcatggcggggtgggatggtgcagagcggggtgatgtgaccg cattctgcccttgcagGGCTGCATGGAGAAGGTGCAGGCGTGGCTGCAGGAGAACC TGGGCATCATCCTCGGCGTGGGCGTGGGTGTGGCCATCATCGAGgtctgagccccc tcccccatcccttcccatcccaggtcctcctgggttgtctctgttctgctgatctc cttggggaggtggggcc

Primers binding sites for KAI1 Genomic Sequence Exon1:

Exon10: tggttgtgaggcTCAAGTTGAGGATCCACTTAATCcccatgtaaacctggatggtg aggctggggcgtctgaggccgggacacccagcctccctctgactctccgcctctcc ccacagCTCCTGGGGATGGTCCTGTCCATCTGCTTGTGCCGGCACGTCCATTCCGA AGACTACAGCAAGGTCCCCAAGTACTGAGGCAGCTGCTATCCCCATCTCCCTGCCT GGCCCCCAACCTCAGGGCTCCCAGGGGTCTCCCTGGCTCCCTCCTCCAGGCCTGCC TCCCACTTCACTGCGAAGACCCTCTTGCCCATCCTGACTGAAAGTAGGGGGCTTTC TGGGGCCTAGCGATCTCTCCTGGCCTATCCGCTGCCAGCCTTGAGCCCTGGCTGTT CTGTGGTTCCTCTGCTCACCGCCCATCAGGGTTCTCTTAGCAACTCAGAGAAAAAT GCTCCCCACAGCGTCCCTGGCGCAGGTGGGCTGGACTTCTACCTGCCCTCAAGGGT GTGTATATTGTATAGGGGCAACTGTATGAAAAATTGGGGAGGAGGGGGCCGGGCGC GGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACGA GGTCAGGAGATCGAGACCATCCTGGCTAACATGGTGAAACCCCGTCTCTACTAAAA ATACAAAAAAAATTTAGCCGGGCGCGGTGGCGGGCACCTGTAGTCCCAGCTACTTG GGAGGCTGAGGCAGGAGAATGGTGTGAACCCGGGAGCGGAGGTTGCAGTGAGCTGA GATCGTGCTACTGCACTCCAGCCTGGGGGACAGAAAGAGACTCCGTCTCaaaaaaa aaaaaaaaaaaaaaaaaaattggggagggaagggcgttagataaggcactctgggc tgtc

cgggggcgaggctgGTTGGGGTACGGCCATAGTGggcggggcctggccggcgggag cgcaccgccttcccaaagggctcgggggcggggccggcggagggggcgtgtcttct gggggcggGGCCTGCCGAGTCCGCGGCGTTCCCCGGCTGCAGCCGGGAGGGGGCCG AGGAGTGACTGAGCCCCGGGCTGTGCAGTCCGACGCCGACTGAGGCACGAGCGGGT GACGCTGGGCCTGCAGCGCGGAGCAGAAAGCAGAACCCGCAGgtgagcaagggggc agcgggccggggtagcctgccggacaaccataggcaaagttagttttagcccccgc ttctgcggtccggctcggacaactttcctcggaactagtgacaggtcggggcagct ggtgggtgaaaaggagctttagccaccagtcctgaacccccagttctcacactcgg ccgtgcacagctgcgtgatcctgggtccgtcc

Exon2: tctgagccaaggcttgtgggaaaatacgggaaaagagtaattctggcagagaaacc cttttgcaaaggccctcaggtaggtataaggacctggggcctgatataccaggaca acaggaaagaggctacatgagcctgtggggggaagaggacagaggaatggcaaccc tggcggggccccgccggccacacgGGGAGCCTGGATTTAAAGTGAgtatgtcttca gccacattgcctttggagagtcacagacaggagtgacgagatctgggtcgtgtttt tcagAGTCCTCCCTGCTGCTGTGTGGACGACACGTGGGCACAGGCAGAAGTGGGCC CTGTGACCAGCTGCACTGGTTTCGTGGAAGgtaagtcctgggctgagaggagtggt gggttggaggggacacacttgggctgtgaggcgaggaaggtggagaccgagcatgg ccctcaggtgttcagtctgagccaccaggtgggtggagggaccacttcttcccctt ctctggctcctacagcatttagagtcctggagtgct

Figure 4. Primer binding sites on KAI1 genomic sequence. Exon sequences are in upper case over orange background and introns in plain text. Forward primers are marked in bold underlined upper case over a grey background. Reverse primers are in bold underlined lower case over a grey background.

Exon3: agccccagccccagccccagccccagtgcagtggctgatccggatcctaactgcca agcatcaagtgcctggcacacaagactgctcgtgggacctcatttcctagctgtgt ggctttgggctagttgtctaacctctctgggcctctgcatcctctaaggcagggAC AGGGTTAGTACCCACCTCCTggggttgctgcaagggcagactgagctgatcccctc actggcctgcctgccttctctcttccagGAAGCTCCAGGACTGGCGGGATGGGCTC

243

Malik et al: Lack of KiSS-1 and KAI-1 mutation in breast cancer somatic mutations also impart their substantial contribution. Genome instability can be the result of either progressive genome damage within a tumour cell or accumulation of all sorts of mutation resulting in the formation of cells with a metastatic potential (Rubin et al, 2001). 4-10% of breast cancer cases are due to germ line mutations in various genes. The current study assessed the potential involvement of possible mutations of KAI1 and KISS1 in breast cancer. We did not observe any mutation in these genes but this does not overlook involvement of these MSGs in metastasis suppression. There are several techniques used for mutation detection including Single Strand Conformational Polymorphism (SSCP), Allele Specific Oligonucleotides, protein truncation test, Temperature Gradient Gel Electrophoresis (TGGE), Denaturing Gradient Gel Electrophoresis, Hetro duplex analysis and DHPLC (Gasser, 1997). These techniques generally rely on altered chemical or physical features of DNA due to variations in one or more nucleotides in the sequence. We used single strand confirmation polymorphism technique because of its simplicity, sensitivity, high throughput and low cost efficiency. The main principle of SSCP technique is largely based on the knowledge that the mobility of single strand DNA molecule depends on its size and structure in a non denaturing gel. During electrophoresis single strand DNA usually forms secondary and tertiary structures. Several factors may influence the mobility pattern of DNA strands including temperature, buffer pH during electrophoresis, percentage of polyacrylamide gel, specificity of the amplified product and sequences. These factors may influence results interpretation and have been taken into consideration which ultimately leads to an increase in SSCP sensitivity as mentioned in previously published reports (Kukita et al, 1997; Noullau and Wagener, 1997). After following a stringent parameter for technique optimization and also sequencing the suspected samples, we concluded that the presence of any germ line mutations of these MSGs in samples from patients with breast is unlikely. The study thus strongly suggests that frequent down-regulation of these proteins in cancer progression is unlikely due to any germ line mutations. Mutations of functional domains and antagonist variants have all been reported in a number of cancer related genes. For example, CHEK2 germ line mutations have adverse affects on prognosis of breast cancer patients (Meyer et al, 2007). Similarly, germ line mutations of several other genes responsible for metastasis like p53, SIPA1 (signal induced proliferation Associated gene1) are also shown to increase disease progression and metastasis in mammary tumours patients (Hsieh et al, 2006; GuĂŠnard et al, 2007). The mutational spectrum of CDH1 gene encoding E-Cadherin (another well known MSG) has also substantially increased the risk (70-80%) of cancer progression (Hsieh et al, 2006). The present study also screened whether there are any mutated alleles present on these MSGs. In this study, the objective was, first to assess whether there are any germ line mutations prevailing on these genes and second to see what proportion they are present in breast cancer patients. According to the authorsâ&#x20AC;&#x2122; knowledge, no such

study has previously been reported in relation to mammary tumours in any population. Expression dysregulation for both genes was previously observed in several cancers (Stafford et al, 2008). This dysregulation in expression may be attributed to other modulating factors, such as binding of p53 at the promoter binding site along with AP1 and AP2, leading to increased KAI1 protein at the intracellular level (Mashimo et al, 1998; Briese et al, 2008). Although correlation of p53 with KAI1 molecules has not been studied in relation to breast cancer, yet the altered modulation is justified by combination of various proteins bindings at the promoter and upstream binding region of KAI1 molecule (Tonoli et al, 2005). In another study conducted on 52 ovarian carcinomas, only one missense somatic mutation of KAI1 was observed and the authors similarly concluded that the mutation frequency is less likely responsible for protein down-regulation in metastasis progression in ovarian cancer. This lack of germ line mutations also emphasizes the conserved sequence of KAI1, which is also consistent with previous findings in which it has been observed that KAI1 is 76% identical to other members of the tetraspanin family (Nagira et al, 2005). Similarly, signaling of KISS1 is regulated by CRSP1 gene located at chromosome 6 which is frequently lost during cancer progression. Up-regulation of CRSP1 protein results in inhibition of metastasis by elevating the level of KISS1 (Goldberg et al, 2003). KISS1 has two dibasic cleavages and an amidation cleavage site. Posttranslational processing results in formation of KP54 and a matured and processed form of KISS1 gene termed as metastin, leading to its role in regulating metastasis (Rouille et., 1995; Karges et al, 2005). The carboxy terminus of KISS1 protein was found to be conserved among human and mouse, resulting in an increase in specific binding for its cognate G protein coupled receptor (Terao et al, 2004). The protease responsible for posttranslational modification KISS1 has yet to be identified. Hence, the mechanism of transcriptional regulation is an area that requires further investigation. Breast cancer cells may be predisposed to high tendency of metastasis, via control mechanisms such as the loss of balance between metastasis stimulating and inhibiting genes. Although germ line mutations of different genes lead to impaired expression of downstream regulatory genes (Marreeiros et al, 2005), levels of proteins and transcripts from these genes are also important in the control of metastatic behaviour of cancer cells. No partial or complete loss of coding region of KAI1 and KISS1 as shown in the present study provides a strong evidential support that down-regulation of these gene products are more likely to be the results of transacting factor rather than mutations. Hence, progression of metastasis with down regulation of both KAI1 and KISS1 proteins is mainly regulated by various cellular proteins rather than germ line mutations in their coding sequences.

V. Conclusion Mutational spectrum of the MSGs, KAI1 and KISS1, is substantially low and their down regulation in breast cancer patients may be attributed to by other regulatory 244

Gene Therapy and Molecular Biology Vol 12, page 245 Huang H, Groth J, Sossey-Alaoui K, Hawthorn L, Beall S, Geradts J (2005) Aberrant expression of novel and previously described cell membrane markers in human breast cancer cell lines and tumours. Clin Cancer Res 11, 43574364. Ichikawa T, Ichikawa Y, Dong J, Hawkins AL, Griffin CA, Isaacs WB, Oshimura M, Barrett JC, Isaacs JT (1992) Localization of metastasis suppressor gene(s) for prostatic cancer to the short arm of human chromosome 11. Cancer Res 52, 3486-3490. Ikeguchi M, Yamaguchi KI, Kaibara N (2004) Clinical Significance of the Loss of KiSS-1 and Orphan G-ProteinCoupled Receptor (hOT7T175) Gene Expression in Esophageal Squamous Cell Carcinoma. Clin Cancer Res 10, 1379-1383. Jackson P, Ow K, Yardley G, Delprado W, Quinn DI, Yang JL, Russell PJ (2003) Downregulation of KAI1mRNA in localized prostate cancer and its bony metastases do not correlate with p53 over expression. Prostate Cancer Prostatic Dis 6, 174–181. Jackson P, Rowe A, Grimm MO (2007) An alternatively spliced KAI1 mRNA is expressed at low levels in human bladder cancers and bladder cancer cell lines and is not associated with invasive behaviour. Oncol Rep 18,1357-63 Karges B, de Roux N (2005) Molecular genetics of isolated hypogonadotropic hypogonadism and Kallmann syndrome. Endocr Dev 8, 67-80. Nash KT, Welch DR (2006) The KISS1 metastasis suppressor: mechanistic insights and clinical utility. Front Biosci 11, 647–659. Köchl S, Niederstätter H, Parson W (2005) DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR. Methods Mol Bio 297, 13-30. Kukita Y, Tahira T, Sommer SS, Hayashi K (1997) SSCP analysis of long DNA fragments in low pH gel. Hum Mutat 10, 400-7. Lallas TA, Buller RE (1998) SSCP techniques in BRCA1 analysis. Molec Genet Metab 64, 173-176. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent J M, Weissman BE, Welch DR (1996) KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. J Nat Cancer Inst 88, 1731-1737. Lee JH, Park SR, Chay KO, Seo YW, Kook H, Ahn KY, Kim YJ and Kim (2004) KAI1 COOH-Terminal interacting Tetraspanin (KITENIN), a member of the tetraspanin family, interacts with KAI1, a tumor metastasis suppressor and enhances metastasis of cancer. Cancer Res 64, 4235-4243. Liu FS, Dong JT, Chen JT, Hsieh YT, Ho ES, Hung MJ (2000) Frequent down-regulation and lack of mutation of the KAI1 metastasis suppressor gene in epithelial ovarian carcinoma. Gynecol Oncol 78, 10-15. Malik F, Sanders AJ, Douglas-Jones A, Mansel RE, Jiang WG (2008) Kai-1 is aberrant expressed in human breast cancer and its link with cancer invasiveness. Brit J Cancer in press. Marreiros A, Dudgeon K, Dao V, Grimm MO, Czolij R, Crossley M, Jackson P (2005) KAI1 promoter activity is dependent on p53, junB and AP2: evidence for a possible mechanism underlying loss of KAI1 expression in cancer cells. Oncogene 20, 637-49. Martin TA, Watkins G, Jiang WG (2005) KiSS-1 expression in human breast cancer. Clin Exp Metastasis 22, 503-511. Martins CMO, Fernandes BF, Antecka E, Di Cesare, S, Mansure JJC, Marshall J-C, Burnier MN (2008) Expression of the metastasis suppressor gene KISS1 in uveal melanoma. Eye 22, 707-711. Mashimo T, Watabe M, Hirota S, Hosobe S, Miura K, Tegtmeyer P J, Rinker-Shaeffer C W, Watabe K (1998) The expression of the KAI1 gene, a tumor metastasis suppressor,

mechanisms. Results are consistent with a previously conducted research on KAI1 molecules in squamous cell carcinoma of oesophagus (Miyazaki et al, 2000) and KISS1in breast cancer (Martin et al, 2005). Further studies regarding the expressional variation detection will be helpful in defining a better prognostic marker for early prognosis of mammary tumor patients. Involvement of regulatory proteins as major contributor in the suppression of these vital genes leading to progression of metastasis and also a poor prognosis on patient survival is an area requisite of further investigation. These regulatory pathways also provide the opportunity for potential therapeutic intervention.

Acknowledgement We are extremely grateful to all patients and Oncologists for their immense help and support in this research. The authors also like to acknowledge the Higher Education Commission of Pakistan for providing funds to carry out this collaborative research work and Dr Richard J Ablin for critical reading of the manuscript. In the last but not least we also extend our gratitude to the reviewers for their valuable time and suggestions for this manuscript.

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Gene Therapy and Molecular Biology Vol 12, page 247 Gene Ther Mol Biol Vol 12, 247-252, 2008

A new potential radiosensitizer- multi-walled carbon nanotubes modified by ammonium persulfate Research Article

Jian-She Yang1,2,*, Xigang Jing1, Wen-Jian Li1, Xiang-Kai Hu1, Wei Wei1, ZhuanZi Wang1 1 2

Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China Life Science School of Northwest Normal University, Lanzhou 730070, PR China

__________________________________________________________________________________ *Correspondence: Jian-She Yang, Life Science School of Northwest Normal University, Lanzhou 730070, PR China; Tel.: +86-9314969234; fax: +86-931-4969201; E-mail address: yangjs@impcas.ac.cn Key words: ammonium persulfate, multi-walled carbon nanotubes Abbreviations: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, (MTT); American Type Culture Collection, (ATCC); Dulbcco's Modifed Eagle Medium, (DMEM); infrared, (IR); phosphate buffer solution, (PBS); pure MWCNTs, (p-MWCNTs); singlestranded DNA, (ssDNA) Received: 29 April 2008; Revised: 2 June 2008 Accepted: 6 June 2008; electronically published: October 2008

Summary Here we prepare carbon nanotubes modified with ammonium persulfate, very short carbon nanotubes with 50-100 nanometer length was obtained, and the higher ! potential of 52 mV was detected, these supporting the successful modification. HeLa cells were irradiated with " rays via adding or absent above functionalized carbon nanotubes (fWCNTs) into cell culture medium with different concentration and radiation dosage. Confocal microscopy images and fluorescence-labeled DNA detection verified the successfully pure multi-walled carbon nanotubes (p-WCNTs) and f-WCNTs penetrated into cells. Compared with pure radiation, by MTT test, f-WCNTs induced cell death markedly with about 8.7 times higher than former one under little dose of radiation; meanwhile, no obvious toxicity was observed both in p-WCNTs and f-WCNTs without of radiation exposure. We hypothesized that large amount of hydroxyl and carbonyl organs on the surface of very short f-WCNTs changed into free radicals result from radiations led cell damage. These implied that f-WCNTs could be regarded as a new radiosensitizer.

2003; Savic et al, 2003; Pantarotto et al, 2004) to transport across cell membranes has received significant interest. Because of itsâ&#x20AC;&#x2122; non-toxic property, carbon nanotubes have been used as a new tool to deliver poorly penetrating drug, peptides, peptidomimetics, protein or small organic probe molecules into cancerous cells (Mattson et al, 2000; Kueltzo and Middaugh, 2003; Pantarottoet al, 2003; Savic et al, 2003; Pantarotto et al, 2004; Shi Kam et al, 2004). However, there is not any studies report that carbon nanotubes have been used in radiobiology experiments as radiosensitizer. In present study, we describe preparation of MWCNTs modified with ammonium persulfate. The modified carbon nanotubes are not only able to readily enter cervix cancer cells (HeLa cells), but also to effectively kill the cancerous cells under low dose of ! radiations. Furthermore, both pure MWCNTs (pMWCNTs) and f-MWCNTs themselves appear non-toxic for HeLa cells. With the increasing radiation dosage and

I. Introduction As is well known, radiotherapy plays a very important role in cancer treatment, and the cure effect strictly relied on the intrinsic radiosensitivity of target cancer cells. Besides of very fewer sensitive examples, such as ataxia telangiectasia (Maria et al, 2008) cells, most malignancy cells are of moderate radiosensitivity, even some of them are radioresistant (Kaliberov et al, 2007). In order to achieve better treatment effect, radiosensitizers (Bump et al, 1982; Cuneo et al, 2007; Kaliberov et al, 2007; Kinsella et al, 2007; Lally et al, 2007; Specenier et al, 2007; Zhang et al, 2007) are frequently used in vivo or in vitro performances. Radiosensitizer introduce a higher radiotherapy effect mainly through capturing electrons in target volume under irradiations to prevent them from recombination with radiation-injured samples (Bump et al, 1982). In recent years, ability of carbon nanotubes (Mattson et al, 2000; Kueltzo and Middaugh, 2003; Pantarottoet al, 247

Yang et al: A new potential radiosensitizer- multi-walled carbon nanotubes modified by ammonium persulfate concentration of f-MWCNTs in growth medium, there is a significant decrease of the cell survivals.

III. Results To examine the dispersion state of the MWCNTs in water solutions, one drop of the water solution with MWCNTs (1 mg/mL) is dropped on a silicon-oxide substrate for scanning electron microscope analysis, the image reveal mostly short (about 100 nm-1 "m) pMWCNTs and 30-100nm f-MWCNTs with diameters of 30 nm corresponding to mostly isolated individual MWCNTs (Figure 1a, b). No significant amount of particles is observed on the substrate, suggesting good purity of short MWCNTs in water solution. In pure water, the suspension of the black MWCNTs is stable for extending periods of time and does not agglomerate, which is likely relative to amount of shortened MWCNTs. This phenomenon is in accordance with what reported in literatures (Liu et al, 1998; Sano et al, 2001). In PBS containing ~0.2 M salt, the suspension of MWCNTs is less stable and start to aggregate after 2 h. We use #sizer 3000HS (Malvern Instruments Ltd, UK) to obtain the $ potential of p- and f-MWCNTs, which is 42 and 54mV. This indicates the more negatively charged groups existed on the surface of f-MWCNTs than that of p-MWCNTs (Figure 1c, d). Furthermore, infrared (IR) analysis shows that both carbonyl and hydroxyl peak number of fMWCNTs are higher than that of p-MWCNTs (Figure 1e, f). These groups will change into free radicals in aqueous atmosphere when exposed to ion radiation, and consequently induce cell damage (Albano, 2006). To visualize the interaction of p-MWCNTs, and fMWCNTs with cells, the HeLa cells are incubated with these nanomaterials (50"g/mL) for 4 h at 37 °C. After the cells are carefully washed with PBS and digested by steapsin, and a fresh culture medium is added. Subsequently, the cells are observed directly in glassbottomed dishes under confocal microscope. Figure 2a, b show the cells with dark cytoplasm and apparent nuclei free of MWCNTs, indicative of intracellular and not extracellular localization of the MWCNTs. To further verify the cellular uptake of both pMWCNTs and f-MWCNTs, negatively charged singlestranded DNA (ssDNA) labeled with 6-carboxy fluorescein is bound to the sidewall of MWCNTs via hydrophobic interaction. The dispersive complexes of the MWCNTs/ssDNA are dialyzed for 2 h with constant stirring in PBS to eliminate free ssDNA. The confocal images indicate that the stable complexes of MWCNTs/ssDNA in PBS reveal green fluorescence, which confirms the ssDNA can be strongly absorbed on sidewall of MWCNTs. We then study the interactions of these resulting complexes with the HeLa cells. Figure 2c shows that these complexes appear to uniformly accumulate in the cytoplasm in the HeLa cells after internalization, and not adhering to the cells extracellularly, which further confirm the intracellular uptake of the complexes. As negative-control experiment, the cells are incubated with a solution that contained only fluorescently labeled ssDNA. No fluorescence of the cells is detected, which means that the MWCNTs can traverse the cell membranes and transport adsorbed ssDNA into the cells.

