Ribograma new directions

New Directions in Cancer Research RIBOGRAMA: - new relevant biomolecular concept.

- Its application in cancer research.

The research, which now presents itself, is part of the XV Chapter of the book "COLORECTAL CANCER - DIAGNOSIS BASES OF BIOMOLECULAR.

Has been prepared in the framework of a doctoral thesis at the Faculty of Medicine of the Universidad Complutense de Madrid, under the direction of Professor JLBalibrea Cantero, and aims to develop the concept RIBOGRAMA. Starting on a based development with techniques and current concepts of Molecular Biology, it was possible to arrive to the concept of RIBOGRAMA, which is a method of diagnosis, screening and monitoring of disease of the colon and rectum cancer (or of any other organ).

This method allows the diagnosis in the stage of "pre-carcinoma in situ" since it is based on the phenotype of the cells of the mucosa showing very high amounts of free ribosomes in their cytoplasm, which can be quantified by a technique of counting particles with flow cytometry after its isolation and labeling with fluorochromes.

With the successive records of such quantities of free ribosomes it can be drawn a graphic curve (RIBOGRAMA), which above a certain level of concentration allows to say that there is a trend of malignant tendency of the underlying tissue/ cells (the colon and rectum, for example.). It can be considered that we are facing a process of development of carcinoma of the colorectal mucosa, before any gross direct visualization (endoscopy).

In other words, this constitutes a new method, at biomolecular level, evident, as demonstrated in this research, which allows a good advance of time on the microscopy.

This method gives a previous and advanced information over other noninvasive colorectal cancer diagnostic methods (test for fecal occult blood test and DNA mutations in the same stool), because it gives a vision of the way and the direction of the behavior of the phenotype.

Thanks to this method it is possible to design a screening program (with a noninvasive method, with strong backing of the population), by which all people with significant elevation RIBOGRAMA curve should be checked with a mandatory endoscopy.

You can then move on to a therapeutic solution, because we act with the security of a diagnosis - and at the same time can keep the patient under observation and therapy (post-diet food surveys and NSAIDs and aspirin).

1

The NSAIDs have proven efficacy in preventing and decreasing the tendency to the formation of precancerous polyps or lesions, so the RIBOGRAMA method represents savings in the investment and treatment of the cancer relatively to the cost implications of treatment of oncological disease.

This study starts from the observation of several lines of evidence, some wellestablished and new, who hold sharply the idea that RNA content is elevated in cancer cells and genetic events that lead to cancer are often linked, directly or indirectly, the ribosome biogenesis (1,2).

On the other hand, the inverse problem is checked in the cases of "idiopathic ineffective anemia (idiopathic Ineffective erythropoiesis-IIE), in which there are insufficient rates of cell proliferation, and patients have erythroblasts containing only 70% of normal levels rRNA (3).

Researchers have shown that cancer cells with high expression of ribosomal proteins have higher content of ribosomes. The genetic alterations associated with cancer development involves, very often, changes in signaling pathways that lead to the rDNA effect (4.5). The scenario described above reiterates what is known by cancer biologists: ribosome biogenesis and tumorigenesis are closely linked.

Our purpose with this basic knowledge of molecular biology is to define some guiding principles about the value of the increased number of free ribosomes, as a clinical manifestation of the dynamics of biochemical process toward malignant cells in a specific cellular community, or organ tissues, by checking and verification in the field of electron microscopy observations of the quantities of free ribosomes in neoplastic tissues, or pre-malignant, and construction of a graph curve (RIBOGRAMA) of the quantities of free ribosomes in these tissues under study, which can define the tendency of malignant cell community under certain conditions that predispose to the growth of tissue in the direction of the malignancy.

MATERIAL AND METHODS It was done a retrospective study of 205 cases of electron microscopic observation on cells or tissues in a state of "premalignizaci贸n", or malignant phenotype, collected from the database of PubMed, relatively to descriptions of cases of well-established pathological diagnoses, published in appropriate scientific journals and credible in scientific community to address issues of molecular biology, oncology and biomedical science issues existing in the literature of the life and health sciences.