II. Experimental The stable aqueous suspensions of purified, shortened, and functionalized carboxylic acid nanotubes is obtained by oxidation and polishing (Chen et al, 1998; Liu et al, 1998; Sano et al, 2001) of laser-ablated raw multi-walled carbon nanotubes (purchased from Shenzhen Nanotech Port Co. Ltd.). In order to eliminate metal catalysts, the carbon nanotubes was afterward dispersed in 6 M HCl under ultrasonic agitation, washed with sodium hydroxide solution and deionized water to neutrality and dried. The purified MWCNTs are suspended in 500 mL concentrated H2SO4/HNO3 (V/V=3:1) solution and sonicated in a water bath for 24 h at 35-40 °C. Centrifugation (7000 rpm, 5 min) removed larger unreacted impurities from the resultant suspension to afford a stable suspension of MWCNTs. The cut nanotubes are recovered by filtration with polytetrafluoroethylene membrane with a pore size of 0.22 "m and rinsed with deionized water. Subsequently, they are then further polished by suspension in a 4:1 mixture of concentrated H2SO4/30% aqueous H2O 2 and stirring at 70 °C for 30 min. After filtering and washing again, the resulting MWCNTs can be relatively dispersed in water, this resulting material was regarded as p-MWCNTs. Then, 50mg p-MWCNTs are added into 10ml deionized water, sonicated for 10 minutes. Thereafter, ammonium persulfate is added into upper solution with the terminated concentration of 0.5M, and stirring for 48 hours at 50 °C. The rinse and filtration process is repeated as described above, at the end, we get f-MWCNTs. All of the tested materials are conducted in sterile phosphate buffer solution (PBS), and kept the cells from contaminant. HeLa cells purchased from American Type Culture Collection (ATCC) are cultured in Dulbcco's Modifed Eagle Medium (DMEM, Invitrogen-Gibco), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 "g/mL streptomycin. The cells are incubated in a humidified atmosphere of 5% CO2-95% air at 37 °C in a 75 cm2 flask, and supplied with fresh medium every three days. Incubation of cells is done by adding PBS of the pMWCNTs, f-MWCNTs into the culture medium (concentration ranges from 0 to 50"g/mL in the culture medium), and the incubation duration is always 4 h. After incubation, the cells are washed with PBS and resuspended in fresh culture medium. All confocal images are taken immediately after the incubation and washing steps except for the radiation experiment and cell viability assay. The cell suspension (20 "L) is dropped onto a glass-bottomed dish and image by a Zeiss LSM 510 confocal microscopy. Cells are randomly divided into three groups: two are adding p-MWCNTs, f-MWCNTs into cell culture medium, respectively; and another group is regarded as control with no other materials added into. Cells are irradiated with 60Co ! rays with dosage range from 0 to 6 Grays (Dose rate was 1Gray per minute). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT) is used to determine cell survival in a quantitative colorimetric assay. Various dehydrogenase enzymes in active mitochondria, forming a bluecolored insoluble product, cleave its tetrazolium ring formazan. The HeLa cells are incubated with MTT (5 mg/mL) added to the culture medium for 4 h at 37 °C. The medium is then aspirated and the formazan product is dissolved in dimethyl sulfoxide and quantified spectrophotometrically at 490 nm with control of 650 nm. The results are expressed as a percentage of control culture viability.

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Gene Therapy and Molecular Biology Vol 12, page 249 Having discovered the ability of p-MWCNTs and fMWCNTs to enter cells, we further seek to examine their potential toxicity due to the delivery of these nanomaterials into cells. Toward this end, the p- and fMWCNTs are used as the toxic control assay, and the cell survival is conducted by the observed MTT experiments. The cells are separately incubated with pure DMEM culture medium (contain 10% BSA), p-MWCNTs and fMWCNTs for 4 h, and rinsed with sterile PBS.

Subsequently, these cells are transferred into 96 bores board. After 48 h incubation, the MTT is added into the each bore in the board. After 5 h, the clear solution into each bore is obtained by centrifugation, and respectively transferred into the other 96 bores board. As show in Figure 3, along with increasing the concentration of pand f-MWCNTs in the incubation solution (concentration ranges from 0 to 50"g/mL in the culture medium) survival rate of the HeLa cells is stable at about 95%, which is

Figure 1. Physical and chemical properties of p-MWCNTs and f-MWCNTs, (a) SEM image of p-MWCNTs, (b) SEM image of fMWCNTs, (c) $ potential of p-MWCNTs, (e) $ potential of f-MWCNTs, (e) IR spectrum diagram of p-MWCNTs and f-MWCNTs.

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Yang et al: A new potential radiosensitizer- multi-walled carbon nanotubes modified by ammonium persulfate

Figure 2. Confocal images of HeLa cells after incubation in solution of p-MWCNTs and f–MWCNTs: (a) after incubation in pMWCNTs, (b) after incubation in f–MWCNTs, (c) after incubation in the MWCNTs/ssDNA complexes at 37 °C.

Figure 3. Confocal images of HeLa cells after incubation in solution of p-MWCNTs and f–MWCNTs: (a) after incubation in pure DMEM culture medium with 10% fetal bovine serum, (b) after incubation in p–MWCNTs, (c) after incubation in f–MWCNTs, (d) cell viability showed by OD value. P<0.05.

similar with that of control group, no significant changes of cell survival are observed. These results indicate that pand f-MWCNTs themselves are little toxic to HeLa cells after the cells are incubated even in highly concentrated solutions. The p- and f-MWCNTs are conducted under high pressure and high temperature, and that all of the tested materials are conducted in sterile PBS, and kept the cells from contaminant. Thus, the observed endotoxin is nearly nonexistent when these cells are exposed to the p- and fMWCNTs in our experiment. Since these materials are immediately dispersed by sonication within a few seconds,

the cell survivals are not likely influenced by the residual transition metal contaminants into the MWCNTs. When absent of radiations, p- and f-MWCNTs have been verified no toxicity to HeLa cells, regardless of its concentration. After irradiation, cell viability was decreased along with the increase of radiation dosage and material concentration. For f-MWCNTs group, there is a sharp decrease, compared with p-MWCNTs and control group; and the cell survival of p-MWCNTs group is lower than that of control group. Cell viability is similar with our previous report (Yang et al, 2005), under 6 Gy ! radiation, there is a 40 percent cell survive with no MWCNTs addition, and the cell survival curves are linear-quadratic, 250

Gene Therapy and Molecular Biology Vol 12, page 251 which is consistent with the classical low linear energy transfer radiation model. After further 72 hours incubation in fresh DMEM culture medium, there is an obvious repair effect in control group (Figure 4d), and this repair effect is not observed in p- and f-MWCNTs groups (Figure 4e, f), it indicates that the MWCNTs induces radiation damage is lethal attack to cells.

with special tumor cell antibody, which will introduce fMWCNTs into tumor cells, thereafter lead a targeted radiosensitizing effect.

V. Conclusions In conclusion, the ammonium persulfate functionalized MWCNTs are soluble, and can enter cancer cells, exhibiting dose-dependent cytotoxicity under radiation. Thus, because of the unique biocompatibility, and bioabsorption of the MWCNTs, it provides the basis for new classes of materials for enhancing the cancer cell radiosensitivity, though the targeting nature of MWCNTs is not discussed in present study. This indicates that MWCNTs are of possibility to be prepared for a new kind of radiosensitizer.

IV. Discussion The ammonium persulfate oxidized and polished areas of the MWCNTs contain large amount of negatively charged carbonyl, hydroxyl groups along the sidewalls, such groups are of feasibility to be radioanalyzed to become hydroxylic and other free radicals under radiation. These free radicals are able to kill cancer cells directly, possibly including cell membrane, plasma, nuclei and other substructures. The unoxidized areas of the MWCNTs may still afford regions of appreciable hydrophobicity, and the DNA molecules can be strongly adsorbed on surface of MWCNTs presumably via hydrophobic interaction with sidewalls of the carbon nanotubes (Pantarotto et al, 2004; Shi Kam et al, 2004). The MWCNTs can nonspecifically associate with hydrophobic regions of the cell surface and internalize by endocytosis (Silverstein et al, 1977; Vida and Emr, 1995). The unoxidized areas can also afford regions to combine

Acknowledgements Financial support from the 973 program of MOST (Grant No. 2006CB705600), National Natural Science Foundation (Grant No. 10475109), K. C. Wong Education Foundation, Hong Kong (Grant No. 20060037) and China Postdoctoral Science Foundation (Grant No. 20060400686) are greatly acknowledged.

Figure 4. MTT analysis of HeLa cell survival ratio after gamma radiation. (a) 4Gy radiation; (b) 4Gy+25"g p-MWCNTs; (c) 4Gy+25"g f-MWCNTs; (d) 4Gy irradiation and 72 hours repair; (e) 4Gy+25"g p-MWCNTs and 72 hours repair; (f) 4Gy+25"g f-MWCNTs and 72 hours repair; (g) Cell violability curves. p<0.05.

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Yang et al: A new potential radiosensitizer- multi-walled carbon nanotubes modified by ammonium persulfate Mattson MP, Haddon RC, Rao AM (2000) Molecular functionalization of carbon nanotubes and use as substrates for neuronal growth. J Mol Neurosci 14, 175-82. Pantarotto D, Briand J, Prato M, Bianco A (2004) Translocation of bioactive peptides across cell membranes by carbon nanotubes. Chem Commun (Camb) 1, 16-7. Pantarotto D, Partidos CD, Hoebeke J, Brown F, Kramer E, Briand JP, Muller S, Prato M, Bianco A (2003) Immunization with peptide-functionalized carbon nanotubes enhances virus-specific neutralizing antibody responses. Chem Biol 10, 961-6. Sano M, Kamino A, Okamura J, Shinkai S (2001) Ring closure of carbon nanotubes. Science 293, 1299-301. Savic R, Luo L, Eisenberg A, Maysinger D (2003) Micellar nanocontainers distribute to defined cytoplasmic organelles. Science 300, 615-8. Shi Kam NW, Jessop TC, Wender PA, Dai H (2004) Nanotube molecular transporters: internalization of carbon nanotubeprotein conjugates into mammalian cells. J Am Chem Soc 126, 6850-1. Silverstein SC, Steinman RM, Cohn ZA (1977) Endocytosis. Annu Rev Biochem 46, 669-722. Specenier PM, Van den WD, Van Laer C, Weyler J, Van den BJ, Huizing MT, Dyck J, Schrijvers D, Vermorken JB (2007) Phase II feasibility study of concurrent radiotherapy and gemcitabine in chemonaive patients with squamous cell carcinoma of the head and neck: long-term follow up data. Ann Oncol 18, 1856-60. Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128, 779-92. Yang JS, Li WJ, Zhou GM, Jin XD, Xia JG, Wang JF, Wang ZZ, Guo CL, Gao QX (2005) Comparative study on radiosensitivity of various tumor cells and human normal liver cells. World J Gastroenterol 11, 4098-101.

References Albano E (2006) Alcohol, oxidative stress and free radical damage. Proc Nutr Soc 65, 278-90. Bump EA, Yu NY, Brown JM (1982) Radiosensitization of hypoxic tumor cells by depletion of intracellular glutathione. Science 217, 544-5. Chen J, Hammon MA, Hu H, Chen YS, Rao AM, Eklund PC, Haddon RC (1998) Solution properties of single-walled carbon nanotubes. Science 282, 95-8. Cuneo KC, Tu T, Geng L, Fu A, Hallahan DE, Willey CD (2007) HIV protease inhibitors enhance the efficacy of irradiation. Cancer Res 67, 4886-93. Kaliberov SA, Market JM, Gillespie GY, Krendelchtchikova V, Manna DD, Sellers JC, Kaliberova LN , Black ME, Buchsbaum DJ (2007) Mutation of Escherichia coli cytosine deaminase significantly enhances molecular chemotherapy of human glioma. Gene Ther 14, 1111-9. Kinsella TJ, Kinsella MT, Seo Y, Berk G (2007) 5-Iodo-2Pyrimidinone-2 -Deoxyribose-Mediated cytotoxicity and radiosensitization in U87 human glioblastoma xenografts. Int J Radiat Oncol Biol Phys 69, 1254-61. Kueltzo LA, Middaugh CR (2003) Nonclassical transport proteins and peptides: an alternative to classical macromolecule delivery systems. J Pharm Sci 92, 1754-72. Lally BE, Geiger GA, Kridel S, Arcury-Quandt AE, Robbins ME, Kock ND, Wheeler K, Peddi P, Georgakilas A, Kao GD, Koumenis C (2007) Identification and biological evaluation of a novel and potent small molecule radiation sensitizer via an unbiased screen of a chemical library. Cancer Res 67, 8791-9. Liu J, Rinzler AG, Dai H, Hafner JH, Bradley RK, Boul PJ, Lu A, Iverson T, Shelimov K, Huffman CB, Rodriguez-Macias F, Shon YS, Lee TR, Colbert DT, Smalley RE (1998) Fullerene pipes. Science 28, 1253-6. Maria LC, Angelika O, Okay S, Jocelyn DS, Katherine FP, Cornel F, Xandra OB (2008) Targeted integration of functional human ATM cDNA into genome mediated by HSV/AAV Hybrid amplicon vector. Mol Ther 16, 81-8.

Zhang AL, Russell PJ, Knitte T, Milross C (2007) Paclitaxel enhanced radiation Sensitization for the suppression of human prostate cancer tumor growth via a p53 independent pathway. Prostate 67, 1630-40.

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Decreased risk of bladder cancer in men treated with quinazoline-based !1-adrenoceptor antagonists Research Article

Frances M. Martin1, Andrew M. Harris1, Randall G. Rowland1, William Conner1, Matthew Lane2, Erik Durbin3, Andre T. Baron4,5, Natasha Kyprianou1,6,* 1

Division of Urology/Department of Surgery, University of Kentucky College of Medicine, Lexington Veterans Affairs Medical Center, 3 Kentucky Cancer Registry, Cancer Bioinformatics Division, Markey Cancer Center, 4 Division of Hematology Oncology, Blood and Marrow Transplantation/Department of Internal Medicine, Markey Cancer Center, University of Kentucky College of Medicine, 5 Department of Epidemiology, University of Kentucky College of Public Health, 6 Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 2

__________________________________________________________________________________ *Correspondence: Dr. Natasha Kyprianou, Division of Urology, MS 283, University of Kentucky College of Medicine, Lexington, KY 40536, USA; Tel: (859)-323-9812; Email: nkypr2@uky.edu Key words: Bladder Cancer, Prevention, !1-adrenoceptor Antagonists, Apoptosis Abbreviations: benign prostatic hypertrophy, (BPH); Fas-associated death domain, (FADD); Kentucky Cancer Registry, (KCR); Surveillance, Epidemiology, and End Results, (SEER); transgenic adenocarcinoma of mouse prostate, (TRAMP); vascular endothelial growth factor, (VEGF); Veterans Administration, (VA) Received: 4 June 2008; Revised: 4 July 2008 Accepted: 8 July 2008; electronically published: October 2008

Summary Previous studies documented that human bladder cancer cells are sensitive to the apoptotic effects of quinazolinederived !1-adrenoreceptor antagonists and bladder tumors exhibit reduced tissue vascularity in response to terazosin. More recent evidence suggests that exposure to quinazoline !1-adrenorecptor antagonists leads to a significant reduction in prostate cancer incidence. This retrospective observational cohort study was conducted to determine whether male patients treated with quinazoline !1-adrenoceptor antagonists for either benign prostate hyperplasia (BPH) or hypertension have a decreased risk of developing bladder cancer. Review of the medical records of all male patients enrolled at the Lexington Veterans Administration (VA) Medical Center identified men exposed to quinazoline-based !1-adrenoceptor antagonists (Jan 1, 1998-Dec 31, 2002) for either hypertension and/or benign prostate obstructive symptoms. The whole group of 27,138 male patients was linked to the Markey Cancer Centerâ&#x20AC;&#x2122;s Kentucky Cancer Registry (KCR), part of the NCIâ&#x20AC;&#x2122;s Surveillance, Epidemiology, and End Results (SEER) Program, to identify all incident bladder cancer cases diagnosed in this population. Measures of disease incidence, relative risk, and attributable risk were calculated to compare the risk of developing bladder cancer for !1-blocker-exposed versus unexposed men. A two-by-two contingency table of !1-antagonist exposure versus bladder cancer diagnoses was constructed and the relative risk was calculated. Our analysis revealed a cumulative bladder cancer incidence of 0.24% among the !1-blocker-exposed men compared to 0.42% in the unexposed group. Thus, there was a risk difference of -0.0018, which indicates that 1.8 fewer bladder cancer cases developed per 1000 exposed men. Alternatively stated, 556 men would need to be treated with quinazoline !1-blockers to prevent one case of bladder cancer. Exposure to quinazoline !1-blockers thus may have prevented 7 to 8 bladder cancer cases among the 4173 treated men during the study period .The data yield an unadjusted risk ratio of 0.57 (95% CI: 0.30, 1.08) and therefore, men treated with !1-adrenoreceptor antagonists have a 43% lower relative risk of developing bladder cancer than unexposed men (p=0.083). Our inability to determine person-years at risk of developing bladder cancer for each unexposed control patient, was a limitation for calculating an incidence ratio and rate difference. These results offer an initial indication that exposure to doxazosin and terazosin decreases the incidence of bladder cancer. This is the first epidemiological evidence that the anti-tumor action of quinazoline-based !1antagonists may potentially translate into a protective effect from bladder cancer development.

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Martin et al: Bladder Cancer Prevention by Quinazoline !1-Blockers epithelial and endothelial cells by promoting TGF-"1 signaling via I#B induction (Garrison and Kyprianou, 2004), and by inhibiting protein kinase B/Akt activation to promote anoikis (Grossmann, 2002; Keledjian and Kyprianou, 2002; Shaw et al, 2004). The quinazolines suppress angiogenesis by targeting vascular endothelial growth factor (VEGF)- mediated endothelial tube formation (Panet al, 2003; Keledjian et al, 2005). Tahmatzopoulos and colleagues recently reported that bladder tumors treated with terazosin had significantly decreased tissue vascularity and an increased apoptotic index as compared to untreated bladder tumors (Tahmatzopouloset al, 2005). Our previous studies established that human bladder cancer cells are susceptible to the apoptotic effect of the quinazolines in vitro (Kyprianou and Jacobs, 2000). Taken together this in vitro data, with our recent retrospective analysis indicating the quinazoline alpha1-adrenorecptor antagonists lowered the incidence of prostate cancer prompted the current epidemiological study. In this observational cohort study, we investigated whether use of the quinazoline-based !1-adrenoceptor antagonists is associated with a decreased risk of bladder cancer. Our retrospective analysis suggests that men treated with this class of !1-adrrenoceptor antagonists have a reduced risk of developing bladder cancer, suggesting that the apoptotic and anti-angiogenic action of these drugs at the cellular level might be a potential mechanism contributing to the prevention of clinical disease.

I. Introduction Carcinoma of the bladder is projected to be the fourth most common cancer in males and ninth in females in 2006 (Jemal et al, 2006). The incidence and mortality of transitional cell carcinoma of the bladder has increased in recent years with an estimated 61,420 new cases and 13,060 deaths in 2006 and 67,160 new cases and 13,750 deaths in 2007 (Jemal et al, 2006, 2007). Loss of apoptosis is causally linked in the development of bladder cancer (Reed, 1999) as illustrated by the observation that in vitro bladder cancer cells eventually become resistant to cytotoxic drugs (Kerret al, 1994); therefore, induction of apoptosis is an attractive therapeutic target. Uncontrolled angiogenesis also plays a role in bladder cancer development because without generating blood supply, bladder tumors are unable to grow over 2-3mm (Folkman, 1971; Streeter and Harris, 2002). Angiogenesis and microvessel density parallel disease progression as well as overall survival in bladder cancer, which supports targeting therapies that inhibit angiogenesis (Bochner et al, 1995). Quinazoline !1-adrenoreceptor antagonists have been shown to promote apoptosis and to inhibit angiogenesis (Garrison et al, 2007). The quinazoline-based !1-adrenoreceptor antagonists, doxazosin and terazosin, are FDA-approved drugs characterized by a few, well-tolerated side effects, primarily dizziness, used clinically for the treatment of benign prostatic hypertrophy (BPH) and systemic hypertension. The !1-adrenoceptor antagonists exert their effect via directly targeting !1-adrenoceptors in smooth muscle cells in the prostate gland and bladder neck (Walden et al, 1997; McConnellet al, 2003), causing a decrease in smooth muscle tone to relieve bladder obstruction secondary to periurethral prostatic enlargement (Caine, 1990). Growing evidence from retrospective clinical studies demonstrates that in addition to causing smooth muscle relaxation and a decrease in vascular pressure, the quinazoline-based !1-adrenoceptor antagonists also can induce apoptosis and suppress angiogenesis in benign and malignant prostate tumors (Kyprianou et al, 1998; Chon, et al, 1999; Kyprianou, 2003). Pharmacologically-relevant levels of the two leading !1-adrenoceptor antagonists used in the US, doxazosin and terazosin, selectively induce apoptosis in benign and malignant prostate epithelial cells, as well as stromal smooth muscle cells, without affecting cell proliferation in vitro or in clinical tumor specimens (Chon et al, 1999). The apoptotic action of quinazolines engages an !1-adrenoceptor-independent mechanism, and affects both androgen-independent and androgen-dependent prostate cancer cells (Benning. and Kyprianou, 2002; Garrison and Kyprianou, 2004; Kyprianou and Benning., 2004). Apoptosis induction proceeds via two classic pathways, the extrinsic death-receptor pathway involving caspase 8 activation, and the intrinsic pathway involving mitochondrial cytochrome C and caspase 9 activation (Wolf and Green, 1999). We recently demonstrated that doxazosin (quinazoline-!1-adrenoceptor antagonist) activates the receptor-mediated pathway of apoptosis via Fas-associated death domain (FADD) and caspase-8 activation (Garrison and Kyprianou, 2006) in both prostate

II. Patients and Methods A. Patient Cohort construction A retrospective observational study was performed on a cohort of male patients seen at the Lexington Veterans Affairs (VA) Hospital between January 1, 1998 and December 31, 2003. The total number of men seen at the VA during this 5-yr period (n = 27,138) was determined from the VA’s electronic hospital registry, and the total number of bladder cancer cases diagnosed at the VA between 1998 and 2002 (n= 107) was obtained from the Markey Cancer Center’s Kentucky Cancer Registry (KCR), a statewide population-based central cancer registry that is part of the NCI’s Surveillance, Epidemiology, and End Results (SEER) Program. Information about age at diagnosis, race (Caucasian or non-Caucasian), disease stage at diagnosis (I, II, III, IV, not applicable, and unknown), tumor grade (1, 2, 3, 4, unknown), and tumor histology (carcinoma NOS, small cell carcinoma, adenocarcinoma NOS, mucinous adenocarcinoma, infiltrating duct carcinoma; NOS, not otherwise specified) was obtained from the KCR. All men exposed to a quinazoline-based !1adrenoceptor antagonist, doxazosin (1-8mg/day), prazosin (210mg/day), or terazosin (1-10mg/day) for either systemic hypertension or BPH between 1998 and 2002 (n = 4,173) were identified from the VA’s electronic pharmacy records and linked to the KCR’s database. The data identified all quinazoline !1blocker-exposed bladder cancer cases diagnosed at the VA greater than 2 months after treatment (n = 10) and exposed patients without bladder cancer (n = 4,163). Bladder cancer cases diagnosed less than 2 months after initiating quinazoline !1blocker treatment were assumed to have pre-existing cancer and, therefore, classified as unexposed bladder cancer cases. The number of unexposed patients with bladder cancer (n = 97) and without bladder cancer (n = 22,868) was calculated subsequently

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Gene Therapy and Molecular Biology Vol 12, page 255 by subtraction from the margin totals of a two-by-two contingency table (Table 1).

median survival of one year (Hussain and James, 2003). The concept that the quinazoline-based !1-adrenoreceptor antagonists may play a role in both preventing tumor initiation as well as mitigating progression to metastatic disease by targeting anoikis and angiogenesis is of potentially significant therapeutic value. Experimental studies have established the apoptotic and anti-angiogenic action of quinazoline-based !1-adrenoceptor antagonists (doxazosin and terazosin) against bladder cancer cells, benign and malignant prostatic epithelial cells, as well as endothelial cells via a mechanism independent of !1adrenoceptor action (Kyprianou and Jacobs, 2000; Benning and Kyprianou, 2002; Keledjianet al, 2005; Garrison and Kyprianou, 2006). In vitro, the quinazolines trigger anoikis in prostate cancer cells, directly inhibit endothelial cell adhesion, migration, invasion, and induce apoptosis of vascular endothelial cells by potentially targeting VEGF signaling (Keledjian and Kyprianou, 2003; Pan et al, 2003; Keledjian et al, 2005). In vivo, administration of doxazosin prior to tumor initiation has been shown to reduce prostate tumor weight and suppress metastasis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model (Chiang et al, 2005). Furthermore, bladder tumors treated with terazosin exhibited a significantly decreased tissue vascularity and increased apoptic index compared to untreated bladder tumors (Tahmatzopoulos et al, 2005). These observations establish a biologically plausible role for the quinazolines as chemotherapeutic and chemopreventive agents of bladder cancer. The well-established safety profile and wide-spread clinical use of these FDA-approved drugs supports their suitability and feasibility as long-term chemopreventive agents (Lepor et al, 1992; Chapple et al, 1994). The present study provides initial epidemiologic evidence of a potential chemopreventive effect for quinazoline !1-adrenoreceptor antagonists on human bladder cancer. Men exposed to quinazoline !1adrenoreceptor antagonists had a cumulative bladder cancer incidence of 0.24% compared to 0.42% for unexposed men, yielding an unadjusted relative risk of 0.57 (95% CI: 0.30, 1.08).

III. Results A. Quinazoline-based !1-adrenoceptor antagonist exposure is associated with reduced bladder cancer incidence A two-by-two contingency table of bladder cancer and non-cancer cases versus quinazoline !1-adenoceptor antagonist-treated and untreated men seen at the Lexington VA was constructed (Table 1). These data were used to calculate measures of disease incidence (cumulative incidence), relative risk, attributable risk (risk difference), and % attributable risk (% risk difference) and to determine whether significant differences exist between !1-blocker-exposed versus unexposed (control) men in developing bladder cancer using a !2-test and 95% confidence intervals. As shown on Table 1, the !1adrenoceptor antagonist-exposed group had a bladder cancer cumulative incidence of 0.24% compared to 0.42% in the unexposed group, which yields a risk difference of 0.0018 and an unadjusted relative risk of 0.57 (95% CI: 0.30, 1.08) for !1-adrenoceptor exposed versus unexposed men. This risk ratio indicates that men treated with !1adrenoceptor antagonists have a 1.76 times lower risk (p = 0.083) and a relative risk reduction of 43.3% meaning that 43.3% of the bladder cancer incidence in the control group might have been prevented by giving the medication. Interpretation of the risk difference indicates that 1.8 fewer bladder cancer cases developed per 1000 treated men; i.e., 7 to 8 additional bladder cancer cases would have been expected among the 4173 treated men in the Lexington VA cohort during the study period, had they not been exposed to quinazoline-based !1-adrenoceptor antagonists. This can be â&#x20AC;&#x153;translatedâ&#x20AC;? into 556 men needed to be treated with quinazoline !1-blockers to prevent one case of bladder cancer.

IV. Discussion Carcinoma of the bladder is a heterogeneous disease that progresses from carcinoma in situ to metastatic disease. Treating advanced metastatic disease has few options other than chemotherapy and radiation with

Table 1. Two-by-two contingency table constructed using data the from VA population illustrating incidence of cancer in treated versus untreated men. Treatment group $1-alpha Blockerexposed Unexposed Total %2 p value Rirk Ration (95% Cl) Risk Difference % Risk Difference

Positive for Bladder Cancer 10

Negative for Bladder Cancer 4163

Total

97 107

22868 27031

22965 27138

0.083 0.567 (0.299, 1.077) -0.0018 -43.3

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4172

Cumulative incidence 0.0024 0.0042

Martin et al: Bladder Cancer Prevention by Quinazoline !1-Blockers Our inability to determine person-years at risk of developing bladder cancer for each unexposed control patient prevented the calculation of an incidence ratio and rate difference, and might be considered as an obvious limitation of this retrospective study. In addition, two sources of misclassification bias might have impacted the data; first, patients in the unexposed group may have been prescribed quinazoline !1-blockers outside the VA system that might interfere with the impact of the defined period of exposure (2 mos) to the drug in the study. Such a misclassification would result in underestimating the protective effect of the !1-adrenoceptor-antagonist treatment. Second, patients in either the exposed or unexposed groups may have received a diagnosis of bladder cancer outside the VA. However, this type of misclassification should have had a minimal effect on the relative attributable risks observed here, assuming equal bladder cancer misclassification rates in both groups of patients. In addition, we were unable to collect information about potential confounders or effect modifiers such as age, race and ethnicity, smoking history, alcohol consumption, and body mass index; co-morbidity of BPH, hypertension, obesity, and other diseases; and the use of other medications for the unexposed control group. As such, we were not able to assess confounding or effect modification by these variables on bladder cancer incidence. The possible protective effect by !1-adrenoceptor antagonists on bladder cancer incidence (43% decrease), calls for nested case-control cohort studies to confirm that quinazoline-based !1-blockers are preventive agents of bladder cancer, prior to considering implementation of a randomized chemoprevention trial. Such studies should minimize misclassification bias, adjust for confounding factors, and assess effect modification by relevant covariates and longer drug-exposure. Ongoing retrospective studies at our center focus on investigating whether non-quinazoline !1-adrenoceptor antagonists (such as the sulfonamide, tamsulosin) can confer protection as well. The additional actions of these drugs including the ability of doxazosin to hinder chemotaxis in human monocytes (Kintscher et al, 2001), inhibit cell cycle progression in human coronary artery smooth muscle cells (Kintscher et al, 2000), and reduce cellular proliferation and migration of vascular smooth muscle cells (Hu et al, 1998), should be â&#x20AC;&#x153;factored-inâ&#x20AC;? when designing multi-center chemoprevention trials. Considering the cancer cell types sensitive to the apoptotic effect of quinazolines in vitro (Kyprianou and Jacobs, 2000), the potential chemopreventive action of !1adrenoreceptor antagonists in other human malignancies calls for investigation. In summary, our findings offer some indication that treatment of men with quinazoline-based !1-adrenoceptor antagonists for BPH and/or hypertension could have a substantial additional public health benefit by reducing the incidence of bladder cancer by 43%. The translational link between inhibition of angiogenesis and induction of bladder tumor cell anoikis by quinazoline !1adrenoreceptor antagonists, provides a biological basis for the development of effective chemoprevention strategies

for bladder cancer. Nested case-control, cohort, and randomized trials are required to confirm or reject the preventive effect of this class of quinazolines on bladder cancer incidence.

Acknowledgements This work was supported by a grant from the National Institutes of Health R01 CA10757-04 (NK). The authors acknowledge the expert assistance of Lorie Howard in the submission of the manuscript.

References Benning CM, Kyprianou N (2002) Quinazoline-derived alpha1adrenoceptor antagonists induce prostate cancer cell apoptosis via an alpha1-adrenoceptor-independent action. Cancer Res 62, 597-602. Bochner BH, Cote RJ, Weidner N, Groshen S, Chen SC, Skinner DG, Nichols PW (1995) Angiogenesis in bladder cancer: relationship between microvessel density and tumor prognosis. J Natl Cancer Inst 87, 1603-1612. Caine M (1990) !-adrenergic blockers for the treatment of benign prostatic hyperplasia. Urol Clin North Am 17, 641649. Chapple CR, Carter P, Christmas TJ, Kirby RS, Bryan J, Milroy EJ, Abrams P (1994) A three month double-blind study of doxazosin as treatment for benign prostatic bladder outlet obstruction. Br J Urol 74, 50-56. Chiang CF, Son EL, Wu GJ (2005) Oral treatment of the TRAMP mice with doxazosin suppresses prostate tumor growth and metastasis. Prostate 64, 408-418. Chon JK, Borkowski A, Partin AW, Isaacs JT, Jacobs SC, Kyprianou N (1999) !1-adrenoceptor antagonists terazosin and doxazosin induce prostate apoptosis without affecting cell proliferation in patients with benign prostatic hyperplasia. J Urol 161, 2002-2008. Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182-1186. Garrison JB, Kyprianou N (2004) Novel targeting of apoptosis pathways for prostate cancer therapy. Curr Cancer Drug Targets 4, 85-95. Garrison JB, Kyprianou N (2006) Doxazosin induces apoptosis of benign and malignant prostate cells via a death receptormediated pathway. Cancer Res 66:464-472. Garrison JB, Shaw YJ, Chen CS, Kyprianou N (2007) Novel quinazoline-based compounds impair prostate tumorigenesis by targeting tumor vascularity. Cancer Res 67, 1134411352. Grossmann J (2002) Molecular mechanisms of "detachmentinduced apoptosis--Anoikis". Apoptosis 7, 247-260. Harris AM, Warner BW, Wilson JM, Becker A, Rowland RG, Conner W, Lane M, Kimbler K, Durbin EB, Baron AT, Kyprianou N (2007) Effect of !1-adrenoceptor antagonist exposure on prostate cancer incidence: an observational cohort study. J Urol 178, 2176-2180. Hu ZW, Shi XY, Hoffman BB (1998) Doxazosin inhibits proliferation and migration of human vascular smoothmuscle cells independent of !1-adrenergic receptor antagonism. J Cardiovasc Pharmacol 31, 833-839. Hussain SA, James ND (2003) The systemic treatment of advanced and metastatic bladder cancer. Lancet Oncol 4, 489-497. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ (2007) Cancer statistics, 2007. CA Cancer J Clin 57, 43-66.

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Gene Therapy and Molecular Biology Vol 12, page 257 Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, Thun MJ (2006) Cancer statistics, 2006. CA Cancer J Clin 56, 106130. Keledjian K, Kyprianou N (2003) Anoikis induction by quinazoline based !1-adrenoceptor antagonists in prostate cancer cells: antagonistic effect of bcl-2. J Urol 169, 11501156. Keledjian K, Garrison JB, Kyprianou N (2005) Doxazosin inhibits human vascular endothelial cell adhesion, migration, and invasion. J Cell Biochem 94, 374-388. Kerr JF, Winterford CM, Harmon BV (1994) Apoptosis. Its significance in cancer and cancer therapy. Cancer 73, 20132026. Kintscher U, Kon D, Wakino S, Goetze S, Graf K, Fleck E, Hsueh WA, Law RE (2001) Doxazosin inhibits monocyte chemotactic protein 1-directed migration of human monocytes. J Cardiovasc Pharmacol 37, 532-539. Kintscher U, Wakino S, Kim S, Jackson SM, Fleck E, Hsueh WA, Law RE (2000) Doxazosin inhibits retinoblastoma protein phosphorylation and G(1)-->S transition in human coronary smooth muscle cells. Arterioscler Thromb Vasc Biol 20, 1216-1224. Kyprianou N (2003) Doxazosin and terazosin suppress prostate growth by inducing apoptosis: clinical significance. J Urol 169, 1520-1525. Kyprianou N, Benning CM (2000) Suppression of human prostate cancer cell growth by alpha1-adrenoceptor antagonists doxazosin and terazosin via induction of apoptosis. Cancer Res 60, 4550-4555. Kyprianou N, Jacobs SC (2000) Induction of apoptosis in the prostate by !1-adrenoceptor antagonists: a novel effect of "old" drugs. Curr Urol Rep 1, 89-96. Kyprianou N, Litvak JP, Borkowski A, Alexander R, Jacobs SC (1998) Induction of prostate apoptosis by doxazosin in benign prostatic hyperplasia. J Urol 159, 1810-1815. Lepor H, Auerbach S, Puras-Baez A, Narayan P, Soloway M, Lowe F, Moon T, Leifer G, Madsen P (1992) A randomized, placebo-controlled multicenter study of the efficacy and

safety of terazosin in the treatment of benign prostatic hyperplasia. J Urol 148, 1467-1474. McConnell JD, Roehrborn CG, Bautista OM, Andriole GL Jr, Dixon CM, Kusek JW, Lepor H, McVary KT, Nyberg LM Jr, Clarke HS, Crawford ED, Diokno A, Foley JP, Foster HE, Jacobs SC, Kaplan SA, Kreder KJ, Lieber MM, Lucia MS, Miller GJ, Menon M, Milam DF, Ramsdell JW, Schenkman NS, Slawin KM, Smith JA; Medical Therapy of Prostatic Symptoms (MTOPS) Research Group (2003) The long-term effect of doxazosin, finasteride, and combination therapy on the clinical progression of benign prostatic hyperplasia. N Engl J Med 349, 2387-2398. Pan SL, Guh JH, Huang YW, Chern JW, Chou JY, Teng CM (2000) Identification of apoptotic and antiangiogenic activities of terazosin in human prostate cancer and endothelial cells. J Urol 169, 724-729. Reed JC (1999) Dysregulation of apoptosis in cancer. J Clin Oncol 17, 2941-2953. Shaw YJ, Yang YT, Garrison JB, Kyprianou N, Chen CS (2004) Pharmacological exploitation of the !1-adrenoreceptor antagonist doxazosin to develop a novel class of antitumor agents that block intracellular protein kinase B/Akt activation. J Med Chem 47, 4453-4462. Streeter EH, Harris AL (2002) Angiogenesis in bladder cancer-prognostic marker and target for future therapy. Surg Oncol 11, 85-100. Tahmatzopoulos A, Lagrange CA, Zeng L, Mitchell BL, Conner WT, Kyprianou N (2005) Effect of terazosin on tissue vascularity and apoptosis in transitional cell carcinoma of bladder. Urology 65, 1019-1023. Walden PD, Durkin MM, Lepor H, Wetzel JM, Gluchowski C, Gustafson EL (1997) Localization of mRNA and receptor binding sites for the !1-adrenoceptor subtype in the rat, monkey and human urinary bladder and prostate. J Urol 157, 1032-1038. Wolf BB, Green DR (1999) Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 274, 20049-20052.

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Gene Therapy and Molecular Biology Vol 12, page 259 Gene Ther Mol Biol Vol 12, 259-266, 2008

Hematopoietic growth factors in the elderly Review Article

Wassim Mchayleh*, Rajesh Sehgal, James Natale, Gurkamal Chatta University of Pittsburgh Cancer Institute; Division of Hematology and Oncology.

__________________________________________________________________________________ *Correspondence: Wassim Mchayleh MD, University of Pittsburgh Cancer Institute; Division of Hematology and Oncology, 5150 centre avenue, Pittsburgh PA 15232, USA; Tel: 412-648-6431; Fax: 412-745-2245; e-mail: Mchaylehw@upmc.edu Key words: Elderly, Growth Factors, G-CSF, Erythropoeitin Abbreviations: acute myeloid leukemia, (AML); American Society of Clinical Oncology, (ASCO); bone marrow, (BM); end stage renal disease, (ESRD); erythropoietin, (EPO); granulocyte colony-stimulating factor, (G-CSF); Granulocyte-macrophage colony-stimulating factor, (GM-CSF); hematopoietic growth factors, (HGFs); hematopoietic stem cell, (HSC); hemoglobin, (Hgb); idiopathic thrombocytopenic purpura, (ITP); macrophage colony-stimulating factor, (M-CSF); mature leukocytes, (PMNs); megakaryocyte growth and differentiation factor , (MGDF); recombinant human G-CSF, (rhG-CSF); thrombopoietin, (TPO)

The authors have no relevant financial relationships.

Received: 13 May 2008; Revised: 17 June 2008 Accepted: 24 July 2008; electronically published: November 2008

Summary The Hematopoietic System is subject to the aging process. This translates in a blunted response to Hematopoietic stress in the elderly population. The clinical use of Hematopoietic growth factors (HGFs) has helped transform the care of the elderly cancer patient. The indications for the use of hematopoietic growth factors in the elderly population are no different from the general population. In fact, given the increased susceptibility of the elderly cancer patient to treatment related morbidity and mortality, there may be even more compelling reason for the use of growth factors, to obviate complications of myelosuppression. We review the biology of aging and hematopoiesis, and the indications for the use of HGFs in the elderly.

cancer patient. In this review, we will discuss: 1) the biology of aging and hematopoiesis, and 2) indications for the use of HGFs in the elderly.

I. Introduction Senescence of the lympho-hematopoietic system is associated with an increased incidence of neoplasia, autoimmune diseases and infections (Ben-Yehuda and Weksler, 1992). In fact, cancer and infections constitute the top two causes of mortality in the population over 65 years (Saltzman and Peterson, 1987!Ben-Yehuda and Weksler, 1992). Myelosuppression, either in the context of cancer chemotherapy or as a consequence of severe infections, is a particularly vexing problem in the elderly (Begg and Carbone, 1983). Pancytopenia is a common manifestation of myelosuppression and negatively impacts the prognosis in elderly patients with cancer by i) increasing infection and bleeding-related morbidity, and ii) preventing the administration of optimal dosages of chemotherapy. Although the physiologic basis of this blunted hematopoietic response remains unclear (Baldwin Jr, 1988; Lipschitz et al, 1984), recent insights into the biology of hematopoiesis, together with the availability in the clinic of a number of hematopoietic growth factors (HGFs), has helped transform the care of the elderly

II. Hematopoiesis and aging The orderly development of the hematopoietic system and the maintenance of homeostasis require that a strict balance be maintained between self-renewal, differentiation, maturation, and cell loss (Metcalf, 1988). Thus a small pool of stem cells can either self-renew or differentiate along one of several lineages to form mature leukocytes (PMNs), erythrocytes or platelets. One of the major questions with regard to the aging hematopoietic system is whether or not the pluripotent hematopoietic stem cell (HSC) has a finite replicative capacity. Evidence for a finite replicative capacity of the stem cell has been obtained both in in vitro long-term bone marrow (BM) cultures (Reincke et al, 1982), as well as in an elegant in vivo mouse model, where repeated total body irradiation was eventually able to induce hematopoietic exhaustion (Mauch et al, 1982). Thus, although finite, the lifespan of

259

Mchayleh et al: Hematopoietic growth factors in the elderly HSCs, is thought to be well in excess of the potential life span of a species. In humans, marrow progenitors can be enriched on the basis of surface markers expressed at sequential stages of maturation. Thus CD33 is found on most cells of the myeloid lineage in the marrow, and CD34 is expressed only by more primitive progenitors (1%-4% of the marrow cells). Precursors of myeloid colony-forming cells (preCFC) express CD34 and lack expression of CD33 and other antigens expressed by mature lymphoid and myeloid cells. Since CD34+ marrow cells can engraft and reconstitute hematopoiesis in lethally irradiated baboons and humans (Berenson et al, 1988, 1991), surface expression of CD34 on marrow and circulating cells, serves as a surrogate for stem cell function. Recent studies in murine and human models, however, have indicated that CD34(-) HSC exist as well, which possess engraftment potential and distinct HSC characteristics. These studies challenge the dogma that HSC are uniformly found in the CD34(+) subset, and question whether primitive HSC are CD34(+) or CD34(-). The question of whether HSCs are CD34+ or CD34remains unanswered (Engelhardt et al, 2002). The aging process primarily affects stimulus-driven hematopoiesis, with little or no impact on the basal state (Baldwin Jr, 1988). The blunted hematopoietic response to stress has been ascribed to age-related deficits in marrow progenitor cell numbers, changes in the marrow microenvironment, decreased production of regulatory growth factors, or a combination of these mechanisms (Hirota et al, 1988; Lee et al, 1989). However, in a number of areas the data are conflicting. This is partly due to tremendous heterogeneity of the aging process and partly a result of the difficulty in separating the effects of age per se from the effects of occult diseases (Pinto et al, 2003). Data from inbred aging animals reveal consistent agerelated defects, but human studies tend to show more variable results.

population. In a comprehensive review of the growth factor literature between 1987-1991, Shank Jr and Balducci, reported in 1992 that there was no age-related difference either in the mean time to response or in the level of absolute hematopoietic response at different doses of the growth factors in cancer clinical trials. In a prospective randomized study, the effects of recombinant human G-CSF (rhG-CSF) on the blood and marrow in 19 young and 19 healthy elderly volunteers were also evaluated (Chatta et al, 1994). No age-related compromise, either in the magnitude or in the timing of the PMN response to rhG-CSF, was found. PMN activation, both via opsonin receptor-dependent and receptor-independent pathways, was preserved with aging, and PMN kinetics were identical in the young and the elderly. Thus, the indications for the use of hematopoietic growth factors in the elderly population are no different from the general population and are briefly summarized (Table 1) (Repetto et al, 2003; Smith et al, 2006). In fact, given the increased susceptibility of the elderly cancer patient to treatment related morbidity and mortality, there may be even more compelling reason for the use of growth factors, to obviate complications of myelosuppression. Currently, of the various hematopoietic growth factors available clinically, the following may have special relevance for use in the elderly:

A. Granulocyte colony stimulating factor (Smith et al, 2006) G-CSF is a 24kD glycoprotein promoting the growth and maturation of myeloid cells and in particular, the proliferation and differentiation of neutrophil progenitors both in vitro and in vivo (Demetri and Griffin, 1991; Lieschke and Burgess, 1992a,b). There are two recombinant forms of G-CSF currently available. Filgrastim (NeupogenÂŽ) is a non glycosylated, smaller molecule than its endogeneous counterpart, but has the same biological activity (Osslund and Boone, 1994). Pegfilgrastim (Neulasta!) is pegylated formulation of GCSF, allowing for an increased plasma half-life permitting once a chemotherapy cycle (every 14 to 21 days) administration as opposed to daily administration with non-pegylated G-CSF. Pegfilgrastim has shown a comparable safety and efficacy profile to filgrastim in three randomized clinical trials (Holmes et al, 2002; Green et al, 2003; Vose et al, 2003). Current indications for the use of G-CSF include:

III. Aging and Hematopoietic Growth Factors Proliferation and differentiation of progenitor cells to become mature blood cells requires intimate contact between stem cells, stromal cells and the extracellular matrix, and is mediated by the (HGFs) (Clark and Kamen, 1987; Metcalf, 1988; Bagby and Segal, 1995). The HGFs, on the basis of their action, are characterized either as multi-lineage hematopoietins, e.g., stem cell factor (SCF) (Williams et al, 1990; Broudy, 1997) or as lineagerestricted hematopoietins, e.g., granulocyte colonystimulating factor (G-CSF), macrophage colonystimulating factor (M-CSF), erythropoietin (EPO), and thrombopoietin (TPO) (Kaushansky et al, 1994; Spivak, 1998). In addition to the above growth factors, lymphohematopoiesis is modulated by an ever-expanding list of other cytokines, i.e., the interleukins. Age-related deficits tend to be subtle and are of clinical import either when present cumulatively or under conditions of hematopoietic stress (Baldwin Jr, 1988; Pinto et al, 2003). Relatively few studies have specifically addressed the use of growth factors in the elderly

1. Treatment neutropenia

chemotherapy-related

Crawford and coleagues in 1991 and Trillet-Lenoir and coleagues in 1993 were among the first to show that in patients receiving chemotherapy for lung cancer, concurrent administration of G-CSF (5 Âľg/kg/day) reduced the duration of neutropenia, decreased the incidence of febrile neutropenia, infections, antibiotic use, and hospitalization by approximately 50%. The current 2006 American Society of Clinical Oncology (ASCO) guidelines suggest reserving primary prophylaxis (ie, the use of hematopoetic growth factors with the first cycle and all subsequent cycles of chemotherapy) with HGFâ&#x20AC;&#x2122;s 260

Gene Therapy and Molecular Biology Vol 12, page 261 Table 1. Growth Factors in the Elderly Growth Factor 1-Granulocyte Colonystimulating factor(G-CSF):

Dosage and Administration

1-Filgrastim(Neupogen!)