These descriptions have reference to cells or malignant tumor tissues (or potentially to equivalent growing tissues compared with malignant). Those descriptions of selected cases were collected from key data describing the morphology found in the respective electron microscopy observation, especially 2

in the morphological elements of the texture of the cell, such as cellular organelles.

In its global aspect every cell of a malignant clone has a good idea on the morphology of a malignant cell phenotype. The organization and registration of data describing the electron microscopic observations, collected from PubMed for the database, comprise the enrollment which drew five columns, corresponding to the following five items (TABLE I - ANNEXES) A) - Order number of the case under study; B) - Quantitative description of free ribosomes of the cells of a particular tissue or organ under study; C) - Brief description of the main generic aspects in terms of electron microscopy observation of the cell organelles; D) - Information of the pathological diagnosis relative to the tissue or organ under study, E) - Reference to the respective references in the scientific medical literature (credible and accepted by the scientific research media and/or institutions of I+D).

OBJECTIVE Description of the quantitative aspects relatively to the free ribosomes of the cells of a particular tissue or organ under study: In the column of the item “quantitative description of the free ribosomes” are found some expression groups, in an universe of 205 observations, which have been used repeatedly by researchers of electron microscopy, which essentially are distributed in six groups: I - NUMEROUS FREE RIBOSOMES………………… ……………..36 cases II - RICH IN FREE RIBOSOMES……………………………….………. 5 cases III - PLENTY OF FREE RIBOSOMES………………….……………….36 cases IV - MANY FREE RIBOSOMES………………………………………... 19 cases V - INCREASED NUMBER OF FREE RIBOSOMES………..……… 28 cases VI - FREE RIBOSOMES…………………………………………..……...70 cases The terms "numerous free ribosomes" "rich in free ribosomes" “abundant free ribosomes”, "many free ribosomes" and "increased number of free ribosomes”, used in the descriptions of electron microscopy, like TABLE 1 – ANNEXES, are equivalent, but are subjective, because they do not allow reproducible numerical/mathematical results. Some other expressions have not been highlighted (have been used in TABLE 1 – Annex), because are not so common in all the 205 cases under study.

In group VI, with 70 cases (corresponding to 33.7% of the 205 selected cases), in which is used the expression "free ribosomes", there is no idea of the amount (increased or decreased) of free ribosomes descriptions of electron microscopy, despite the pathological diagnoses described in Table VI - ANNEXES.

3

Through the other 124 cases (corresponding to 59.6% of the 205 selected cases with electron microscopic morphological descriptions), in which are used the terms "numerous free ribosomes," "rich in free ribosomes", "abundant free ribosomes", "many free ribosomes" and "increased number of free ribosomes", it is not possible to quantify numerically/mathematically the increase in free ribosomes.

From the subjective nature relatively to the amount of free ribosomes, as shown in Tables I, II, III, IV, V (see Annex) a diagnosis of malignancy only can be made just with the finding that corresponds to the idea of the constancy property that consists of an increase in free ribosomes in cells that are in the process of multiplication without self control, as in the case of malignant cells.

In the TABLE 1 - ANNEX is the description of electron microscopy of cell organelles obtained through a search on PubMed database, using as search parameters the terms "free ribosomes" and "cancer cells" In each of the "groups of words" was counting the number of cases of tissues or organs with malignant phenotype unequivocally confirmed and, relative to total sample of that group. Was obtained and the percentage of tumors and malignant tissues for each of the six "groups of words", as shown in TABLE II: TABLE II Table of sample of each "group "groups of of terms" expressions"

malignant phenotypes for each "group expression"

% of malignant tissue

I

36

33

92 %

II

5

5

100 %

III

36

34

94,4 %

IV

19

16

84,2 %

V

28

22

78,6 %

VI

70

59

84,29 %

With the results presented in Table II one verifies that the amount/density of free ribosomes is one of the fundamental and obvious phenotypic aspects of a cell with characteristics of malignancy. So, the important thing is finding a language not subjective, reproducible, with mathematical translation, through a curve at which RIBOGRAMA call.

Such a curve reflects a dynamic idea of the trends of changes in numerical values or quantities of all the free ribosomes. The amount/density of free ribossomas is the phenotype of the trend, of the malignancy of a cell, relatively

4

to a normal range, of a community of cells under observation in regard to biomolecular behavior, representing a malignant growth of the cell phenotype.