- Neutropenia: - 5mcg/kg/d sc (rounded to the nearest vial size), starting 24 to 72 hours after stopping hemotherapy and continuing until ANC > 1000 (shorter durations have been suggested) - PBSCT mobilization 10mcg/kd/d or 58mcg/kg/ bid have been used. Optimal duration and timing not established.

2-Pegfilgrastim(Neulasta!):

- Neutropenia: - 6mg sc once per cycle (minimal 2 weeks between doses) - PBSCT mobilization: Ongoing studies.

Indications

• Chemotherapy related neutropenia • Chronic and drug induced neutropenia • Peripheral blood stem cell transplantation • Myelodysplasia

Growth Factor 2-Erythropoietin( EPO) : 1-RhEpo (Procrit!):

Dosage and Administration

Indications

150 units/kg sc/IV three times weekly or 40,000-60,000 units weekly

2-Darbepoetin (Aranesp!):

2.25 mcg/kg sc/IV weekly or 500mcg sc/IV every three weeks

• Renal disease anemia • Anemia in cancer patients • Myelodysplasia

3-Granulocyte-Macrophage Colony-stimulating factor (GMCSF): Sargramostim (Leukine!):

250-500 mcg/kd/d sc/IV (rounded to the nearest vial size), starting 24 to 72 hours after stopping chemotherapy and continuing until ANC > 1500/microL for two consecutive days.

administration for: a) high risk patients with an expected incidence of febrile neutropenia of 20%; b) patients at risk of increased complications from prolonged neutropenia (ie, elderly, poor performance status); and c) patients receiving dose dense chemotherapy (Smith et al, 2006). This is a change from ASCO’s previous published guidelines which used a 40% incidence of febrile neutropenia as the trigger for primary prophylaxis (Vogel et al, 2005; Smith et al, 2006). In regards to patients at greater risk of complications due to prolonged neutropenia, aging (> 60-70yo) has been shown to be an independent risk factor for development of febrile neutropenia (Dees et al, 1984; Crivellari et al, 2000; Gelman and Taylor, 2004; Kim et al, 2007), as well as incurring a higher mortality rate from the complications of neutropenic infections (Armitage and Potter, 2003; Doordujn et al, 2007; Gomez et al, 2007). Moreover, in two large retrospective reviews of lymphoma and breast cancer, age > 60-65 yo was an independent predictor of receiving less than 85% relative dose intensity (RDI-

with

• As for G-CSF • Vaccine adjuvant

defined as the delivered dose intensity over the standard dose intensity multiplied by 100) (Lyman et al, 2003; 2006). Thus, these studies have shown elderly cohorts to receive either lower doses or the same dose over a longer period of time than their younger counterparts. The significance of these dose reductions and delays, at least in the lymphoma and breast cancer populations, has been relevant reductions in overall survival (Kwak et al, 1990; Bonadonna et al, 1995). Based on these data, the latest ASCO guidelines have added recommendations for the use of HGF’s in older patients. The recommendation includes the use of primary prophylaxis with HGF’s in patients with lymphoma over the age of 65 receiving chemotherapy with a curative intent, regardless of the threshold risk of neutropenia based on the individual regimen used. The guidelines also comment on the practice of dose reducing or delaying in this population of lymphoma patients, stating this practice is no longer recommended. Instead the use of HGF’s to maintain dose intensity (giving the planned dose at the planned time

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Mchayleh et al: Hematopoietic growth factors in the elderly interval), is a more reasonable strategy even in elderly patients receiving moderate-low myelosuppressive chemotherapy regimens.

AML in the elderly. The use of GM-CSF as a vaccine adjuvant, in particular its ability to recruit dendritic cells to the site of injection is under investigation.

2. Mobilization of PBSC for hematopoietic reconstitution (Fennelly et al, 1994; Tricot et al, 1995)

D. Erythropoietin (Rizzo et al, 1993; Spivak, 1998) EPO has been in clinical use since 1985 for patients with end stage renal disease (ESRD). Erythropoiesis can be stimulated by exogenous administration of two FDA approved agents: the recombinant human erythropoietin (Epogen®or Procrit®), and darbepoetin alfa (Aranesp®). The latter is more heavily glycosylated and longer acting than the former. In the elderly, EPO may be indicated under the following circumstances:

The use of G-CSF for the purpose of mobilizing stem cells into peripheral blood for subsequent use in autologous bone marrow transplantation is now established. In the elderly, hematopoietic support with PBSC may prove useful in the setting of high-dose chemotherapy for cancers resistant to standard chemotherapy, i.e., multiple myeloma and non-hodgkin's lymphoma. The dose of G-CSF for mobilizing PBSC ranges between 10-30 µg/kg/day. A study analyzed 150 patients with AML comparind CD34+ cells mobilization in patients older or younger than age 60. The successful mobilization rate (>2 x 10(6) CD34+ cells/kg) was comparable between the two groups (87% vs. 80%, p = 0.29). In addition, no statistically significant difference was found in terms of either median number of CD34+ cells collected (Ferrara et al, 2007).

1. ESRD on dialysis (Paganini and Miller, 1993; Locatelli et al, 2001) The usual dose of EPO is 75-100 U/kg; administered three times per week during the last 5 minutes of dialysis. On this dose, 95% of patients reach the target hemoglobin (> 11gm% and < 12 gm%) in 10-12 weeks, have marked reduction in transfusion dependency, and have beneficial cardiac, neurovascular, immunologic, and psychosocial effects. For a normal response to EPO, the patient must have adequate iron stores and red cell folate. Although EPO is well tolerated, dialysis patients on EPO have a higher risk of hypertension, seizures, cerebrovascular disease, and thrombo-embolic episodes. Since the elderly are more prone to all the above diseases, older patients on EPO need to be monitored closely. The less frequent darbepoetin alfa dosing schedule of once weekly or once every two weeks, with the possibility of monthly dosing in some patients, offers many potential benefits to both patients and caregivers.

3. In hematological malignancis (Rowe et al, 1991; Dombret et al, 1994; Hiddemann et al, 1995; Stone et al, 2004) Because of the presence of growth factor receptors on malignant myeloid cells, exacerbation of the underlying leukemia is a concern. However, in a number of studies, where G-CSF was used in the setting of myelodysplasia and acute myeloid leukemia (AML), there was no evidence of tumor stimulation. Most cases of AML occur in patients over the age of 60 years, usually in the setting of complex and unfavorable cytogenetic abnormalities. Compared with younger patients, the elderly have a lower response rate to induction therapy, a reduced probability of remaining in remission, and a lower cure rate. Furthermore, mortality in the cytopenic phase of treatment is also in the order of 30-40% in the elderly. The experience of three multi-center clinical trials using either G-CSF or GM-CSF in elderly patients with AML has been reported. The results of these studies are conflicting, and at this point no firm recommendations can be made. However, stimulation of the leukemic clone was not noted with either of the growth factors.

2. Anemia in cancer patients (Spivak, 2001, 2005) Anemia is a common finding in patients with cancer. Usually it is multifactorial, being related to chronic disease, as well as cancer progression (marrow involvement) and cancer treatment (chemotherapy). Multiple studies have shown that patients with a hemoglobin (Hgb) level of 11 gm% benefit from epoetin therapy: they require fewer blood transfusions and have an enhanced sense of well-being. However Hgb levels consistently > 12 gm%, have been associated with vascular side-effects and a shorter life expectancy. More recent data on the effects of recombinant human erythropoietin and darbepoetin and tumor progression and thrombosis (Henke et al, 2003; Leyland-Jones et al, 2005; Bohlius et al, 2006; Wright et al, 2007; Unpublished results of the DAHANCA 10 Trial, 2007), have lead to changes in the guidelines from the American Society of Clinical Oncology and the American Society of Hematology. This data clearly shows that targeting Hb concentrations of greater than 12 gm/dl increases the risk of thromboembolic events and potentially stimulates tumor growth. At this point, no conclusive evidence exists that these potential deleterious effects occur if current ASCO/ASH and FDA-approved dosing guidelines and

B. Granulocyte-macrophage colonystimulating factor (GM-CSF) (Rowe et al, 1991; Nemunaitis et al, 2002; Witz et al, 2004) GM-CSF, is a glycosylated peptide of 22kD, which has a broader range of cellular targets than G-CSF. The two forms of recombinant human GM-CSF currently in use are sargramostim(Leukine®), and molgramostim (nonglycosylated or E coli -derived). The effects of GM-CSF both in causing neutrophilia and in mobilizing PBSC are very similar to those of G-CSF. GM-CSF was first reported to enhance marrow recovery in the setting of autologous marrow transplantation for lymphoid malignancies. Currently GM-CSF is approved for use in 262

Gene Therapy and Molecular Biology Vol 12, page 263 adults with de novo acute myeloid leukemia. Blood 94, 3694-3701. Armitage JO, Potter JF (1984) Aggressive chemotherapy for diffuse histiocytic lymphoma in the elderly: Increased complications with advancing age. J Am Geriat Soc 32, 269-273. Bagby G, Segal G (1995) Growth factors and the control of hematopoiesis. In: Hoffman R, Benz EJ Jr, Shanttil SJ, et al, eds. Hematology Basic Principles and Practice. 2nd edition.New York, NY: Churchill Livingstone, 207. Baldwin JG Jr (1988) Hematopoietic function in the elderly. Arch Intern Med 148, 2544-2546. Begg CB, Carbone PP (1983) Clinical trials and drug toxicity in the elderly. The experience of the Eastern Cooperative Oncology Group. Cancer 52, 1986-1992. Ben-Yehuda A, Weksler ME (1992) Host resistance and the immune system. Clin Geriatr Med 8, 701-711. Berenson RJ, Andrews RG, Bensinger WI, Kalamasz D, Knitter G, Buckner CD, Bernstein ID (1988) Antigen CD34+ marrow cells engraft lethally irradiated baboons. J Clin Invest 81, 951-955. Berenson RJ, Bensinger WI, Hill RS, Andrews RG, GarciaLopez J, Kalamasz DF, Still BJ, Spitzer G, Buckner CD, Bernstein ID (1991) Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 77, 1717-1722. Bohlius J, Wilson J, Seidenfeld J, Piper M, Schwarzer G, Sandercock J, Trelle S, Weingart O, Bayliss S, Djulbegovic B, Bennett CL, Langensiepen S, Hyde C, Engert A (2006) Recombinant human erythropoietins and cancer patients: updated meta-analysis of 57 studies including 9353 patients. J Natl Cancer Inst. 98, 708. Bonadonna G, Valagussa P, Moliterni A, Zambetti M, Brambilla C (1995) Adjuvant Cyclophosphamide, Methotrexate and fluorouracil in node-positive breast cancer. N Engl J Med. 332, 901-906. Broudy VC (1997) Stem cell factor and hematopoiesis. Blood 90, 1345-1364. Bussel J, Kuter D, George J, Aledort L, Lichtin A, Lyons R, Nieva J, Wasser J, Bourgeois E, Kappers-Klunne M, Lefrere M, Schipperus M, Kelly R, Christal J, Guo M, Nichol J (2005) Long-term dosing of AMG 531 is effective and well tolerated in thrombocytopenic patients with immune thrombocytopenic purpura (Abst 511). Blood 106, 68a. Chatta GS, Price TH, Allen RC, Dale DC (1994) Effects of in vivo recombinant methionyl human granulocyte colonystimulating factor on the neutrophil response and peripheral blood colony-forming cells in healthy young and elderly adult volunteers. Blood 84, 2923-2929. Clark SC, Kamen R (1987) The human hematopoietic colonystimulating factors. Science 236, 1229-1237 Crawford J, Ozer H, Stoller R, Johnson D, Lyman G, Tabbara I, Kris M, Grous J, Picozzi V, Rausch G (1991) Reduction by granulocyte colony-stimulating factor of fever and neutropenia induced by chemotherapy in patients with smallcell lung cancer. N Engl J Med 325, 164-170. Crivellari D, Bonetti M, Castiglione-Gertsch M, Gelber RD, Rudenstam CM, Th端rlimann B, Price KN, Coates AS, H端rny C, Bernhard J, Lindtner J, Collins J, Senn HJ, Cavalli F, Forbes J, Gudgeon A, Simoncini E, Cortes-Funes H, Veronesi A, Fey M, Goldhirsch A (2000) Burdens and benefits of adjuvant cyclophosphamide, methotrexate, and fluorouracil and tamoxifen for elderly patients with breast cancer: The International Breast Cancer Study Group Trial VII. J Clin Oncol. 18, 1412-1422. Dees EC, O'Reilly S, Goodman SN, Sartorius S, Levine MA, Jones RJ, Grochow LB, Donehower RC, Fetting JH (2000) A prospective pharmacologic evaluation of age-related toxicity

modifications are followed. The guidelines recommend use of epoetin as a treatment option for patients with chemotherapy-associated anemia with a Hgb concentration at or below 10 gm%. Subcutaneous epoetin can be used thrice weekly (150 IU/kg) for a minimum of 4 weeks. Alternatively, a weekly (40,000 IU/wk) dosing regimen is also reasonable. Current FDA-approved starting dose of darbepoetin is 2.25mcg/kg weekly or 500mcg every 3 weeks. However, the ASCO/ASH guidelines recognize alternative darbepoietin dosing as every two weeks at a dose of 200 mcg, or every 3 weeks at a dose of 300 to 400 mcg. Both agents should be titrated to a target hemoglobin concentration at or near 12 g/dL. Evidence from one randomized controlled trial supports use of epoetin for patients with anemia associated with low-risk myelodysplasia. Anemic patients with hematologic malignancies should be first treated with conventional therapy and monitored for a hematologic response, prior to considering the use of recombinant erythropoietic agents (Rizzo et al, 1993).

4. Thrombopoietin (Kaushansky et al, 1994; Archimbaud et al, 1999) The gene for thrombopoietin was cloned 12 years ago, but the protein has not yet been approved for clinical use. It is likely that thrombopoietin will be useful for accelerating platelet recovery after intensive chemotherapy. It can augment the number of hematopoietic stem cells mobilized by G-CSF, increase the number of platelets available for apheresis from platelet donors, and stimulate platelet production in patients with myelodysplastic syndromes, idiopathic thrombocytopenic purpura (ITP), or liver disease. Thrombopoietin was initially called megakaryocyte growth and differentiation factor (MGDF) and was studied in several clinical trials prior to 1998. However its use was associated with the production of antibodies against MGDF that cross-reacted with endogenous thrombopoietin, causing severe thrombocytopenia (Li et al, 2000). This adverse event led to the abandonment of the use of MGDF and full-length forms of thrombopoietin as therapeutic proteins. Recent studies using small-molecules that mimic thrombopoietin in their ability to bind and stimulate the thrombopoietin receptor in ITP are ongoing (Bussel et al, 1984).

IV. Conclusions The feverish pace of growth factor research and the arrival in the clinic of various growth factors have transformed the practice of modern day hematologyoncology. For the elderly cancer patient, there are now available newer, more effective and safer therapies with tremendous potential.

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Gene Therapy and Molecular Biology Vol 12, page 267 Gene Ther Mol Biol Vol 12, 267-276, 2008

HSP70 variations in the acute treatment with mood stabilizers in patients with bipolar disorder: results of a preliminary work Research Article

Chi-Un Pae1,2,*, Antonio Drago3, Jung-Jin Kim1, Laura Mandelli3, Ashwin A. Patkar2, Diana De Ronchi3, Alessandro Serretti3,* 1

Department of Psychiatry, Kangnam St. Mary’s Hospital, The Catholic University of Korea College of Medicine Department of Psychiatry and Behavioural Sciences, Duke University 3 Institute of Psychiatry, University of Bologna, Italy 2

__________________________________________________________________________________ *Correspondence: Chi-Un Pae, MD, Department of psychiatry, Kangnam St. Mary’s Hospital, The Catholic university of Korea College of Medicine, 505 Banpo-Dong, Seocho-Gu, Seoul 137-701, Korea; Tel: 82-2-590-1532; Fax: 82-2-536-8744; Email: pae@catholic.ac.kr Alessandro Serretti, MD, Institute of Psychiatry, University of Bologna, Viale Carlo Pepoli 5, 40123 Bologna, Italy; Tel: 39-0516584233; Fax: 39-051-521030; e-mail: alessandro.serretti@unibo.it Key words: Pharmacogenetics, lithium, heat shock proteins, bipolar disorder, manic Abbreviations: analysis of covariance, (ANCOVA); Analysis of variance, (ANOVA); bipolar I disorder, (BID); brain derived neurotrophic factor, (BDNF); Clinical Global Impression, (CGI); DSM-IV Axis I disorders-Clinical Version, (SCID-CV); endoplasmic reticulum, (ER); heat shock protein, (HSP); HSP70 family, (HSPA1L); hystone deacetylase, (HDAC); linkage disequilibrium, (LD); small heat-shock proteins, (sHSPs); Young Mania Rating Scale, (YMRS) Received: 7 November 2008; Revised: 11 November 2008 Accepted: 12 November 2008; electronically published: November 2008

Summary A pharmacogenetic approach was used to investigate the role of heat shock protein (HSP) 70 on the effect of mood stabilizers since a line of evidence has proposed a possible involvement of its chaperone activity in the pathophysiology of bipolar disorders. Forty five patients with bipolar I disorder were treated for an average of 36.5 (±19.9) days with mood stabilizers (lithium, valproate, or carbamazepine), were evaluated with using the Clinical Global Impression (CGI) scale and the Young Mania Rating Scale (YMRS), and were genotyped for their HSP 70 variants (rs2227956 C/T, rs2075799 A/G, rs1043618 C/G, rs562047 C/G, rs539689 C/G). Results: No association was found between the investigated variations and response to mood stabilizer treatments even considering possible stratification factors. The small number of subjects is an important limitation to the present study, nonetheless HSP 70 gene variants seem not to be involved in acute antimanic effect. Adequately-powered study would properly address the potential role of HSP 70 gene variants for the effect of mood stabilizers.

effect of lithium has recently been reported (Maj, 1992; Grof et al, 1994; Alda, 1999; Serretti et al, 2002a; Serretti et al, 2002b; Serretti and Artioli, 2003). A list of pivotal genes that are probably involved in the action mechanism of mood stabilizers such lithium has been recently identified. Although such results are still inconsistent, the findings are presented in Table 1. The antimanic effects of carbamazepine and valproate have been less widely investigated comparing with lithium. Nevertheless, their prophylactic action has been associated with some genes or proteins as listed in Table 2, giving some rational for a pharmacogenetic approach

I. Introduction Lithium, valproate and carbamazepine are the first line agents for both acute and long-term treatments of bipolar disorder (American Psychiatric Association, 2002). Given the paucity of clinical predictors of treatment response (Maj, 1992; Kleindienst et al, 2005), genetic predictors would be of a great help to clinicians. Bipolar disorder itself as well as antidepressant and antipsychotics may be influenced by genetic factors (Malhotra et al, 2004; Althoff et al, 2005; MacQueen et al, 2005; Serretti et al, 2005; Savitz and Ramesar, 2006). Moreover, a partial genetic control over the long term prophylactic 267

Pae et al: HSP70 variations in the acute treatment with mood stabilizers in patients with bipolar disorder Table 1. Pharmacogenetic studies on lithium prophylactic action References

(Mamdani et al, 2007)

(Michelon et al, 2006) (Szczepankiewicz et al, 2006) (Masui et al, 2006b) (Masui et al, 2006a)

(Benedetti et al, 2005) (Rybakowski et al, 2005a) (Rybakowski et al, 2005b)

Gene

Gene Variant

BDNF, INPP1, AP2!, SERTPR

rs3799990; rs3818281; rs1149320; rs6902415; rs12192054; rs1028792; rs9486069; rs9500087; rs720225 G196A, C973A, VNTR, s-l

GSK-3!

T-50C

BDNF

Val66Met

Xbp-1

-116C/G

PREP

GSK3-!

-50 T/C

SERTPR

s-l

BDNF

Val66Met and 270C/T

IMPA2

8 SNPs !

(Dimitrova et al, 2005)

(Serretti et al, 2004) (Washizuka et al, 2003) (Serretti et al, 2002b) (Serretti et al, 2001)

(Serretti et al, 2000) (Serretti et al, 1999b) (Serretti et al, 1999a) (Steen et al, 1998) (Serretti et al, 1998)

Subjects

Length of observation

Results

Caucasian 249 BID 126 controls

13.25Âą7.26 years

No significant association

Caucasian 134 BID Caucasian 89 BID Japanese 83 BPI 78 BD II Japanese 83 BPI 78 BD II Caucasians 88 BID Caucasians 67 BID Caucasians 88 BID Caucasian 237 parentsoffspring trios 174 BID 170 controls Caucasian 83 BID

2 years 5 years 1 year 1 year More than 2 years before and 2 years on lithium treatment More than 5 years 5-27 years (mean 15 years)

3 years

SERTPR

s-l !

mtDNA

10398 A/G

COMT, MAO-A, G!3

G158A, 30-bp repeat, C825T

SERTPR

s-l

HTR2A, HTR2C, HTR1A

T102C, C-1420T, Cys23Ser, C1019G

Caucasian 102 BID

4.3 years

TPH

A218C; A779C

Caucasian 90 BID

4.2 years

DRD2, DRD4, GABRA1

SerCys, VNTR, VNTR

INPP1

C973A

DRD3

BalI

Japanese 54 BID Caucasian 160 BID Caucasian 167 BID

Caucasian 100 BID Caucasian pilot bipolar sample Caucasian 43 BID

3 years

4.4 - 5.6 years 4.9 years 4.85 years

4.41 years / 4.08 years

No significant association No significant association No significant association C associated with better response C associated with better response S associated with worse response Met and T associated with better response No significant association l/l associated with poor response l/s associated with better response A associated with better response No significant association s/s associated with worse response No significant association TPH*A/A variant showed a trend toward a worse response No significant association C973A transversion was present in responders No significant association

AP-2 = activator protein-2; BDNF = brain-derived neurotrophic factor; COMT = catechol-O-methyltransferase; DRD2 = dopamine receptor D2; DRD3 = dopamine receptor D3; DRD4 = dopamine receptor D4; GABRA1 = GABA(A) receptor "1 subunit; GSK3-! = glycogen synthase kinase !3; HTR1A = serotonin receptor 1A; HTR2A = serotonin receptor 2a; HTR2C = serotonin receptor 2C; IMPA2 = Inositol Monophosphatase 2; INPP1 = inositol polyphosphate 1-phosphatase INPP1 = inositol polyphosphate 1-phosphatase; MAO-A = monoamine oxidase A; mtDNA = mitochondrial DNA; PREP = prolyl endopeptidase; SERTPR = promoter of serotonin transporter gene; TPH = tryptophan hydroxylase; Xbp-1 = X-box binding protein 1

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Gene Therapy and Molecular Biology Vol 12, page 269 Table 2. Potential candidate targets for the action mechanism of mood stabilizers. References (Kazuno et al, 2007) (Rao et al, 2007) (Montezinho et al, 2006) (Phiel et al, 2001) (Sharma et al, 2006) (Gurvich et al, 2005) (Dokucu et al, 2005) (Zhou et al, 2005) (Chen et al, 2005) (Shaltiel et al, 2004) (Chetcuti et al, 2006) (Ju and Greenberg, 2003) (Bown et al, 2002) (Shao et al, 2006) (Okada et al, 2004) (Lagace et al, 2004) (Sullivan et al, 2004) (Du et al, 2004) (Nelson-DeGrave et al, 2004) (Yildirim et al, 2003) (Sands et al, 2000) (Chen et al, 1999b)

Drugs Valproate Carbamazepine Carbamazepine; Valproate Valproate Valproate Valproate Valproate; Lithium Valproate; Lithium Valproate Valproate Valproate Valproate Valproate Valproate; Lithium Valproate Valproate Valproate Valproate; Lithium Valproate

Targets Mitochondrial DNA AP-2 DNA-binding activity; AP-2 " protein expression dopamine D2-like and !-adrenergic receptors HDACs acH3; acH4 HDACs GSK-3! BAG-1 B56! regulatory subunits; transcriptional coactivator p300 inositol biosynthesis ZIC1; SFMBT2; SCM4L1; PAR-4 Inositol; phospholipid biosynthesis ER stress proteins ER stress proteins Pc-G leptin secretion; leptin messenger ribonucleic acid HTR2A AMPA glutamate receptor androgen

Valproate Valproate Valproate

acH; 5-lipoxygenase TH mRNA AP1 family of transcription factors

Abbreviations: acH = acetylated Histone protein; AP-2 = phospholipase A2; BAG-1 = glucocorticoid receptor cochaperone protein; ER = endoplasmic reticulum; GSK-3!= glycogen synthase kinase !3; HDACs = histone deacetylases; HTR2A = serotonin receptor 2A; PAR-4 = prostate apoptosis response-4; Pc-G = Polycomb group; SCM4L1 = structural maintenance of chromosome 4-like 1; SFMBT2 = Scm-related gene containing four mbt domains; TH = tyrosine hydroxylase; ZIC1 = zinc finger protein of the cerebellum 1

onto their antimanic effects. This field of research is not only difficult but also intriguing since the specific action mechanisms leading to mood stabilization still need to be identified (Chen et al, 1999a; Brunello and Tascedda, 2003; Harwood and Agam, 2003; Einat and Manji, 2006; Kazuno et al, 2007). Generally, the inheritance of acute antimanic response remains still unclear (Dooley and Andermann, 1989; Skarpa et al, 1994; Hwang et al, 1998). Even though the prophylactic action of mood stabilizers has been widely investigated, pharmacogenetic studies investigating the acute antimanic effect are lacking till today. The first step in this research is to identify which molecular mechanisms are relevant to the antimanic effects of mood stabilizers. Within the variety of theories dealing with mood stabilizers pharmacodynamic aspects (Harwood and Agam, 2003), the neuroprotective pathway seems to play a relevant role (Jope, 1999). Consistently, early effects of lithium and valproate over cellâ&#x20AC;&#x201C;life promoting mechanisms have been reported as follows: influence on apoptotic or antiapoptotic mechanisms, action on glutamate induced reactions, induction of brain derived neurotrophic factor (BDNF) cascade (i.e., BDNF, TrkB), stimulation of neuroblasts proliferation, stabilization of lysosomal membrane, hystone deacetylase (HDAC) inhibition, as well as stimulation of heat shock factor 1, and inhibition of glycogen synthase kinase 3. Chaperones associated with endoplasmic reticulum (ER) including

GRP78, GRP94, PDI, calreticulin, caspase 3 together with the cytosolic chaperones belonging to the heat shock protein (HSP) family, may be involved in the pathways where are related to the effects of mood stabilizers (Bown et al, 2000, 2002; Chen et al, 2000; Ren et al, 2003, 2004; Chuang, 2004, 2005; Hiroi et al, 2005; Kim et al, 2005; Pan et al, 2005; Sinn et al, 2007). Moreover, neuroprotective action of lithium and valproate has been also demonstrated in vivo (Ren et al, 2003; Chuang, 2004; Chuang, 2005; Xu et al, 2006; Sinn et al, 2007). All the above mentioned proteins could represent good targets for pharmacogenetic research on the acute antimanic effect of mood stabilizers. In fact, even not a classical neurodegenerative disease, bipolar disorder has been recently found to be associated with a neurodegenerative pathophysiology (Savitz et al, 2005). Moreover, cellâ&#x20AC;&#x201C;life promoting events are at reasonably responsible for the neuronal plasticity and resilience, which have been also recognized to be relevant with bipolar disorder (Chen et al, 1999a; Ikonomov and Manji, 1999; Manji et al, 1999, 2000). In this direction, HSPs are essential neuroprotective proteins, they have been proven to be influenced by lithium and valproate and they were recently proposed as possible state markers of acute manic episode (Shen et al, 2006). Moreover, there is some evidence that HSPs are associated with other psychiatric disorders as well (Shimizu et al, 1996; Pae et al, 2005; Pae et al, 2007).

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Pae et al: HSP70 variations in the acute treatment with mood stabilizers in patients with bipolar disorder (Biotage AB, Sweden) and one primer of each primer set was biotinylated. Genotyping was confirmed by J.J.K.

Taken together above mentioned, we employed a pharmacogenetic approach to investigate the hypothesis of an involvement of HSPs in acute antimanic effects of mood stabilizers. A set of genetic variations (rs2227956 C/T, rs2075799 A/G, rs1043618 C/G, rs562047 C/G, rs539689 C/G) was studied on the basis of previous results (Pae et al, 2005).

D. Statistical analyses Haploview 3.2 was used to generate a linkage disequilibrium (LD) map and to test for Hardy-Weinberg equilibrium. Single genotype associations with YMRS and CGI scores were analyzed by the Analysis of variance (ANOVA); when including covariates or other factors, the analysis of covariance (ANCOVA) and the multivariate analysis of co/variance (MANOVA/MANCOVA) were employed. Baseline scores were included as covariates plus the clinical variables associated with genotypes. Associations with other clinical variables in the subjects were performed by the ANOVA or the Chi-square test. The “R” software (“A Programming Environment for Data Analysis and Graphics” Version 2.2.1) was employed to analyze haplotype with both discrete and continuous traits and to include covariates. Permutation (50,000 permutations) was used to estimate the global significance of the results for haplotype analyses to confirm the expectationmaximization values. Results were considered significant with an " level lower than 0.05. With this level of significance, for single marker allele analyses, we had a power of 0.80 to detect a medium-large effect size of d=0.86, which corresponded to a difference of approximately 4.5% in the YMRS improvement between two main genotype variants and corresponded to an explained variance of about 15.6% (Cohen, 1988).

II. Materials and Methods A. Subjects The sample was composed by 45 patients (20 males) suffering from bipolar I disorder (BID, diagnosed with a consensus between C.U.P. and J.J.K.), and scoring at least 13 in YMRS at baseline. The patients were assessed using a Structured Clinical Interview, DSM-IV Axis I disorders-Clinical Version (SCID-CV) and patients with comorbid Axis I disorder other than BID were excluded. Subjects with neurological and current medico-surgical illness were also excluded. Patients were administered the Clinical Global Impression (CGI) (Guy, 1976) scale and the Young Mania Rating Scale (YMRS) (Young et al, 1978) for evaluation of the effectiveness of mood stabilizers. Patients were assessed with such effectiveness measures at the time of admission and discharge. All subjects were biologically unrelated, native Korean descendants residing in Korea. Written informed consent was provided by the subjects after being explained the purpose and method of the study. The institutional review board of Kangnam St. Mary’s Hospital approved the study that was conducted in accordance with the Declaration of Helsinki.

III. Results Subjects description is presented in Table 3. A significant % reduction in YMRS score was reported (64.2 ± 4.6) for the whole subjects; lithium, valproate and carbamazepine treated subjects with reduction in YMRS score of 64.9±4.7, 64.2±2.8 and 60.7±5.8, respectively. The present study was not designed to investigate the different efficacy of the single drugs, so we did not perform any statistical investigation in that direction, however the effects were very similar. Correlation analysis was performed between clinical variables and % reductions in the scores of YMRS and CGI. We observed a significant positive correlation (R Spearman = 0.63; p < 0.0001) between YMRS scores at baseline and % reduction in YMRS score by the end of treatment. We also observed an inverse association with age at onset and the % reduction in YMRS score (R Spearman = -0.29 p = 0.05). Otherwise were all nonsignificant. When considering the dichotomic variable “remitters” or “non remitters” results were also similar (data not shown). As regard to the allelic analysis on the HSPs, all 45 subjects were successfully genotyped. All markers were in Hardy-Weinberg equilibrium (rs2227956 p=1.0, rs2075799 p=1.0, rs1043618 p=1.0, rs562047 p=1.0, rs539689 p=0.6). Comparing the allele frequencies with the international databases, all the investigated SNPs reported expected frequencies. No significant association was found between the antimanic effect or manic score at baseline and the investigated genetic variations even after covariate analyses (sex, age, age at onset, YMRS scores at baseline). A strong LD was found between SNPs rs2227956, rs2075799, rs1043618 and between SNPs rs562047 and rs539689 (>0.8). Haplotype analysis gave no

B. HSP variants investigated HSPs are coded by different genes and are clustered by their molecular weights: the most important classes are HSP100, HSP90, HSP70, HSP60, HSP33, and the small heat-shock proteins (sHSPs). One of the best-characterized chaperones belong to the HSP70 family (Pilon and Schekman, 1999; Hartl and Hayer-Hartl, 2002). HSP70 have structural and functional properties in common, but vary in their inducibility in response to metabolic stress. In the class III region of the major histocompatibility complex on 6p21.3 was identified a duplicated HSP70 locus (Sargent et al, 1989), named HSPA1A and HSPA1B. Both encode identical 641-amino acid proteins but the 3’ untranslated regions of these genes are divergent. Northern blot analysis of HeLa cell RNA detected an approximately 2.4-kb HSPA1B transcript that was expressed at elevated levels following heat shock. Sargent et al. also identified a region with similarity to HSPA1A located approximately 4 kb telomeric to HSPA1A; this homologous region has been defined as a gene of the HSP70 family (HSPA1L) (Milner and Campbell, 1992). In the present paper we investigated a set of polymorphisms concerning HSP70, within the mentioned HSPA1A, HSPA1B and HSPA1L genes.

C. Genetic analyses Genomic DNA was extracted from venous blood by standard methods and quantified. The high-throughput genotyping method using pyrosequencer (Biotage AB, Sweden) was used for genotyping 5 HSP 70 SNPs (rs2227956 C/T, rs2075799 A/G, rs1043618 C/G, rs562047 C/G, rs539689 C/G). A set of genetic variations were selected based on public database (National Center for Biotechnology Information, dbSNP, http://www.ncbi.nlm.nih.gov/SNP/). PCR primers (Bioneer, Daejeon, Korea) and sequencing primers (Bioneer, Daejeon, Korea) used for the Pyrosequencing assay were designed by using the Pyrosequencing Assay Design Software v1

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Gene Therapy and Molecular Biology Vol 12, page 271 Table 3. Demographics of the subjects in the study. Variable

Results Male Female

Sex Age (years) Age of onset (years) Treatment (subjects in treatment with one drug)

32.7 (±10.9) 26.7 (±10.0) Lithium Valproate Carbamazepine Other

Duration of treatment (days) Treatment Dose (mg/day)

20 (44.4) 25 (55.6)

30 (66) 9 (20) 3 (6) 3 (6)

36.5 (±19.9) Lithium Valproate Carbamazepine

1066.7 (±174.9) 1166.7 (±251.2) 600 (±200)

Suicide (suicide attempters)

3 (6.7)

Psychotic features (subjects with psychotic features)

21 (46.6) 3 missing values

YMRS at baseline YMRS at retest % reduction in YMRS CGI at baseline CGI at retest % reduction in CGI

42.9 (±4.0) 15.2 (±1.5) 64.2 (±4.6) 5.2 (±0.7) 3.7 (±1.2) 28.4 (±20.6)

Data represent mean±standard deviation or number and frequency; Abbreviations: CGI, Clinical Global Impression scale; YMRS, Young Mania Rating Scale.

significant association results with antimanic acute effect. No correlation, even after covariation analysis, was found for YMRS at baseline or at final tests.

future research. In fact, also under physiological conditions, those proteins support folding of non-native and misfolded proteins, and prevent aggregation during proliferation and cellular differentiation. HSPs demonstrate extremely high conservation of the genetic code sequence (Karlin and Brocchieri, 1998): this is probably due to their essential role in cellular molecular homeostasis (Aufricht, 2005). This important cellular role is like-minded with the hypothesis that HSPs can represent a promising pharmacological target in mood stabilizing action, consistently, there is some evidence that bipolar disorder can be partially dependent on a neurodegenerative pathophysiology (Savitz et al, 2005). The negative result in this study could be due to the some limitations: the small sample size, heterogeneity and different length of treatment and different doses of drugs. Drug plasma levels are an important missing variable. The lack of genomic control which is liable for stratification bias should be another limit of the study, however Korean population is considered genetically homogenous (Cavalli Sforza, 1994). We investigated only a portion of the HSPs coding sequence, on the basis of above mentioned literature evidence. In this study, five SNPs of HSP70 genes were selected. The distance between those SNPs were not evenly distributed and not enough to cover the

IV. Discussion In the present paper we failed to find an association between the variations within the investigated HSPs coding sequence and the acute antimanic effect of lithium, valproate and carbamazepine. Minor clinical significant results were observed: a significant positive correlation between YMRS scores at baseline and % reduction in YMRS score by the end of treatment. We also observed an inverse association with age at onset and the % reduction in YMRS score. This finding is in line with some recent reports focusing on the age of onset of bipolar disorder: Lin and colleagues proposed to use it to identify more homogeneous groups of bipolar disorder families (Lin et al, 2006). This is consistent with other reports (Mick et al, 2003; Kennedy et al, 2005a,b; Leboyer et al, 2005). Age of onset has also been correlated with the severity of symptomatology (Patel et al, 2006) and poorer outcome (Carter et al, 2003). Nevertheless, even though we reported negative association results, HSPs still represent good candidate for

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Pae et al: HSP70 variations in the acute treatment with mood stabilizers in patients with bipolar disorder Cavalli Sforza L (1994) The History and Geography of Human Genes. Princeton, New Jersey, USA: Princeton University Press. Chen G, Hasanat KA, Bebchuk JM, Moore GJ, Glitz D, Manji HK (1999a) Regulation of signal transduction pathways and gene expression by mood stabilizers and antidepressants. Psychosom Med 61, 599-617. Chen G, Masana MI, Manji HK (2000) Lithium regulates PKCmediated intracellular cross-talk and gene expression in the CNS in vivo. Bipolar Disord 2, 217-236. Chen G, Yuan PX, Jiang YM, Huang LD, Manji HK (1999b) Valproate robustly enhances AP-1 mediated gene expression. Brain Res Mol Brain Res 64, 52-58. Chen J, St-Germain JR, Li Q (2005) B56 regulatory subunit of protein phosphatase 2A mediates valproic acid-induced p300 degradation. Mol Cell Biol 25, 525-532. Chetcuti A, Adams LJ, Mitchell PB, Schofield PR (2006) Altered gene expression in mice treated with the mood stabilizer sodium valproate. Int J Neuropsychopharmacol 9, 267-276. Chuang DM (2004) Neuroprotective and neurotrophic actions of the mood stabilizer lithium: can it be used to treat neurodegenerative diseases? Crit Rev Neurobiol 16, 83-90. Chuang DM (2005) The antiapoptotic actions of mood stabilizers: molecular mechanisms and therapeutic potentials. Ann N Y Acad Sci 1053, 195-204. Cohen J (1988) Statistical power analysis for the behavioral sciences. Hillsdale, New Jersey: Lawrence Erlbaum Associates. Dimitrova A, Milanova V, Krastev S, Nikolov I, Toncheva D, Owen MJ, Kirov G (2005) Association study of myo-inositol monophosphatase 2 (IMPA2) polymorphisms with bipolar affective disorder and response to lithium treatment. Pharmacogenomics J 5, 35-41. Dokucu ME, Yu L, Taghert PH (2005) Lithium- and valproateinduced alterations in circadian locomotor behavior in Drosophila. Neuropsychopharmacology 30, 2216-2224. Dooley JM, Andermann F (1989) Startle disease or hyperekplexia: adolescent onset and response to valproate. Pediatr Neurol 5, 126-127. Du J, Gray NA, Falke CA, Chen W, Yuan P, Szabo ST, Einat H, Manji HK (2004) Modulation of synaptic plasticity by antimanic agents: the role of AMPA glutamate receptor subunit 1 synaptic expression. J Neurosci 24, 6578-6589. Einat H, Manji HK (2006) Cellular plasticity cascades: genes-tobehavior pathways in animal models of bipolar disorder. Biol Psychiatry 59, 1160-1171. Grof P, Alda M, Grof E, Zvolsky P, Walsh M (1994) Lithium response and genetics of affective disorders. J Affect Disord 32, 85-95. Gurvich N, Berman MG, Wittner BS, Gentleman RC, Klein PS, Green JB (2005) Association of valproate-induced teratogenesis with histone deacetylase inhibition in vivo. Faseb J 19, 1166-1168. Guy W (1976) ECDEU Assessment manual for psychopharmacology: revised. Rockville, MD. Hartl FU, Hayer-Hartl M (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852-1858. Harwood AJ, Agam G (2003) Search for a common mechanism of mood stabilizers. Biochem Pharmacol 66, 179-189. Hiroi T, Wei H, Hough C, Leeds P, Chuang DM (2005) Protracted lithium treatment protects against the ER stress elicited by thapsigargin in rat PC12 cells: roles of

whole genetic variation. Therefore, it cannot be confirmed which SNPs have a critical role in the pathogenesis of bipolar disorder and possible interaction with mood stabilization by the result of this study. More SNPs on HSP70 genes in larger sample are needed in future. Epigenetic studies between possibly interconnected gene variants should be also investigated in order to cover minor gene effects. Finally, given that therapeutic agents used in bipolar disorder are heterogeneous in action mechanism, and thus unified mood stabilizer trials should be more proper for bipolar pharmacogenetic researches. In addition, to narrow down the homogeneity of subjects, study with endophenotypic diagnosis for bipolar disorder patients may be also valuable to converge the findings in this field. The strength of this study is that it is the second one to our knowledge that focused on the pharmacogenetics of acute response of antimanic mood stabilizers treatment, and the first one that investigated a portion of the HSP coding sequence on this topic. Hence, the present paper should be considered with clear limitations as well as some potential advantages of preliminary work, highlighting an interesting but not yet deeply investigated pharmacogenetic field.

Acknowledgement This work was supported by a financial support of the Catholic Medical Center Research Foundation made in the program year of 2008 and by a grant from the Medical Research Center, Korea Science and Engineering Foundation, Republic of Korea (R13-2002-005-04001-0).

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Chi-Un Pae

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Gene Therapy and Molecular Biology Vol 12, page 277 Gene Ther Mol Biol Vol 12, 277-292, 2008

Recombinant adeno-associated virus as vaccine delivery vehicles Review Article

Komal Vig1, Roland Herzog2, Douglas Martin3, Eddie G. Moore4, Vida A. Dennis1, Shreekumar Pillai1, Shree R. Singh1,* 1

Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL 36101, USA Department of Pediatrics, University of Florida, Gainesville, FL 32610, USA 3 Scott-Ritchey Research Center, Auburn University, Auburn, AL 36832, USA 4 Department of Biological Sciences, Alabama State University, Montgomery, AL 36101, USA 2

__________________________________________________________________________________ *Correspondence: Shree R. Singh, PhD, Center for NanoBiotechnology Research, Alabama State University,915 S. Jackson St., Montgomery, AL 36101, USA; Tel: +1-334- 229- 4168; Fax: +1-334- 229-4955; e-mail address: ssingh@alasu.edu Key words: Adeno-associated virus, Vector, Vaccine, Transgene, Cellular response, Humoral response Abbreviations: Adeno-associated virus, (AAV); adenovirus, (AdV); antigen presenting cells, (APC); dendritic cells, (DCs); hemagglutinin, (HA); Herpes simplex virus, (HSV); human immunodeficiency virus, (HIV); human papillomavirus, (HPV); Intramuscular, (IM); Intranasal, (IN); intraperitoneally, (IP); Intravenous, (IV); inverted terminal repeats, (ITRs); open reading frames, (ORFs); recombinanat adeno-associated virus, (rAAV); severe acute respiratory syndrome, (SARS); single stranded DNA, (ssDNA) Received: 7 November 2008; Revised: 22 November 2008 Accepted: 25 November 2008; electronically published: November 2008

Summary Adeno-associated viruses (AAV) are non-enveloped, replication defective, single-stranded DNA virus and require co-infection with a helper virus, such as adenovirus or herpes virus, to undergo a productive infection. The AAV genome is 4.7 kb in size and is framed by two inverted terminal repeats (ITRs) at both ends of the DNA strand, and contains two open reading frames (ORFs): Rep and Cap. In total, 11 strains of AAV have been isolated and characterized from humans and primates, and new serotypes are continuously discovered. All serotypes share similar structure, genome size and organization. Serotype 2 (AAV2) has been the extensively studied and presents natural tropism towards skeletal muscles, neurons, retinal cells, vascular smooth muscle cells and hepatocytes. The most divergent serotype is AAV5 with notable differences at the level of the ITR size. AAV`s natural defectiveness, its lack of pathogenicity, and the ability to infect cells in vivo have led to the study of its potential use as a gene therapy vector. Recent studies have begun to test AAV vectors as vaccine carriers against human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis, severe acute respiratory syndrome (SARS), and many other viruses. rAAV vectors can evade the immune response and mediate a durable expression of transgene in vivo. However, evidence has been gathering that in some circumstances, the rAAV vector may initiate a cellular and humoral response to the expressed gene product in vivo. It is therefore important to understand the factors, which influence the establishment of these immune responses in order to design safe and efficient procedures for AAVbased gene therapies or vaccine delivery. Various factors seem to influence the immune response of AAV vaccine vectors. These include the AAV serotype, the transgene, route of administration, dose of vector, transgene expression levels, immune responses to the viral capsid, and others. A more thorough understanding of the interplay between rAAV and their encoded transgenes and the host immune system is necessary for the optimal development of rAAV vaccine system.