DISCUSSION ACCUMULATION OF RIBOSOMES IN THE PROCESS ONCOGENESIS In the decades of 60 and 70 were published studies on free ribosomes and ribosomes bound to membranes (22, 23) and accumulation during induction of growth in many organs and tissues (17-20).

In the sequence of these investigations were published studies that have attempted to quantify the accumulation of ribosomes in interfollicular areas of the dorsal skin of mice during chemically induced neoplastic growth in two phases, initiation by 7,12-dimethylbenz (a )anthracene and promotion caused by 12-O-tetradecanoyl-phorbol-13-acetate (25).

The epidermis is a surface epithelium, and other epithelia, which are in direct contact with the external environment, such as the linings of the respiratory and gastrointestinal tract, shows a high incidence of neoplasms (21). Thus, the epidermis has served as a useful model to establish a comprehensive development of the role of ribosome accumulation during neoplastic growth (24).

It will be very important to produce predictive mechanistic models (counting free ribosomes) based on tumor dynamics, with the translation in the cell phenotype, for example, the count of free ribosomes in a cell as the phenotypic alteration process, under the oncogenic stimulus.

It will be useful for clinical practice that clinical and basic researchers conceive meaningful tests instead of the relatively advanced biological details of the oncogenesis and tumor progression, depending on countless factors variables.

Like many fields of life sciences, biology of cancer is an exponentially growing field, complex, involving work which has a range from molecular biology of the oncogenes to the environment epidemiology. Survival rates for various cancers, once they manifest clinically, have shown modest improvement in last decades.

The identification of changes in the structure of the cytoplasm, such as the sharp increase in the number of ribosomes (quantitative/numerical method) accumulated in the cytoplasm of a community of cells of a given tissue may be one of the phenotypic expression of malignancy of the fine structure of the transformed cells, which may be essential to characterize the evolution of cell behavior.

With this counting process of ribosomes will be possible to follow the evolution of changes in the quantity of the ribosomes, in a sense of monitoring the behavior of a community of cells, as well as the influence of the composition and concentration of mutagens of the medium in which are these cells.

5

In this sense, for example, one could study graphic profile of changes in the number of ribosomes per cell (or per unit volume) in a defined time period, within an average of the exfoliated cells of colorectal mucosa a patient whose records will form a sequential curve pattern/average number of free ribosomes per unit volume (RIBOGRAMA) in relation to the cells lining the colon and rectum, isolated and separated from the feces.

This may allow a strong motivation to integrate different fields of knowledge in cancer biology (and other fields of the biosciences and also life sciences), to introduce a new conceptual and theoretical framework that can improve the understanding of researchers on the dynamics of tumor formation to develop better methods of prevention, diagnosis and therapeutic.

While it is true that cancer is a multifaceted disease with a variety of close "triggers" in different tissues and different patients, there is also a strong possibility that cancers share a central feature originating from a common cellular machinery from which cells depend for their proliferation (11).

The most visible aspect of aggressive malignant neoplasms is increased cell proliferation, which has at its core a marked increase in protein synthesis. In the process of normal mitogenic response there is a transient and cyclical increase in the rate of general protein synthesis.

The overall increase in protein synthesis is a necessary phenomenon observed controlled before cell division, leading to duplication of content and the increase in size before the normal mitosis. Thus, the average size of the cells is maintained during physiological proliferative response. One of the key mechanisms of loss of control of protein synthesis in transformed cells is the inability to decrease the number of ribosomes that is correlated with cell proliferation in fresh culture media without addition of serum growth factors (6).

Many researchers have observed, in tissue cultures, differences in growth properties between normal cells and their malignant counterparts, one of which points to the failure of these to show a cyclic variation of cellular and biochemical parameters through the cell cycle or growth cycle (7-10).

Previously, other researchers have given attention to changes that occur in the rate of protein synthesis and function of "translational" machines of the cell, relative to the cell cycle, once such changes are necessary transitions for growth and multiplication in normal conditions.