The virus particle is composed of an icosahedric capsid and one single molecule of the viral genome of either positive- or negative-sense. They belong to the Parvoviridae family and are classified in the Dependovirus genus. AAVs are replication defective and require co-infection with a helper virus, such as

I. Introduction A. AAV genome structure Adeno-associated virus (AAV) is the smallest of known human viruses. AAV are non-enveloped, singlestranded (ss) DNA viruses with a diameter of 18-25 nm.

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Vig et al: Recombinant adeno-associated virus as vaccine delivery vehicles adenovirus or herpes virus, to undergo a productive infection in the cultured cells. These common human viruses are naturally defective and non-pathogenic. AAVs are very resistant to extreme conditions of pH, detergent and temperature, making them easy to manipulate. AAVs are frequently found in the human populations, 70-80% of individuals having been exposed to an infectious event. So far there are no diseases associated with AAV. The virus causes only a very mild innate immune response and can infect non-dividing cells. The wild-type virus integrates into the host cell`s genome, but there is no evidence that it causes malignant transformation. Because of these features it presents a very attractive choice for creating vectors for gene therapy and drug delivery. The single-stranded DNA genome of AAV is 4.7 kb and is framed by two inverted terminal repeats (ITRs) at the ends of the DNA strand, and two open reading frames (ORFs): Rep and Cap (Figure 1). The ITRs are basepaired hairpin structures of 145 nucleotides length. They were named so because of their symmetry, which is required for efficient multiplication of the AAV genome (Bohenzky, 1988). Their ability to form a hairpin contributes to self-priming that allows primaseindependent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it (Weitzman et al, 1994; Wang et al, 1995) as well as for efficient encapsidation of the AAV DNA combined with generation of a fully-assembled, deoxyribonuclease-resistant AAV particles (Zhou and Muzyczkz, 1998). The ITRs contain the only necessary regulatory cis acting sequences required by the virus to complete its life cycle, namely the origin of replication of the genome, the terminal resolution site and, the packaging and the integration signals. In this context, Young and colleagues have shown in 2000 that

the complete ITRs are not required for integration of AAV or plasmid into the chromosome 19 site. The left ORF or Rep is composed of four overlapping genes encoding four regulatory Rep proteins called Rep78, 68, 52 and 40 (Figure 1). The two major Rep proteins, Rep78 and Rep68, are involved in viral genome excision, rescue, replication and integration (Weitzman et al, 1994) and also regulate gene expression from AAV and heterologous promoters (Horer et al, 1995; Pereira et al, 1997). The minor Rep proteins, Rep52 and Rep40, are involved in replicated ssDNA genome accumulation and packaging (King, 2001). The right ORF or Cap is initiated at the p40 promoter and encodes the 3 structural proteins VP1, VP2 and VP3 which interact together to form a capsid of an icosahedral symmetry (Carter, 2000). Finally, all the transcripts share the same polyadenylation signal and equal amounts of virions are found containing strands of plus or minus polarity. Once inside the cell, the genome is converted to double-stranded transcriptionally active DNA, which is stabilized as a predominantly nonintegrated episomal form (Duan et al, 1998; Nakai et al, 2001).

B. AAV infection cycle The AAV infection cycle involves various steps from infecting the cell to producing new infectious particles (Ding et al, 2005; Kwon and Schaffer, 2008) including: (1) viral binding to a receptor, (2) endocytosis of the virus, (3) intracellular trafficking of the virus through endosomes, (4) endosomal escape of the virus, (5) intracellular trafficking of the virus to the nucleus and nuclear import, (6) virion uncoating, and (7) viral genome conversion from a single-stranded to a double-stranded genome capable of expressing an encoded gene (Figure 2).

Figure 1. AAV genome organization. The transcripts for Rep78 and Rep68 are initiated at the p5 promoter. The p19 promoter produces Rep40 in spliced form and Rep52 in unspliced form. Messenger RNAs encoding the capsid proteins VP1, VP2 and VP3 are transcribed from the p40 promoter. ITR, inverted terminal repeats. Poly A, polyadenylation site. Reproduced from Merten et al, 2005 with kind permission from Gene Therapy.

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Figure 2. rAAV transduction. The various steps involved are: (1) viral binding to a receptor, (2) endocytosis of the virus, (3) intracellular trafficking of the virus through endosomes, (4) endosomal escape of the virus, (5) intracellular trafficking of the virus to the nucleus and nuclear import, (6) virion uncoating, and (7) viral genome conversion from a single-stranded to a double-stranded genome capable of expressing an encoded gene. Reproduced from Ding et al, 2005 with kind permission from Gene Therapy.

Some of these steps may look different in various types of cells, which in part, contributes to the defined and quite limited native tropism of AAV. Replication of the virus can also vary in one cell type, depending on the cell`s current cycle phase (Rohr et al, 2002). The characteristic feature of AAV is its deficiency in replication and thus the inability to multiply in unaffected cells. The first factor ascribed as providing successful generation of new AAV particles, was the adenovirus, from which the name AAV originated. It has been shown that AAV replication can be facilitated by selected proteins from the adenovirus genome (Matsushita et al, 1998; Myers et al, 1980), by other viruses such as the Herpes simplex virus (HSV) (Handa and Carter, 1979), or by genotoxic agents, such as UV irradiation or hydroxyurea (Yakobson et al, 1987, 1989; Yalkinoglu et al, 1988). An important step in AAV viral production was achieved when the adenovirus (Ad) helper virus step was replaced by a plasmid construct containing a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes (Matsushita et al 1998; Xiao et al 1998a). This discovery allowed for new production methods of recombinant AAV, which do not require adenoviral co-infection of the AAV-producing cells. ITRs and either Rep78 or Rep68 are sufficient for replication of the AAV genome in the presence of helper virus. In particular, Rep78 and Rep68 bind to specific sequence within the ITRs called the rep binding site (RBS) (McCarty et al, 1994; Ryan et al, 1996), and cleave in a site-and strand-specific manner at the terminal resolution site located 13 nucleotides upstream of the RBS (Brister and Muzyczka, 1999; Im and Muzyczka, 1989; Snyder et al, 1990). The RBS and terminal resolution site act as a

minimum origin of Rep-mediated DNA replication (Ward and Berns, 1995; Ward et al, 2001). In the absence of the helper virus, ITRs and either Rep78 or Rep68 are also sufficient to mediate the integration of the AAV genome into the host cell genome, preferentially into a site termed AAVS1 on chromosome 19 of human cells (Kotin et al, 1990, 1992; Linden et al, 1996; Surosky et al, 1997).

C. AAV serotypes In total, 11 strains of AAV have been isolated and characterised from humans and primates, and new serotypes are continuously discovered. All serotypes share similar structure, genome size and organization, i.e, structure and location of ORFs, promoters, introns and polyadenylation site. Serotype 2 (AAV2) has been the most extensively examined so far (Bartlett et al, 1998; Rabinowitz et al, 1999; Wu et al, 2000). AAV2 presents natural tropism towards skeletal muscles (Manno et al, 2003), neurons (Bartlett et al, 1998), vascular smooth muscle cells (Richter et al, 2000) and hepatocytes (Koeberl et al, 1997). AAV-2 based vectors use heparin sulfate proteoglycans as the primary receptor (Summerford and Samulski, 1998), a co-receptor fibroblast growth factor 1 receptor (Qing et al, 1999) and !v"5 integrin (Summerford et al, 1999), giving access to a wide range of tissue types (Table 1). Although AAV-2 is the most popular serotype in various AAV-based approaches, it has been shown that other serotypes can be more effective as gene delivery vectors. AAV6 for e.g., appears much better in infecting airway epithelial cells, AAV7 presents very high transduction rate of murine skeletal muscle cells (similar to AAV1 and AAV5), AAV8 is excellent in transducing

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Vig et al: Recombinant adeno-associated virus as vaccine delivery vehicles Table 1. Receptors of AAV serotypes on the cell membrane AAV serotype AAV-1 AAV-2

AAV-3 AAV-4 AAV-5 AAV-6 AAV-7 AAV-8 AAV-9 AAV-10, AAV11, AAV-12

Receptors Heparan sulphate proteoglycan (HSPG)a, Fibroblast growth factor receptor 1 (FGFR1)b, hepatocyte growth factor receptorb, cmetb, Laminin receptorb, !v"5b !v" b a b HSPG , FGFR-1 , Laminin receptorb O-linked sialic acid a N-linked sialic acida, Platelet derived growth factor receptor-! b Sialic acida, HSPGb Laminin receptorb Laminin receptorb -

References Qing et al, 1999; Summerford et al, 1999; Kashiwakura et al, 2005; Asokan et al, 2006; Akache et al, 2006 Handa et al, 2000; Rabinowitz et al, 2002; Akache et al, 2006; Blackburn et al, 2006 Kaludov et al, 2001 Kaludov et al, 2001; Di Pasquale et al, 2003 Halbert et al, 2001; Seiler et al, 2006 Akache et al, 2006 Akache et al, 2006

a -Primary receptor b- Co-receptor

hepatocytes (Halbert et al, 2001; Gao et al, 2002; Rabinowitz et al, 2004) and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells (Chen et al, 2005). AAV6, a hybrid of AAV1 and AAV2 (Rabinowitz et al, 2004), also shows lower immunogenicity than AAV2 (Halbert et al, 2001). Serotypes can differ with respect to the receptor they are bound to e.g., AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (Kaludov et al, 2001), and AAV5 was shown to enter cells via the platelet-derived growth factor receptor (Di Pasquale et al, 2003). The most divergent serotype is AAV5 with notable differences at the level of the ITR size (167 nucleotides for AAV5 compared to 143-146 for AAV1 to 4 and AAV6) and function. In addition, at the biological level, they are all dependent on the presence of a helper virus for their replication and gene expression.

there is evidence that rAAV vectors also are efficient in genetic vaccination (Sun et al, 2003). Most reports have described the durability of transgene expression in the tissues of AAV vector-infected animals and have demonstrated that the use of AAV vectors does not result in an immune response (especially a cell-mediated response) against the vector encoded transgene (Flotte et al, 1993; Xiao et al, 1996; Fisher et al, 1997). However, many people have neutralizing antibodies to AAV due to prior infection (Chirmule et al, 1999). Using different serotypes of AAV may circumvent this problem and allow effective long-term treatment by AAV-based gene therapy. Two major hurdles remain for use of AAV-based gene therapy vectors: the small transgene capacity and the effect of neutralizing antibodies. AAV can package 4.9kb DNA, which is too small for many applications. By utilizing AAV`s capacity to form concatemers, larger inserts can be split over two vectors and concomitantly, leading to transgene expression; however, efficiency is significantly reduced (Duan et al, 2000; Sun et al, 2000).

II. AAV as a gene therapy vector Overall, AAV vectors have shown an excellent safety record in humans in clinical trials. Broad tissue tropism, the ability to infect dividing and quiescent cells, and the long-term expression are attractive properties of this vector system. Using the rAAV vector system, many genes have been efficiently transferred into a number of tissues such as lung (Flotte et al, 1993; Halbert et al, 1997), muscle (Xiao et al, 1996), eye (Lewin et al, 1998), central nervous system (Kaplitt et al, 1994; Peel et al, 1997), gut (During et al, 1998) and liver (Xiao et al, 1998b). AAV has been used to amend genetic and acquired human diseases such as cystic fibrosis, hemophilia, muscular dystrophy or diabetes mellitus (Kaufmann et al, 2001; Grimm and Kay, 2003; Hildinger and Auricchio, 2004; Nathwani et al, 2005). Despite reports that AAV induces only weak immune responses against the vector and the expressed transgene in gene therapy approaches (Sun et al, 2002; Bessis et al, 2004),

III. AAV vaccination and immune response The recombinant adeno-associated virus (rAAV) has attracted tremendous interest as a promising vector for gene delivery. These vectors are simple, versatile and safe and successfully used for the long-term expression of therapeutic genes in animal models and patients. Furthermore, studies have demonstrated that rAAV vectors can evade the immune response and mediate a durable expression of transgene in vivo (Fisher et al, 1997; Xiao et al, 1997). However, evidence has been gathering that in some circumstances, the rAAV vector may initiate adaptive immune responses to the transgene product (Manning et al, 1997; Brockstedt et al, 1999; Lo et al, 1999). It is therefore important to understand the factors, 280

Gene Therapy and Molecular Biology Vol 12, page 281 which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies or vaccine delivery. Viral vectors are detected by the immune system and generate an immune response that becomes effective before the virus infects the target cells. Vectors induced immune response directed against them may be beneficial when the goal is vaccination or tumor lysis. However, in most cases the immune response is undesirable as it may eliminate the vector and the transfected cells decreasing both the intensity and the duration of transgenic protein expression. The immune response to gene therapy vectors, as with the infection with other microorganisms, involves the production of cytokines and chemokines that have detrimental effects. An adaptive immune response generally follows the innate response. It includes a humoral response characterized by production of neutralizing antibodies specific to the vector or transgene antigen and a cell-mediated response involving T cells and NK cells. Adaptive immunity not only contributes to eliminating the vectors and infected cells from the body but also results in a memory response that impede further efforts to use the same vector or transgene. Even though the mechanisms behind the ability of the rAAV-vectored transgenes to induce an immune response are not very clear, transduction of dendritic cells (DCs) following inoculation of rAAV may induce an immune response (Wang et al, 2004). DCs are key antigen presenting cells (APC) for regulating immune responses. Therefore, a major focus of present-day vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that amend disease states such as malignancy, infection, autoimmunity, and allergy. AAV, however, generate a weaker adaptive cellmediated response compared to the other vectors such as adenovirus (AdV) (Bessis et al, 2004). This could be due to the low efficiency of AAVs to efficiently infect APCs such as DCs and macrophages (Bessis et al, 2004). Nonetheless, AAV vectors are able to infect immature DCs to some degree (Zhang et al, 2000). Furthermore, intramuscular injection of AAV vectors has been shown to direct local immune responses resulting in activation of CTL and B cell responses against the transgene (Sarukhan et al, 2001; Wang et al, 2005a,b). Given the relatively low innate immunity to AAV vectors, however, generating a sufficiently strong adaptive response for vaccine development may remain a challenge (Bessis et al, 2004). In gene replacement therapy a gene that is not expressed in the patient is introduced de novo (Bessis et al, 2004). Therefore, CD4 T cells specific for the therapeutic gene have not been deleted in the thymus resulting in an immune response along with B cell activation producing antibodies. This leads to CD8 T cell activation and a cytotoxic response against the therapeutic gene. T cells can distinguish ‘infectious non-self’ from ‘non-infectious self’. In the gene substitution therapy, the transgene by itself will not likely cause upregulation of co-stimulatory molecules on APCs, but the viruses used as vectors or sequences of nonhuman origin present in the plasmid

vector, when using naked DNA, can induce a strong host immune reaction (Onodera et al, 1999). Tolerance can thus be disrupted in such situations. Maintained expression of the transgene or repeated administration of the vector carrying the therapeutic gene is required to boost the immune system in such situations. Humoral responses can also be generated by AAV vectors. Infection by the nonpathogenic AAV2 is common, and the prevalence of anti-AAV2 antibodies ranges from 35 to 80% according to the age group and geographic location (Chirmule et al, 1999; Erles et al, 1999; Moskalenko et al, 2000). Several studies have shown that anti-AAV antibodies have neutralizing effects that decrease the efficiency of in vivo vector infection in the liver (Halbert et al, 1997) or lungs, (Moskalenko et al, 2000) and therefore limit the chances of success with repeated administration of these vectors. Other studies, in contrast, have established that this humoral response has no influence on the efficiency of infection with the vector administered within the muscle (Fisher et al, 1997) or lungs (Beck et al, 1999). Similarly, the development of anti-AAV antibodies is minimal or nonexistent after administration of AAV into the brain (Lo et al, 1999; Mastakov et al, 2002) or retina (Anand et al, 2002). However, these studies were conducted in animals, which do not have pre-existing anti-AAV immunity, in contrast to humans. In humans, anti-AAV antibodies are found in serum and other body fluids such as joint fluids (Cottard et al, 2004) and amniotic fluid (Boyle et al, 2003). The intensity of immune response varies with a number of factors, such as vector dose, the route of administration, the nature of transgene and host-related factors responsible for interindividual variability. AAV has been used as a gene delivery virus in various studies (Table 2). A number of investigators have pursued the use of AAV2 as a vaccine carrier. In this article we will be discussing only the immune response generated by AAV vectors expressing other viral genes.

A. AAV mediated vaccination 1. AAV mediated vaccination against human immunodeficiency virus (HIV) rAAV vectors are being used currently in human trials as vaccine carriers for HIV-1. tgAAC09 consisting of ssDNA from Clade C HIV-1 genes for the gag, protease and part of the reverse transcriptase proteins enclosed within a rAAV2 protein capsid, was developed as an HIV vaccine (van Lunzen et al, 2007). In the initial trial, vaccination with tgAAC09 appears to be safe and well tolerated and stimulated a modest immune response against the gag protein. HIV-specific T-cell responses were observed in 20% of vaccine recipients receiving the highest dose of tgAAC09 tested; however antibody responses were not observed. Vectors based on other serotypes of AAV, most notably AAV1, are now entering trials (Pastor et al, 2007). Various genes of HIV have been targeted and cloned for delivery into the cells using rAAV. rAAV-Fab105 vectors were produced by cloning the Fab105 expression cassette of HIV-1 into a AAV shuttle vector (Chen et al, 1996). When this vector was transduced into human 281

Vig et al: Recombinant adeno-associated virus as vaccine delivery vehicles Table 2. immune response to AAV used as a vaccine vector. Transgene HIV-1

Targeted region/ gene Fab 105 expression cassette

Immune response generated The infection of several primary HIV-1 patient isolates was effectively blocked in the transduced lymphocytes IgG, IgA, MHC class-I CTL Cell-mediated immnunity was enhanced

Cell line / animal Human lymphocytes

Reference Chen et al, 1996

HIV-1

env, tat, rev

BALB/c mice

Xin et al, 2001

HIV-1

env

Systemic and regional immunity induced Cellular immune response

BALB/c mice (Oral) BALB/c mice

Xin et al, 2002

HIV-1

HIV-1subtype B

env by introducting ITRs from AAV to regulatory region of DNA plasmid (pITR/CMV-HIV plasmid) gagV3 gene

Increase in CTL response but no increase in antibody level by pCI-gagV3

BALB/c mice

Liu et al, 2004

HIV-1

Gp 120

CTL and IgG

BALB/c mice

HIV-1

Gag

T cell response, IgG, Th2

BALB/c mice

HIV-1

Gag

BALB/c mice

HIV-1

Gag

HIV-1

env, gag, RT

CD8+ T cell response, B cell response CD8+ T cell IFN-# T and B cell response

SIV

rev-gag-PR-$RT-RRE; rev-env; RT-IN RBD RBD

Cynomolgus macaque Rhesus macaques (Indian origin) BALB/c mice BALB/c mice

Feng et al, 2004 Chikhlikar et al, 2004 Lin et al 2007a Lin et al 2007b Calcedo et al, 2006 Johnson et al, 2005 Du et al, 2006 Du et al, 2008

C57BL/6

Liu et al, 2000

BALB/c (Intramuscular) C57BL/6 mice (Intranasal) BALB/c mice Intramuscular or intraportal

Liu et al, 2005

Mouse hepatocyes; BALB/c mice Dendritic cells (DC) BALB/c mice (Intramuscular)

Li et al, 2008

DCs

Sun et al, 2002

H2Kb mice

GallezHawkins et al, 2004

SARS-CoV SARS-CoV HPV16

T cell and Ab High neutralizing Ab Th1 and neutralizing Ab, Th2 and CTL response Cellular response, CD4+ and CD8+ dependent CTL Neutralizing Ab

HPV16

HPV16 E7 fused to heat shock protein L1

HPV 16

Hepatitis B virus Hepatitis B/ woodchuck hepatitis Hepatitis B virus

Surface antigen Woodchuck IFN!

Serum and mucosal Ab Cellular response Humoral immune response High Interferon levels

IFN #1

Immunocytochemical studies

Hepatitis C virus

Full length (aa 1-190); truncated (aa 49-180) Glycoprotein B (gB); glycoprotein D (gD)

CTL

Herpes simplex virus 2 Chronic myelogenous leukemia Cytomegalovirus

varient

gB specific , MHC class I CTL response; Ab titers to gB or gD increased over time Cytotoxic CD4+/Th1; CD8+

Immediate early 1 (IE-1) and pp65 proteins

AB response, CD8 lymphocytes with Cytotoxic function

p210BCR-ABLb3a2 fusion region

282

BALB/c mice

Xin et al, 2003

Kuck et al, 2006 Di et al, 2003 Pedro et al, 2005

Liu et al, 2006 Manning et al, 1997

Gene Therapy and Molecular Biology Vol 12, page 283 lymphocytes, it produced and secreted the Fab105 fragments, while maintaining their normal morphology, growth rates, and responsiveness to mitogen stimulation. The infection of several primary HIV-1 patient isolates was also effectively blocked in the transduced lymphocytes. HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector) was developed (Xin et al, 2001). A single injection of the AAV-HIV vector induced strong production of HIV-1specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When the AAV-HIV vector was coadministered with AAV-IL2 vector (AAV with 0.7 kb murine interleukin 2 cDNA), the HIV-specific cellmediated immunity was significantly enhanced. Also, boosting with the AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin gene showed protective immunity against homologous influenza virus challenge. Similarly, systemic and regional immunity was induced in the mice after oral administration of a rAAV vector expressing HIV-1 env gene (Xin et al, 2002). This study also reported a significant reduction in viral load after an intrarectal challenge with a recombinant vaccinia virus expressing HIV env gene. In order for predetermination of antibody affinity and specificity prior to â&#x20AC;&#x153;immunizationâ&#x20AC;? against HIV envelope protein, rAAV vector was used to deliver the gene for the human monoclonal antibody IgG1b12 to mouse muscle (Lewis et al, 2002). Significant levels of HIV-neutralizing activity were found in the sera of mice for over 6 months after a single intramuscular administration of the rAAV vector. HIV-1 DNA vaccine and rAAV expressing gagV3 gene of HIV-1 subtype B were constructed, and BALB/c mice were immunized by a vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine (Liu et al, 2004). CTL and antibody responses were measured and compared with those induced by DNA vaccine or rAAV vaccine separately. No evident increase in the antibody level induced by pCIgagV3 combined with rAAV was observed, but there was an increased CTL response. The results indicate that HIV1 specific cytotoxicity can be increased by immunization of BALB/c mice with a DNA vaccine combined with rAAV vaccine. The potential of DNA vaccine immunogenicity improvement by introducing ITRs from AAV into the regulatory region of the DNA plasmid was tested (Xin et al, 2003). Mice immunized with pITR/CMV-HIV plasmid generated significantly higher HIV-specific antibody, higher cellular immune responses and lower viral loading than animals immunized with pCMV-HIV plasmid showing that AAV ITRs enhance CMV-dependent upregulation of transgene expression and immunogenicity of the DNA vaccine.

In another study, the immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) was examined for the effect of the addition of the ITR sequences of the AAV to the DNA plasmid construct, and of packaging the LAMP/gag gene as a rAAV (Chikhlikar et al, 2004). The immune responses of mice to immunization with these constructs were examined using DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG(1) isotype resulting from the activation of the Th2 subset of CD4(+) T cells, that was sustained for at least 5 months after immunization. To study the immune effect of rAAV combined with rAdV vaccine in BALB/c mice, the codon-modified HIV1 gp120 gene was inserted into a plasmid containing AAV and AdV separately to construct the rAAV and rAdV vaccines (Feng et al, 2004). Both rAAV and rAdV vaccine could express the gp120 gene in mice immunized with rAAV and rAdV. The mice primed with rAAV at week 0, 2 and boosted with rAdV at week 5, 14 and 20 elicited the strongest gp120 specific CTL and IgG antibody response. Cell mediated T cell response and humoral responses were observed after intramuscular immunization with AAV2, AAV2/7 or AAV2/8 mixture, expressing HIV1W61Dgp140 (env), Gag-Nef and HIV-1 RT in Cynomolgus macaque (Calcedo et al, 2006). Similarly, a rAAV2 vaccine encoding simian immunodeficiency virus (SIV) elicited protective SIVspecific T cells and antibodies in macaques after a single intramuscular dose (Johnson et al, 2005). Furthermore, immunized animals were able to significantly restrict replication of a live, virulent SIV challenge. Potential of AAV vectors based on novel AAVs as vaccine carriers were evaluated for HIV-1 gag in mice (Lin et al, 2007a). Strong immunogenicity in terms of gag CD8+ T-cell and antibody responses was demonstrated by AAV7, AAV8, and AAV9 based vectors. Likewise, Lin and colleagues observed in 2007 rAAV vectors expressing HIV-1 gag stimulated gag-specific response in BALB/c mice. However, CD8+ T cells induced by rAAV vectors failed to efficiently proliferate upon a booster immunization with an Ad vaccine vector or other vaccine modalities carrying the same transgene. Antigen derived from continued transgene expression in skeletal muscle induced an unresponsive phenotype in the activated CD8+ T cells. These results illustrate that additional modification are required to successfully develop an AAV-based vaccine, and that quantitation of T cell frequencies is not sufficient to determine the effectiveness of the vaccine vector. Rather, functionally of the induced T cells and the ability to re-activate these cells has to also be evaluated.

2. AAV Hepatitis

mediated

vaccination

against

The Hepatitis B surface antigen (HBsAg) gene was cloned into the AAV vector pSNAV to form the recombinant pSNAV-HBsAg, which was transfected into 283

Vig et al: Recombinant adeno-associated virus as vaccine delivery vehicles BHK-21 cells (Di et al, 2003). The cells infected with rAAV-HBsAg were capable of HBsAg expression, the amount of which augmented with the increase of multiplicity of infection. BALB/C mice immunized with rAAV-HBsAg produced anti-HBsAg antibodies. rAAVHBsAg could induce a humoral immune response against HBsAg and therefore could be a promising candidate hepatitis B vaccine. Interferon-!2 (IFN!2) is routinely used for antihepatitis B virus (HBV) treatment. AAV-IFN!1 was generated to deliver the IFN!1 gene into hepatocytes (Li et al, 2008). AAV-IFN!1 effectively transduced HBVproducing cells and mouse hepatocytes. A single dose administration of AAV-IFN!1 viral vector displayed prolonged transgene expression and superior antiviral effects both in vitro and in vivo suggesting that, the use of AAV-IFN!1 might be a potential alternative strategy for anti-HBV therapy. Two rAAV vectors, carrying either the full length (aa 1-190) or truncated (aa 49-180) versions of chronic hepatitis C virus (HCV) core gene, were generated for targeting HCV-infected cells (Liu et al, 2006). Both AAV/core (l-190) and AAV/core (49-180) were used to transduce/load DCs. These two genetically altered DC types then stimulated anti-core CTL. The results indicated that the core (49-180) gene is an effective antigen, but has the advantage of stimulating less self-recognition. Thus, core (49-180) may be useful for further translational immunotherapy studies against HCV.

compared with those of VLP. The data suggests that a single intramuscular co-injection with rAAV-16L1/rAdmGM-CSF can achieve the same vaccine effect as a VLP vaccine requiring 3 booster injections, which is currently recognized as a prophylactic vaccine (Harro et al, 2001; Koutsky et al, 2002; Pinto et al, 2003). In another study aiming to develop a vaccine against HPV16 infection, a single dose of rAAV5 L1h administrated intranasally to mice was sufficient to induce high titers of L1-specific serum antibodies, as well as mucosal antibodies in vaginal washes (Kuck et al, 2006). They observed that seroconversion was maintained for atleast one year and also detected a cellular immune response even after 60 weeks of immunization. Furthermore, lyophilized rAAV5 L1h successfully evoked a systemic and mucosal immune response in mice.

4. AAV mediated vaccination against severe acute respiratory syndrome (SARS) AAV vectors are also being utilized for vaccine development against other viruses such as SARS coronavirus (SARS-CoV). A novel vaccine against SARS coronavirus was developed based on the rAAV delivery system by cloning the receptor binding domain (RBD) and evaluated in BALB/c mice (Du et al, 2006). High titers of neutralizing antibodies were observed against SARS-CoV infection after a single dose of RBD-rAAV vaccination. Two more repeated doses of the vaccination boosted the neutralizing antibody to about 5 times of the level achieved by a single dose of the immunization and the level of the antibody continued to increase for the entire duration of the experiment of 5.5 months. The immune responses and protective effects of immunization with RBD-rAAV prime/RBD-specific T cell peptide boost (RBD-Pep) were further assessed (Du et al, 2008). Compared with the RBD-rAAV prime/boost vaccination, RBD-rAAV prime/RBD-Pep boost induced similar levels of Th1 and neutralizing antibody responses that protected the vaccinated mice from subsequent SARS-CoV challenge, but stronger Th2 and CTL responses suggesting this vaccination protocol to be ideal for providing effective, broad and long-term protection against SARS-CoV infection.

3. AAV mediated vaccination against human papillomavirus (HPV) AAV vector based systems have been used for vaccination against HPV infections. A rAAV2 encoding a chimera between HPV 16 with an E7 oncogene (HPV16E7) CTL epitope and a heat shock protein elicited a potent antitumor response against challenge with an E7expressing syngenic cell line in immunocompetent mice (Liu et al, 2000). In vitro analysis indicated induction of both CD4- and CD8-dependent CTL activity. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection was CD4 and CD8 dependent. More recently, a prophylactic vaccination approach against HPV infections was investigated (Liu et al, 2005). The intramuscular application of a rAAV2 vaccine encoding the capsid protein L1 from HPV16, together with rAdV encoding murine granulocyte-macrophage colonystimulating factor, led to induction of neutralizing L1 antibodies in BALB/c mice, when compared to the DNA vaccine (Liu et al, 2005). Immunohistochemistry, however, showed that the accumulation of APCs, such as macrophages and DCs, in rAAV- 16L1 and L1 DNAinjected muscle fibers may be due to L1 protein expression, but not due to AAV infection. When compared to the non-infectious HPV-like particles (VLPs) L1 vaccine, however, the titers of neutralizing L1 antibodies induced by VLP were higher than those induced by rAAV16L1. Co-vaccinating with rAAV-16L1 and AdV encoding murine GM-CSF (rAAV-16L1/rAd-mGM-CSF) induced higher levels of neutralizing L1 antibodies

5. AAV mediated vaccination against other viruses rAAV vector encoding the p210BCR-ABLb3a2 variant fusion region with flanking sequences (CWRBA) was constructed and used to express the BCR-ABL fusion region within primary human DCs, as a strategy for the immunotherapy of chronic myelogenous leukemia (CML) (Sun et al, 2002). CWRBA-transduced DCs elicited CD4+/Th1 and CD8+ responses demonstrating that the developed construct may serve as a vaccine for gene-based antigen-specific immunotherapy of CML. A low titer, helper-free rAAV-pp65mII and rAAVIE1 virus was used to elicit specific humoral and cellular responses to two important cytomegalovirus (CMV) antigens: the immediate-early 1 (IE-1) and pp65 proteins (Gallez-Hawkins et al, 2004). Simultaneous immunization 284

Gene Therapy and Molecular Biology Vol 12, page 285 of both CMV proteins, using DNA vaccine priming followed by rAAV boost, induced antibody response and CD8 lymphocytes with cytotoxic function. The utility of AAV vectors for genetic immunization of herpes virus-2 was examined by Manning and colleagues in 1997. Vectors expressing either HSV-2 glycoprotein B (gB) or glycoprotein D (gD) were constructed and injected intramuscularly in mice. Intramuscular injection of rAAV-gB induced a vigorous gB-specific, MHC class I CTL response and anti-gB antibody both 4 and 11 weeks postimmunization. The ability of rAAV-gB vaccination to prime a helper T-cell response was assessed by using a lymphoproliferation assay. The mice demonstrated gB-specific lymphoproferalition at 4 and 11 weeks postvaccination. The results also show that antibody titers to gB or gD increased over time. Activation of hemagglutinin (HA) -specific CD4+ T cells and target cell destruction was triggered by rAAVmediated gene transfer of the HA gene into the muscle (Sarukhan et al, 2001). Similarly, in a model of gene transfer in muscle, delivery of the influenza HA membrane protein by AAV was impaired by strong immune responses that lead to rapid rejection of the transduced fibers (Gross et al, 2003). However, injection of HAspecific CD4+CD25+ T cells from T-cell receptortransgenic animals, along with gene transfer, downregulated the anti-HA cytotoxic and B-lymphocyte responses and enabled persistent HA expression in muscle. Their results demonstrated that adoptive transfer of antigen-specific CD4+CD25+ regulatory T cells can be used to induce sustained transgene engraftment in solid tissues. In another study using AAV as a vaccine delivery system against two strains of malaria, a single injection of rAAV encoding the malarial antigens MSP4 (Plasmodium falciparum) or MSP4/5 (Plasmodium yoelii) although stimulated long-term antigen-specific antibody responses after intramuscular injection, but the vaccination was not protective against infection (Logan et al, 2007).

B. Factors influencing the response of AAV vaccine vectors

Seroepidemiological studies have demonstrated that AAV1, 2 and 3 antibodies prevalence rises steeply between the ages of 1 and 10, and reaches a peak of 60% by the age of 10 years. In contrast, antibody against AAV4, originally isolated from nonhuman primates, is detected much less frequently, with a peak incidence of about 10% between 2 and 5 years of age (Blacklow et al, 1968, 1971). AAV5, which was originally isolated from a human genital lesion, show average peak titers between 15 and 20 years of age (Georg-Fries et al, 1984). Among the AAV serotypes anti-AAV2 antibodies have potent neutralizing effects compared to other serotypes (Bessis et al, 2004). Xiao and colleagues found in 1999 neutralizing anti-AAV1 antibodies in 20% of individuals compared to 27% for anti-AAV2 in their study of 77 healthy individuals. On the other hand, neutralizing anti-AAV5 antibodies are generally not common in healthy individuals (Hildinger et al, 2001). Studies have also shown that neutralizing antibodies to AAV-7 and AAV-8 are rare in human sera (Gao et al, 2002), making them good vector candidate for humans. AAV serotypes may differ in their affinities for different cell types. AAV-1, -7, -8, and -9 are the most efficient vector for infecting muscle, followed by AAV-5, 3, 2 and 4 (Rabinowitz et al, 2002). AAV-8 is most efficient for infecting liver cells in mice, while AAV-2, -5, -8, and -9 appear more similar in efficacy in hepatic gene transfer in larger animals (Sarkar et al 2006; Nathwani et al, 2007). Similar differences in tropism exist for other tissues and cell types. AAV2, AAV2/7 or an AAV2/8 mixture, expressing HIV-1W61Dgp140 (env), Gag-Nef and HIV-1 RT were intramuscularly adminstered to three groups of Cynomolgus macaque (Calcedo et al, 2006). Peripheral blood mononuclear cells were collected at regular intervals after immunization and T cell mediated immune response was assessed over time. AAV2/7 or AAV2/8 induced a diverse T cell response towards Gag, Rt, Nef but not env whereas the AAV2 group displayed a strong response only towards env and negligible towards Gag, RT and Nef. In conclusion, a single intramuscular injection of AAV2/7 or AAV2/8 induced a diverse and stable T cell response and a quick and robust humoral response in Cynomolgus macaque monkeys. The immune response induced by AAV serotype 1 to 8 vectors expressing HIV-1 env gp160 was evaluated in BALB/c mice (Xin et al, 2006). Higher HIV-specific humoral and cell-mediated immune responses were induced by AAV1, AAV5, AAV7, and AAV8 vectors expressing the env gp160 gene than those the AAV2 vector produced. The AAV5 vector induced the best responses demonstrating that the immunogenicity of AAV vectors depends on serotype tropism and that AAV5 is a better vector than other AAV serotypes. Additionally, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. A truncated gag open reading frame was cloned into an expression cassette driven by a cytomegalovirus promoter between AAV2 inverted terminal repeats (Lin et

immune

1. AAV serotypes and their effect on immunization Humans commonly carry neutralizing antibodies to human serotypes of AAV such as AAV2 and AAV1 as a result of natural infections (Mingozzi et al, 2007a), and such antibodies have been shown in gene therapy trials to reduce gene transfer (Manno et al, 2003; Mingozzi et al, 2007a). AAV-specific neutralizing antibodies would also be expected to reduce the potency of rAAV vaccines therefore; research is focusing towards developing vectors with AAV serotypes that will not generate neutralizing antibodies. Seroprevalence rates of neutralizing antibodies to the capsid of AAV serotypes isolated from nonhuman primates would be expected to be lower than those to human serotypes, which may provide the former with an advantage over the latter. Occurrence of antibodies to AAV serotypes 1, 3, and 5 are common in humans, and they increase with age. 285

Vig et al: Recombinant adeno-associated virus as vaccine delivery vehicles al, 2007a). This vector plasmid was used to generate AAV vector particles with capsids from AAV1, AAV2, AAV5, AAV7, AAV8, and AAV9. Higher numbers of IFN-!expressing Gag CD8+ T cells were observed in vectors based on AAV7, AAV 8, and AAV9 compared to AAVs based on capsids from types 1, 2, and 5, with the lowest frequencies noted for AAV2/2. Vectors were ranked by the levels of Gag antibodies obtained as tracked by B-cell responses ranked as follows: AAV2/8, AAV2/7 and AAV2/9 (equal), followed by AAV2/1, AAV2/5, and AAV2/2. Gag tetramer specific CD8+T cells peaked at about 3 weeks following vaccination and was highest for AAV2/8. Responses were higher with vectors based on AAV1, AAV7, and AAV8 compared to AAV2, AAV5, and AAV9, which may relate to the vectorsâ&#x20AC;&#x2122; ability to efficiently transduce muscle cells.

Other experiments using virus antigens or tumor associated antigens encoded within rAAV2 vectors also demonstrated that the delivery route influenced the type of host immune responses. The env, tat, and rev genes of HIV were inserted into rAAV2 under the control of the CMV-IE promoter. The highest titer of specific serum IgG was observed in BALB/c mice after immunization with these vectors by IM as compared to IN, IP, or subcutaneous routes. In contrast, the highest secretory IgA titer was induced by IN inoculation (Xin et al, 2001). The impact of the route of administration of the AAV vector encoding human factor IX (hFIX) on the induction of an immune response against the vector and its xenogenic transgene product, hFIX was studied (Ge et al, 2001). Increasing doses of AAV-hFIX were administered by different routes to C57Bl/6 mice. The route of delivery had a profound impact on serum hFIX levels as well as the induction of an anti-hFIX humoral immune response. Delivery of AAV-hFIX by an IM route induced an antibody response against the human FIX protein and no hFIX was detected in the serum of animals. However, this was in contrast in mice that received AAV-hFIX by intraportal vein (IPV) administration. When pre-existing neutralizing immunity to AAV was established in mice, AAV-hFIX administration by either the IM or IPV routes did not result in detectable serum hFIX. Although hFIX expression was not observed in mice with pre-existing neutralizing immunity to AAV, an anti-hFIX response was induced in all of the animals that received AAV-hFIX by the IM route. This was not observed in the preimmune mice that received AAV-hFIX by IPV administration. The results suggest that the threshold of inducing an immune response against a secreted transgene product, such as hFIX, is lower when the vector is administered by the IM route even in animals with pre-existing immunity to AAV. The production of proinflammatory cytokines induced by adenoviral vectors depends on the vector dose, both in vitro (Liu et al, 2001) and in vivo, as does the intensity of the specific humoral anti-AAV response. In addition, high doses of the vector can induce tolerance to the transgene product, as shown with an AAV encoding human FIX and injected into mice via the intrahepatic route. Induction of tolerance seems to depend on the amount of transgene expressed, as is often the case with induced tolerance (Mingozzi et al, 2003). Low doses of vector can avoid induction of humoral immunity to the AAV capsid (Halbert et al, 1998; Manning et al, 1998). Neutralizing antibodies were elicited with IV and IM infection in rhesus macaques with AAV2 while intranasal (IN) infection did not elicite any response demonstrating that primary and memory immune responses were dependent upon both the route of infection and the presence of helper virus (Sun et al, 2003). In contrast, IN coinfection with wild-type AAV2 and adenovirus elicited neutralizing antibodies, lymphocyte proliferative responses to AAV2, and cellular infiltration in local tissue (Hernandez et al, 1999). A rAAV encoding woodchuck IFNa (AAV-IFN) to treat animals with chronic woodchuck hepatitis virus infection, a model of chronic hepatitis B was tested (Pedro et al, 2005). The vector was given by IP or IM route.

2. Route of administration and vector dose are critical components for AAV-based immunization Many studies have established that humoral and cell mediated immunity to AAV components or to transgenes varies with the route of administration of the vector. For example, hepatic gene transfer can induce immune tolerance to the transgene product by induction of regulatory CD4+CD25+ T cells and other T cell tolerance mechanisms (Dobrzynski et al, 2004; Cao et al, 2007a,b). On the other hand, muscle-directed gene transfer, resulting in transgene expression in muscle fibers following IM administration of vector, has been demonstrated to initiate a local immune response followed by a systemic response (Nathwani et al, 2001; Wang et al, 2005b). This route has thus far been the most utilized in rAAV-based vaccine studies. Other routes such as subcutaneous or administration to the respiratory tract remains to be studied in detail. Virus doses and the route of administration substantially effect on the magnitude of the host immune response to rAAV2 transduction as demonstrated by murine models. For induction of significant neutralizing antibodies in a C57BL/6 mouse lung model, 106 intrapulmonary rAAV2 particles per mouse were required. However, administration of vector at doses below this threshold dose also led to low level transgene expression (Halbert et al, 1998). In contrast, a higher amount of vector (108 per mouse) is needed to generate anti-vector immune response to rAAV2 given IM to C57BL/6 mice (Manning et al, 1998). Interestingly, lower rAAV doses could transduce some cell types without induction of host antivector immune responses, suggesting that vector readministration might be successful following a lower primary dose. Neutralizing activities against IM (Chirmule et al, 2000) or IV (Xiao et al, 2000) administered rAAV2 were entirely T-cell dependent, as mice lacking functional CD4+ T cells did not generate neutralizing antibodies. These findings were consistent with those obtained in MHC class I and II knockout mice (Manning et al, 1998). However, rAAV2 delivered to the liver of mice or rhesus monkeys induced transient neutralizing humoral immune responses that were T cell independent (Xiao et al, 2000).

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Gene Therapy and Molecular Biology Vol 12, page 287 Long-term transgene expression in the liver of woodchucks was detected after IP administration of an AAV encoding luciferase. In contrast, in the majority of the animals that received AAV-IFN through the portal vein, the expression of IFN! was transient (30-40 days) and was associated with a significant but transient decrease in viral load. Varying routes of vector administration thus may expose the same transgene product to differing immune pathways, which, in turn, may result in different immune presentation and responses.

2b mice recognized an epitope that was conserved between the AAV-2 and AAV-8 capsids. Cross-reactivity of AAV-specific CD8+ T cells induced by different AAV serotypes may have important implications for gene transfer and identification of these epitopes will facilitate studies of immune responses to the AAV capsids.

IV. Conclusions Vaccination has been achieved in numerous animal models; however, the successful use of AAV vectors as vaccines in animal models might not be easily replicated in humans, due to the ability of AAV to induce functionally impaired T cells and tolerance. For rAAV vectors, the magnitude and type of immune response is dependent on route of administration, the transgene itself and also expression levels as determined by serotype, promoter, and vector dose. A more thorough understanding of the interplay between rAAV and their encoded transgenes and the host immune system is necessary for the optimal development of a rAAV vaccine system. Therefore, efficient and successful gene delivery can be achieved by rAAV if the factors involved in generating immune responses are taken into consideration.