In skin tumors in mice induced by the application of tumor promoters, the RNA: DNA and RNA content (percentage of dry mass contributed by RNA) were 2 to 3 times higher than in normal tissues (12) In several types of leukemia, the cell RNA content was strongly correlated with accelerated cell growth kinetics and patient prognosis (13).

6

In a study of gynecologic cancers in the neoplastic tissues were compared with their normal counterparts, the DNA and RNA content in the neoplastic tissues were increased 1.6 and 2.4 times, respectively (14). Similarly, cellular RNA content was increased by a factor of 1.4 in myc-transfected neuroblastoma cells relatively to normal cells (15).

In another study of breast cancer the DNA content cell test was normal, but the cellular RNA content was well correlated with tumor grade, histological type, with the hormonal status and patient survival (16).

Ribosome biogenesis The ribosome biogenesis and translation control are essential cellular processes controlled at many levels. Several tumor suppressor and protooncogenes have been blamed for altering the formation of mature ribosomes and regulate the activity of proteins known as translation factors.

The disturbance in one or more of the steps that control protein biosynthesis has been associated with alterations in cell cycle and in regulating cell growth.

Therefore, certain tumor suppressors and proto-oncogenes can regulate malignant progression by altering the protein synthesis machinery.

The production of mature ribosomes, which are responsible for cellular mRNA translation, requires a process "multistep" that is highly coordinated in eukaryotic cells. The ribosome, which is the main factory of protein synthesis, can be seen as a finely regulated machine that functions as a static component of the ordered central complex at superior levels.

In fact, ribosomes have the task to, correctly and efficiently, produce all the proteins in the cell. Although it is known that in cancer cells the components of the translation machinery are disturbed, or poorly expressed, its role in tumorigenesis has long been forgotten. For example, in the early 70's, the changes in the nucleolus have been recognized as an important marker of cell transformation (26).

Mutations in the genes that encode proteins that are directly involved in ribosome biogenesis are associated with cancer and other diseases. The gene DKC1 (Dyskeratosis Congenita) is mutated in patients with dyskeratosis congenita; a disease characterized by premature aging and increased susceptibility to cancer (27, 28).

The dyskerin DKC1 encodes a pseudouridine synthase that makes the posttranscriptional mediation of ribosomal RNA. Were identified mutations in the gene encoding ribosomal protein S19 in another syndrome that is characterized by increased susceptibility to cancer - Diamond-Blackfan anemia (29).

Growth and cell proliferation are associated with changes in the rate of production of ribosomes. During G1 is a prerequisite that is the increase in 7

rRNA synthesis and assembly of ribosomes for protein synthesis increased during S phase (30).

Moreover, it may be necessary regulate ribosomes in low activity or its formation, or both, during the M phase, to ensure proper output of the cell cycle (31, 32). So, there is a significant relationship between cell cycle and the production of ribosomes.

This balance is maintained in the cell through the checkpoints, which ensure that the translation of mRNA occurring at appropriate levels and during a window of the cell cycle. The rRNA synthesis is the first event in ribosome biogenesis. It is dependent on the regulation of rDNA by RNA polymerase I (Pol I) in the nucleolus. The rRNA synthesis in the cell can be induced by extracellular stimuli at certain times when a cell needs to grow and proliferate.

The concept of regulation of rRNA synthesis was originally tested on cells in that deprivation of an amino acid resulted in a rapid termination of rRNA synthesis (33). Since then, many other articles have shown that the initiation of rRNA transcription is intimately linked to cell cycle progression. The rRNA synthesis is maximal in S and G2 phases and suppressed in mitosis and increased in G1 (32, 34, 35).

These fluctuations in the synthesis of rRNA, dependent of cell cycle, are dependent on the activity of Pol I. The transcription factor UBF (upstream binding factor) is a key regulator of rRNA synthesis having the ability to modulate the transcriptional activity of Pol I (36-38). Furthermore, several proto-oncogenes and tumor suppressors directly regulate rRNA synthesis by enhancing or suppressing the activity of UBF, respectively.