3. Immune response to AAV capsid proteins In vivo administration of AAV vectors leads to presentation of the viral capsid antigens to the B cells present within lymph nodes. This results in CD4+ T-cell activation, which in turn induces differentiation of B cells to plasma cells via cell cooperation mechanisms involving costimulatory molecules CD40-CD40L and cytokines such as IL-6 or IL-4. Antibodies produced by plasma cells are specific for viral capsid proteins; when they have neutralizing effects, these antibodies can prevent infection by the vectors during subsequent gene therapy attempts (Bessis et al, 2004). Results from a recent clinical trial on hepatic gene transfer of AAV2 vector showed that rAAV vector transduction in humans induces a CTL response against the input capsid antigen (Manno et al, 2006; Mingozzi et al, 2007b). The authors suggested that these capsidspecific CTLs eliminated rAAV transduced hepatcoytes in human subjects, thereby causing a loss in transgene expression, and that the CTLs represented memory CD8+ T cells preveiously generated during natural infection of AAV in the presence of helper virus. To address the ability of AAV capsid-specific CTLs to eliminate rAAVtransduced cells in mice, it was demonstrated that AAV2 capsid-specific CTLs could be induced by DCs with endogenous AAV2 capsid expression or pulsed with AAV2 vectors (Li et al, 2007a). Others used adenoviral vectors to generate CTL responses to AAV capsid in mice (Li et al, 2007b). In either case, the investigators were unable to demonstrate elimination of AAV vectortransduced hepatocytes by AAV2 capsid-specific CTLs in vivo in mice, even though the AAV capsid can induce a measurable CTL response. Humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration was characterized by Chirmule and colleagues in 2000. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. Studies strongly suggested that the B-cell response was T cell dependent. To understand the impact of AAV capsid-specific CD8+ T cells on AAV-mediated gene transfer, CD8+ T cell epitopes for AAV-2 and AAV-8 capsid in C57BL/6 (H-2b MHC haplotype) and BALB/c (H-2d MHC haplotype) mice were identified (Sabatino et al, 2005). Mice of both the H-2b and the H-2d haplotypes recognized epitopes on AAV-2 and AAV-8 capsids. T cells from H-

Acknowledgements This work was supported by NSF-CREST (HRD0734232), NSF-HBCU-UP (HRD-0505872) and NIH (2S06 GM008219-200012) grants.

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Manji HK, Moore GJ, Chen G (2000) Lithium up-regulates the cytoprotective protein Bcl-2 in the CNS in vivo: a role for neurotrophic and neuroprotective effects in manic depressive illness. J Clin Psychiatry 61 Suppl 9, 82-96. Masui T, Hashimoto R, Kusumi I, Suzuki K, Tanaka T, Nakagawa S, Kunugi H, Koyama T (2006a) A possible association between the -116C/G single nucleotide polymorphism of the XBP1 gene and lithium prophylaxis in bipolar disorder. Int J Neuropsychopharmacol 9, 83-88. Masui T, Hashimoto R, Kusumi I, Suzuki K, Tanaka T, Nakagawa S, Suzuki T, Iwata N, Ozaki N, Kato T, Kunugi H, Koyama T (2006b) Lithium response and Val66Met polymorphism of the brain-derived neurotrophic factor gene in Japanese patients with bipolar disorder. Psychiatr Genet 16, 49-50. Michelon L, Meira-Lima I, Cordeiro Q, Miguita K, Breen G, Collier D, Vallada H (2006) Association study of the INPP1, 5HTT, BDNF, AP-2! and GSK-3! GENE variants and restrospectively scored response to lithium prophylaxis in bipolar disorder. Neurosci Lett 403, 288-293. Mick E, Biederman J, Faraone SV, Murray K, Wozniak J (2003) Defining a developmental subtype of bipolar disorder in a sample of nonreferred adults by age at onset. J Child Adolesc Psychopharmacol 13, 453-462. Milner CM, Campbell RD (1992) Polymorphic analysis of the three MHC-linked HSP70 genes. Immunogenetics 36, 357362. Montezinho LP, Castro MM, Duarte CB, Penschuck S, Geraldes CF, Mork A (2006) The interaction between dopamine D2like and !-adrenergic receptors in the prefrontal cortex is altered by mood-stabilizing agents. J Neurochem 96, 13361348. Nelson-DeGrave VL, Wickenheisser JK, Cockrell JE, Wood JR, Legro RS, Strauss JF, 3rd McAllister, JM (2004) Valproate potentiates androgen biosynthesis in human ovarian theca cells. Endocrinology 145, 799-808. Okada A, Aoki Y, Kushima K, Kurihara H, Bialer M, Fujiwara M (2004) Polycomb homologs are involved in teratogenicity of valproic acid in mice. Birth Defects Res A Clin Mol Teratol 70, 870-879. Pae CU, Kim TS, Kwon OJ, Artioli P, Serretti A, Lee CU, Lee SJ, Lee C, Paik IH, Kim JJ (2005) Polymorphisms of heat shock protein 70 gene (HSPA1A, HSPA1B and HSPA1L) and schizophrenia. Neurosci Res 53, 8-13. Pae CU, Mandelli L, Serretti A, Patkar AA, Kim JJ, Lee CU, Lee SJ, Lee C, De Ronchi D, Paik IH (2007) Heat-shock protein70 genes and response to antidepressants in major depression. Prog Neuropsychopharmacol Biol Psychiatry 31, 1006-1011. Pan T, Li X, Xie W, Jankovic J, Le W (2005) Valproic acidmediated Hsp70 induction and anti-apoptotic neuroprotection in SH-SY5Y cells. FEBS Lett 579, 6716-6720. Patel NC, Delbello MP, Keck PE, Jr. Strakowski, SM (2006) Phenomenology associated with age at onset in patients with bipolar disorder at their first psychiatric hospitalization. Bipolar Disord 8, 91-94. Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS (2001) Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem 276, 36734-36741. Pilon M, Schekman R (1999) Protein translocation: how Hsp70 pulls it off. Cell 97, 679-682. Rao JS, Bazinet RP, Rapoport SI, Lee HJ (2007) Chronic administration of carbamazepine down-regulates AP-2 DNA-

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Inhibition of focal adhesion kinase with her-2 targeted antibody pertuzumab (Omnitarg®, 2C4) in breast cancer cells Research Article

Emel Canbay1,*, Bala Gur-Dedeoglu2, Betul Bozkurt3, Melih Karabeyoglu3, Bulent Unal4, Osman Yıldırım3, Omer Cengiz3, Isik G Yulug2 1

Istanbul University, Istanbul Medical Faculty, Department of General Surgery, Capa-Istanbul, 34340 Department of Biology, Bilkent University, Faculty of Science, Bilkent- Ankara 3 Ankara Numune Teaching & Research Hospital, II. Surgery Clinic, Sihhiye-Ankara 06100 4 Inonu University, Faculty of Medicine, Department of General Surgery, Malatya-Turkey 2

__________________________________________________________________________________ *Correspondence: Dr. Emel Canbay, Calislar Caddesi, Albay Ibrahim Karaoglanoglu sokak, No: 34/10 Bahcelievler-Istanbul, Turkey 34160; Tel/Fax: + 90 212 642 3357; e-mail: dremelcanbay@gmail.com Key words: Pertuzumab, Omnitarg®, 2C4, focal adhesion kinase, breast cancer Abbreviations: American Type Culture Collection, (ATCC); bovine serum albumin, (BSA); Dulbecco’s modified Eagle’s medium, (DMEM); epidermal growth factor receptor, (EGFR); Focal adhesion kinase, (FAK); phosphatidylinositol 3-kinase, (PI3K) Received: 17 May 2007; Revised: 1 December 2008 Accepted: 5 December 2008; electronically published: December 2008

Summary Pertuzumab (Omnitarg!, 2C4) is a recombinant humanized monoclonal antibody targeted to extracellular region of HER-2. Previous results proved the inhibitory effect of Pertuzumab on the survival of breast cancer cells via MAPK and Akt pathway. Focal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. Here, we aimed to investigate the effects of Pertuzumab on ligand activated total FAK expression and phosphorylation in the HER-2 overexpressing BT-474 breast cancer cell line. Heregulin was used for ligand activation. We have found that FAK expression and phosphorylation were inhibited in with Pertuzumab in breast cancer cells.

activated signaling from HER-2/HER-1 and HER-2/HER3 heterodimers (Agus et al, 2002). It has been shown that the signaling pathways and cellular processes associated with tumor growth and progression could be inhibited with Pertuzumab both in vitro or in vivo models (Agus et al, 2002). Pertuzumab has undergone phase I trials in patients with advanced solid malignancies (Agus et al, 2005; Albanell et al, 2008) and is currently in phase II clinical trials in NSCLC, metastatic breast, ovarian, and prostate cancers (Friess et al, 2005). The invasion and metastasis of cancer is the process that includes changes in cell adhesion and motility that tumor cells gain the ability to invade and migrate through the extracellular matrix. FAK is a tyrosine kinase considered to be a central molecule in integrin mediated signaling, and it is involved in cellular motility and protection against apoptosis (Parsons et al, 2000).

I. Introduction The HER-2 (c-erbB-2, neu) gene encodes a 185-kDa transmembrane glycoprotein that is a member of the epidermal growth factor receptor (EGFR or erbB) family of receptor tyrosine kinases. HER-2 mediates signal transduction, resulting in mitogenesis, apoptosis, angiogenesis, and cell differentiation (Ménard et al, 2000). The HER-2 gene is amplified and overexpressed in 2030% of invasive breast carcinomas, and is associated with increased metastatic potential and decreased overall survival (Slamon et al, 1987; Ménard et al, 2000). Pertuzumab (Omnitarg!, 2C4; Genentech) is a humanized monoclonal antibody against the dimerization domain of HER-2. This agent is the first in a new class of targeted therapeutics known as HER-2 ‘’dimerization inhibitors’’ (Franklin et al, 2004). In contrast to Trastuzumab, Pertuzumab sterically blocks HER-2 dimerization with other HER receptors and blocks ligand293

Canbay et al: FAK Inhibition with Pertuzumab (Omnitarg®, 2C4) difluoride (PVDF) membrane. Total FAK (p125) and PhosphoFAK protein (pY 397) were detected by rabbit anti-human FAK (TaKaRa) and anti-human phospho-FAK (Invitrogen) polyclonal antibodies and were used at a 1:1000 dilution in 4% BSA in PBS-Tween-20. Horseradish peroxidase-conjugated anti-rabbit antibody was obtained from Sigma (USA) and was detected using enhanced chemiluminescence (Amersham-Pharmacia Biotech, Piscataway, NJ).

The aim of the present study was to assess the effects of Pertuzumab on the expression and tyrosine phosphorylation of FAK in HER-2 overexpressing BT-474 breast cancer cells.

II. Materials and Methods A. Materials Pertuzumab (Genentech) was provided in freezed dried powder at 50 mg. Heregulin -! and. Calnexin were purchased from Sigma and the anti-Human FAK antibody (M135) was purchased from TaKaRa laboratories. Anti-phospho FAK (pY397) antibody was purchased from Invitrogen. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were obtained from Sigma (USA).

III. Results A. Immunohistochemical analysis of FAK Figure 1A shows the basal FAK phosphorylation without ligand activation in Her-2 overexpressed BT-474 cell line (+2). HER-2 dimerization with HER-3 or HER-4 was induced with 10 ng/ml Heregulin for 2 hours in BT-474 cell line which is used as an internal positive control of the experiment (Figure 1B). FAK immunostaining revealed strong membranous and cytoplasmic staining with 10 ng/ml of Heregulin treatment for 2 hours in BT-474 cells compared to control cells (+3). Figures 1C and 1D show FAK expression in BT-474 cells with 1"g/ml and 10"g/ml Pertuzumab in the presence of 10 ng/ml Heregulin for 2 hours, respectively. The FAK expression was gradually decreased with the increasing amount of Pertuzumab at a concentration of 1 µg/ml and 10 µg/ml after 2 hours (+1). Figure 2A shows the basal FAK phosphorylation in BT474 cells without Heregulin for 24 hours. Her-2 dimerization was activated with 10ng/ml Heregulin for 24 hours in BT-474 cell line (Figure 2B).Figures 2C and 2D show decrement of FAK expression with either 1"g/ml or 10"g/ml Pertuzumab in the presence of 10 ng/ml Heregulin for 24 hours in BT-474 cell line. The cells in each condition were seeded triplicate and counted from minumum 10 areas of the slides and the ratio of positive cells to whole cell count of three experiments was calculated as percentage. Figure 3 shows the mean value of three experiments ± SD. The decrease of FAK expression was not significant in the cells treated with 1"g/ml and 10 "g/ml Pertuzumab in the presence of 10ng/ml Heregulin for 2 hours when compared to the ones treated only with Heregulin (p>0.05). However, a significant decrease was present in the FAK expression in the cells treated with 1"g/ml and 10"g/ml Pertuzumab for 24 hours compared to treatment with 10 ng/ml Heregulin alone for 24 hours (p=0.04 and p=0.005, respectively).

B. Cell culture BT-474 breast cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 1 mM glutamine, 10U/ml penicillin G and 10ug/ml streptomycin at 37oC in 5% CO 2-containing atmosphere.

C. Immunohistochemistry BT-474 cells were plated in 6 well-plates (150.000 cells/well) on coverslips one day before the experiment. After 24 hrs, cells were treated with 10ng/ml Heregulin to induce HER-2/ HER-3 or HER-4 dimerization in the cells. Heregulin concentration was used as previously described (Gregory et al, 2005). One and 10 mg/ml of Pertuzumab was added to wells and incubated for 2 to 24 hours. Each experiment was performed in triplicate and repeated three times. After 2 and 24 hours of incubation with Pertuzumab cells were fixed with 4% paraformaldehyde for 20 mins at room temperature and permeabilized with Sodium citrate for 5 mins at 4°C. Blocking was done with 10% bovine serum albumin (BSA) for 30 mins. And the cells were incubated with FAK (p125) antibody. HRP conjugated streptavidin was used in order to visualize the signal of the protein. The signal was developed with a substrate DAB (DAKO). Finally counterstaining was performed by hematoxylene staining. The positive staining of the Heregulin treated HER-2 overexpressing BT-474 cells served as the positive internal control of the experiment. FAK-positive cells were counted in each slide and the ratio of the positive cells to the whole cell count was calculated as percentage. Minimum ten areas were counted from each triplicate slides and the standard deviations were calculated from the mean of the counts. If more than 20% of carcinoma cells were stained more intensely than that of untreated cells the sample was classified as strong FAK overexpression (3+) (Langer et al, 2004; Zinner et al, 2004; Krug et al, 2005). When the FAK immunostaining was equal compared to that of control, the sample was classified as intermediate expression (2+). When the FAK immunostaining was weaker than that of internal control, the cells were classified as low FAK expression (+1). The lack of FAK immunostaining was classified as negative (0).

B. Evaluation of expression phosphorylation of the FAK immunoblotting

and with

We further confirmed our immunostaining results with immunoblotting. FAK expression was gradually decreased with the increasing amount of Pertuzumab at a concentration of 1"g/ml and 10"g/ml either in 2 or in 24 hours (Figure 4). We also investigated whether Pertuzumab could modulate protein phosphorylation of the signal transduction molecule- the FAK. FAK phosphorylation was strikingly inhibited with Pertuzumab in dose-dependent manner after 24 hours. Figure 5 shows the decrement of FAK phosphorylation in response to Pertuzumab in BT-474 breast cancer cells.

D. Immunoblotting BT-474 cells were treated with 10 ng/ml Heregulin and at the same time 1 and 10 µg/ml pertuzumab was added onto the cells. Cells were harvested using lysis buffer (10mM Tris-Cl, pH 7.6, 5mM EDTA, 50mM NaCl, 30mM Na-pyrophosphate, 50mM NaF, 100"M Na-ortovanadate, 1% Triton X and 1mM PMSF). Equal amounts of protein extracts were loaded in 8% SDS-polyacrylamide gel and transferred onto polyvinylidene

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Figure 1. Expression of FAK in BT-474 cells for 2 hours. (A) Basal FAK expressionwithout HER-2 dimerization (B) with 10ng/ml of Heregulin (C) with 1µg/ml Pertuzumab+ 10ng/ml Heregulin (D) with 10µg/ml Pertuzumab +10ng/ml Heregulin.

Figure 2. Expression of FAK in BT-474 cells for 24 hours. (A) Basal FAK expression without HER-2 dimerization (B) with 10ng/ml of Heregulin (C) with 1µg/ml Pertuzumab + 10ng/ml Heregulin (D) with 10µg/ml Pertuzumab + 10ng/ml Heregulin.

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Figure-3. Pertuzumab inhibits FAK expression with immunostaing. Each immunostaining experiment was done in triplicates. The cells were counted and the ratio of positive cells to whole cell count was calculated as percentage. Minimum 10 areas from each slide were counted. The standart deviations were calculated according to the whole cell count. The decrease of the FAK expression is significant in the cells treated with 10 µg/ml Pertuzumab for 24 hours when compared to the ones treated with 10ng/ml Heregulin for 24 hours (p=0.005). Likewise a significant decrease is present in the FAK expression in the cells treated with 1 µg/ml Pertuzumab for 24 hours (p=0.04). Decrease in FAK expression was not significant in the cells treated with 1 µg/ml and 10 µg/ml 2C4 in the presence of 10ng/ml heregulin for 2 hours.

Figure 4. Pertuzumab decreases the expression of FAK with immunoblotting.The BT-474 cells were treated with 1 µg/ml and 10 µg/ml of Pertuzumab in the presence of 10ng/ml Heregulin for 2 and 24 hours. The inhibitory effect of Pertuzumab on FAK expression was observed in the cells treated with 10 µg/ml of Pertuzumab for 24 hours. The equal loading was adjusted with calnexin according to the Comassie blue staining.

Figure 5. Pertuzumab decreases the phosphorylation of FAK. The BT-474 cells were treated with 10ng/ml of Heregulin for 2 and 24 hours. Meanwhile they were treated with 1 µg/ml and 10 µg/ml of Pertuzumab. The inhibitory effect of Pertuzumab on phosphorylatedFAK was observed in the cells treated with 10 µg/ml of Pertuzumab for 24 hours. The equal loading was adjusted with calnexin according to the Comassie blue staining.

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Gene Therapy and Molecular Biology Vol 12, page 297 signals (Rozengurt, 1995). In recent studies, Vadlamudi and colleagues utilized human breast cancer cell lines in vitro to establish a novel signaling pathway involving HER-2, phospho-Src Tyr-215 and phospho-FAK Tyr-861 leading to increased cellular motility (Vadlamudi et al, 2002, 2003). The authors showed that heregulin-induced HER-2 activation resulted in phosphorylation of FAK at tyrosine 861. Further support to our study was reported by Schmitz et al. They have reported that HER-2 and FAK associated signaling in tumor tissue of breast cancer patients (Schmitz et al, 2005). A recent study also identified frequent polysomic patterns for chromosome 1, chromosome 8 and chromosome 17 that are indicative for increased tumor malignancy in breast cancer (Nakopoulou et al, 2002). The FAK is located on chromosome 8 and the HER-2 is located on chromosome 17. These polysomic patterns can be lead to the alterations in HER-2 and FAK expression and signaling in breast cancer. In the present study, a significant downregulation of FAK expression and phosphorylation with Pertuzumab was observed, suggesting that Pertuzumab may serve as a potential important anticancer agent for breast cancer. Increased FAK expression and phosphorylation by ligand activated HER-2 signaling and inhibition with Pertuzumab indicating that FAK also could be an important pharmacologic target site and whether FAK is the upstream molecule of MAPK/Akt pathway of apoptosis and/or metastasis remains to be investigated.

IV. Discussion In this report, for the first time we have shown that Heregulin activated total FAK expression and FAK phosphorylation were inhibited with Pertuzumab in BT474 HER-2 overexpressing breast cancer cell line. Even though the surgical techniques and the adjuvant therapies have been proven to be useful in the treatment of primary tumors (Entschladen et al, 2004), invasion and metastasis remain a major cause of poor prognosis and death in cancer patients. Trastuzumab monotherapy offers clinical benefit to a subset of HER-2-overexpressing metastatic breast cancers. However, the majority of breast cancers that initially respond to Trastuzumab-containing regimens begin to progress again within 1 year (Albanell and Baselga, 2001). The recombinant humanized HER-2 monoclonal antibody Pertuzumab sterically blocks dimerization of HER-2 with other HER receptors (Agus et al, 2002) known as ‘’HER dimerization inhibitors’’. Reports from phase I and II trials indicate that Pertuzumab plays an important role in the inhibition of the solid tumors progression including breast cancer (Parsons et al, 2000; Friess et al, 2005; Albanell et al, 2008). Beside these reports, which ligand activated HER-2 signaling molecules inhibited by Pertuzumab are not completely detected. Signaling pathways activated by HER-2 include the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK cascades (Mendoza et al, 2002; Nahta et al, 2004). The reports of studies on the effects of Pertuzumab show that inhibiting the survival of breast cancer cells via MAPK (Agus et al, 2002) and Akt pathway (Nahta et al, 2004). The combination of Trastuzumab and 2C4 reduced the serine phosphorylation of Akt whereas signaling from the MAPK cascade was not inhibited (Nahta et al, 2004). Previous studies also show that Pertuzumab inhibited Heregulin-activated mitogenic signaling in breast and prostate cancer models in vitro and in vivo because of dissociation of HER-2/HER-3 dimers (Agus et al, 2002; Mendoza et al, 2002). FAK is a cytoplasmic tyrosine kinase that plays an important role in integrin-mediated signal transduction pathways closely related to cell adhesion, motility, and growth (Parsons et al, 2000; Parsons et al, 2000; Schlaepfer et al, 2004). Upregulation of FAK expression is associated with oncogenesis (Cance et al, 2000) and decrease in FAK is associated with the loss of ability to attach (Mitra et al, 2005), decreased migration (Schlaepfer et al, 2004) and induction of apoptosis (Parsons, 2003). In our study, the FAK expression and phosphorylation were increased in response to HER-2 dimerization induced by Heregulin. Specifically, FAK is phosphorylated at multiple sites in cells stimulated by mitogenic agonists that act via heptahelical GPCRs including bombesin (Zachary et al, 1992; Salazar and Rozengurt, 2001) and lysophosphatidic acid (Seufferlein and Rozengurt, 1995), ligands of tyrosine kinase receptors, including EGF (Leventhal et al, 1997; Ojaniemi and Vuori, 1997), integrin clustering induced by cell adhesion (Owen et al, 1999; Ruest et al, 2000) and activated variants of pp60src (Guan and Shalloway, 1992; Parsons and Parsons, 1997). It is increasingly recognized that FAK functions as a point of convergence and integration in the action of multiple

Acknowledgements We thank Ms Dina Washington for Pertuzumab (Omnitarg!, Genentech). This work was supported by T.R.State Planing Organization Project No: T-256 (E.C.). EC is rotational specialist in Department of General Surgery, Breast Unit, Istanbul University, Istanbul Medical Faculty.

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