The first protein identified that regulates the activity of UBF has been the CKII Casein Kinase II (41), a serine threonine kinase that has increased expression in many cancers, including leukemias and solid tumors. The CKII has been responsible for contributing to tumorigenesis through direct interaction with the cell cycle machinery. In addition, this phosphorylates UBF in the carboxyl terminus and thus regulates the transcription of rDNA. There are subsequent studies that have examined other UBF kinases that are also disarray in cancer and similarly affect rRNA synthesis (39, 40). It has been known, for over 25 years, that the rate of proliferation and cell growth is proportional to the rate of protein synthesis (42, 43). In addition, increased deregulation of UBF kinases in cancer can stimulate rRNA synthesis and contribute to its oncogenic properties.

Therefore, the disruption of control of protein synthesis may make cells more susceptible to disturb the growth and proliferation. Deregulation increased the rproteins in cancer cells corresponds reasonably to their involvement in the production of ribosomes. Similarly, the increase in Pol I transcriptional activity resulting in increased synthesis of rRNA, r-proteins could also regulate the number of functional ribosomes in the cell. In both cases the cells contain more 8

ribosomes have an increased rate of translation, which promote cell transformation (44, 45).

Growth and cell proliferation are associated with changes in the rate of production of ribosomes and ribosome biogenesis and may serve as a sensor for cells overstep important "checkpoints" during the cell cycle. In the transformed cells, showing increased production of ribosomes in the cell cycle, the number of ribosomes can be altered as a result of alterations in ribosome biogenesis.

This system of ribosomes tight self-regulation and the cell cycle may function to maintain cellular homeostasis. Therefore, an increase in the number of ribosomes, due to some upright effect, affects protein translation and may contribute to the transformation process. However, presently it is not possible to know in particular if there is any "cross-talk" between the ribosomes and the cell cycle.

THEORETICAL CONSIDERATIONS ON A CURVE CHART RIBOGRAMA In the scheme of Fig. 1.15 are designed the two axes, which represent the horizontal and the vertical axis, that represent the coordinates relative to which it is intended to build a graphical curve. This curve is characterized by two variables on the free ribosomes of cells under study (the epithelial cells of colorectal mucosa or colonocytes).

These variables are: a) - the "quantity (number) average of free ribosomes� in the cytoplasm of each cell and b) -"time" over which is observed the behavior of certain variations of the "quantity (number) mean free ribosomes�.

It will be necessary to construct experimental profile graphs of numerical values of "quantity (number) mean free ribosomes" that can be set as normal, from which one can consider a curve-middle-register as normal. Thus, it is possible to make comparative studies of large population groups, from which it will be possible to see a potential multiplicity of malignant disease trends or other diseases (through rising, or a variation, of the ribograma curve) in a community of cells in a tissue under investigation.

In correlated studies between electron microscopy and biochemical data, these results are usually quantitative, while the in the electron microscopy (morphological) data are more limited, or semi-quantitative, whose descriptions are based on subjective criteria of researchers.

This subjective criterion of approaching electron microscopy does not allow a statistical measurement of data, with mathematical rigor, not allowing a parametric correlation between morphological and biochemical data.

In the RIBOGRAMA there is a record of the quantitative assessment of free ribosomes in the static and dynamic sense, as information collected from subcellular fractions, which can be

9

correlated with other quantitative measurement of biological parameters of cell (eg, some tumor markers, as the CEA).

From the knowledge of the range of values of the mean "quantity (number) of free ribosomes" considered within the normal range (range A) one may consider other intervals (levels), which in the Fig. 1.15 are represented by B, C and D. In the horizontal axis are embedded multiple periods of six months (SMT), counted from the beginning of the monitoring records of the values of the "average quantity (number) free ribosomes from the cells under study.

III - RIBOGRAMA CURVE

Fig.1.15 The figure shows an example of the scheme with the axes (horizontal and vertical), where are represented the coordinates. One constructs a curve to represent the change of the quantity of free ribosomes per cell (or per unit volume) in a defined time period, within an average of the exfoliated cells of colorectal mucosa of a patient.

INTERPRETATION OF THE CURVE CHART (RIBOGRAMA) → Curve that starts at the normal level and reaching the increased level, which involves care for a prophylactic study on the reasons or causes that determine its appearance. The cells at this level (increased) still have no security features of malignancy, but their increased number of ribosomas continues growing.

The cell is transformed into a malignant phenotype (alarm level). At this point it is essential to make a dietary study to survey and identify possible mutations responsible for the uncontrolled growth of ribosome biogenesis in the process of carcinogenesis; → Segment of the curve derived from the anterior (segment a-b),

which already corresponds to the phenotypic alterations seen in electron microscopy, in which cells are already present with phenotypic features of malignancy (significant increase in median density of free ribosomes), which is the alarm level; → Segment of the curve, continuing the anterior segment, which represents the level of signs and symptoms of the CRC disease (clinical level);

10

→ This segment of the curve corresponds to a change in direction of the curve a-b that results from preventive action and treatment (medications and special diet); → Corresponds to a curve in which cells grow (multiply or proliferate) in

a self-proliferation mechanisms increased, as in the case of the seminiferous tubules, or the endometrium. This increased level of ribosome biogenesis control is thereof within the physiological (non-malignant); → Normal curve; → This work is

intended to study the item colorectal cancer (CRC), in which ribograma curve is high, so it is not the time to talk about the meaning of it at low level. Still, in this level, it will be useful to study conditions of degeneration and involution of cells and tissues, either the intestinal mucosa, or other extra-intestinal tissue.

CHARACTERISTICS OF MALIGNANCY VERSUS THE COUNTING of FREE RIBOSSOMAS For the contents of Table 1.15 is verified that the descriptions of ME and the behavior of cells with phenotypic characteristics of malignancy vary by greater or lesser degree of differentiation of its texture or morphology, but these descriptions are subjective, depending on the research observer.

In terms of molecular biology of cancer, the malignant transformation of the normal cells is the acquisition of a series of progressive specific genetic changes that act disobeying to the strong antitumor mechanisms that exist in all normal cells, which include: a) - regulation of signal transduction, b) - differentiation, c) - apoptosis, d) - DNA repair; e) - cell cycle progression, f) angiogenesis, g) - cell adhesion, which are not quantifiable.

As the descriptions of ME, these mechanisms of malignant transformation are evaluated only by subjective criteria, in which the described features cannot be quantified mathematically. Similarly, the properties of malignant transformed cells, growing in cell culture or in vivo, as observed by techniques of cell biology or molecular biology do not allow a rigorous quantitative assessment, because they depend on subjective criteria, without concrete numbers or standardized numerical limits The characteristics listed in Table 7.15 are properties of malignant transformed cells growing in cell culture (or in vivo), that are not susceptible of being quantified, while with the free ribosomes it is possible to quantify, through counting them with the use of flow cytometry techniques. Then, with the count of free ribosomes is possible to compare the results with reference to the time variable.

TABLE 7.15

PROPERTIES OF GROWING MALIGNANT TRANSFORMED CELLS IN A CULTURE OF CELLS AND / OR IN VIVO

11

1. Cytological changes similar to cancer cells in vivo, including increased cytoplasmic basophilia, number and size of the nuclei increased, increased nucleus-cytoplasm relation, formation of clusters and cords of cells; 2. Altered growth characteristics; a."Immortality" of the transformed cells in culture. Transformed malignant cells become "immortal" in that they can be transferred in culture indefinitely; b. Decreased dependent inhibition of cell density or loss of "contact inhibition." The transformed cells often grow to a density higher than their normal counterparts, and they can pile up in culture rather than stop growing when they contact each other; c. Decreased serum needs. The transformed cells require decreased concentrations of serum or growth factors to replicate in culture compared to cells not transformed. d. Loss of anchorage dependence and acquisition of the ability to grow in soft agar. The transformed cells may lose their need to grow attached to surfaces and can grow as colonies in semisolid free. e. Loss of cell cycle control. The transformed cells do not stop in G1, or borderline G1 / S cell cycle when subjected to metabolic constraints of growth. f. Resistance to apoptosis (programmed cell death).

3. Changes in the structure and function of the cell membrane - including increased agglutination by plant lectins, altered composition of cell surface glycoproteins, proteoglycans, glycolipids and mucins; appearance of tumor-associated antigens, and increased uptake of amino acids, hexoses and nucleotides.

4. Loss of cell-cell and cell-extracellular matrix that promotes cell differentiation.

5. Loss of response to inducers of differentiation and altered cellular receptors for these agents.

6. Alteration of signal transduction mechanisms, including growth receptors constitutive phosphorylation cascades and mechanisms of dephosphorylation rather than a regulated function.

7. Increased expression of oncogenic proteins due to translocation, amplification and chromosome mutation.

8. Loss of protein products of tumor suppressor genes due to deletion or mutation.

9. Genomic misreading that causes the overproduction of growth-promoting substances, eg, IGF2.

10. Increase, or unruly production, of growth factors, e g, TGF-alpha, tumor angiogenesis factor, PDGF, hematopoietic growth factors (e g, CSFs, interleukins).

11. Genetic instability, leading to progressive loss of regulated cell proliferation, enhanced invasion and increased metastatic potential. Genes "mutator" may be involved in this effect.

12. Altered enzyme profifes. The transformed cells have increased levels of enzymes involved in the synthesis of nucleic acids and produce higher levels of lytic enzymes, e g, proteases, collagenases and glycosidases.

13. Production of oncodeveloping gene products. Many malignant transformed cells growing in culture or in vivo produce increased cuantidades of oncofetal antigen (e g CEA), placental hormones (e g, chorionic gonadotropin), or isoenzymes feto-placental type (e g, placental alkaline phosphatase).

14. Ability to produce experimental animal tumors. This is the condition sine qua non that define the malignant transformation in vitro. If the cells that are thought to have been transformed do not product tumors in appropriate hosts animals, they can not be defined as "malignant". However, the failure to grow in an animal model does not exclude the fact that they can be tumorigenic in a different kind of animal.

15. Ability to avoid host antitumor immune response.

12

Of the characteristics listed above, with the variable time, it is not possible to register a numeric result, which could constitute a set of values, through a graphic expression, to show a mathematic and reproducible dynamic trend growth and proliferation status of a set of cells belonging to a given tissue. This is possible with the RIBOGRAMA concept.

CONCLUSIONS 01 - There is evidence well established (and recent), which states emphatically the idea that RNA content is high in cancer cells and genetic events that lead to cancer are often linked, directly and indirectly, to the ribosome biogenesis ; 02 – Thus, the significant increasing of the number of free ribosomes accumulated in the cytoplasm (by counting them with a flow cytometer, after isolated and marked with fluorescent particles --- fluorochromes), of a community of cells of any tissue, may constitute one of the aspects of phenotypic expression of malignancy of the fine structure of the transformed cells; 03 - In the cells in a malignant process, the increasing number of free ribosomes above a certain level of concentration (by volume), which is established as the standard limit of normal, it means a measurable sign of the trend of the expression of malignancy. The verification of this increase of free ribosomes, as malignant character set, is assumed before the global fine morphological structure of a transformed cell (or tissue to which it belongs) is observed; 04 - The phenotypic expression of the cytoplasm, represented by the significant increase of free ribosomes, may be a feature of neoplastic growth and may represent a predict aspect the rate of cell proliferation and prognosis of patients.

05 - Morphological and biochemical studies suggest that the ribosomes of mammalian cells are free in the cytoplasmic matrix (free ribosomes) or associated with cytoplasmic membranes that are part of the rough endoplasmic reticulum (membrane-bound ribosomes); 06 - There is evidence that morphological and physicochemical distinct tissues differs from the poorly differentiated in that the latter have a higher proportion of free ribosomes; 07 - The production of ribosomes is an important metabolic activity related to cell growth and recent data suggest that the nucleolus also plays an important role in regulating the cell cycle, senescence and stress responses; 08 - The ribosome biogenesis involves the synthesis of rRNA, maturation, and the "assembly" of the RNA and ribosomal proteins in ribosomal subunits, small and large; 09 - The ribosome biogenesis is regulated through the cell cycle, primarily at the level of rRNA synthesis;

13

10 - Oncobiologic investigations are developed, which have shown that the expression of changes in the rDNA and ribosomal protein genes are associated with the development of tumors and cytological studies show that the nucleoli (the sites where the rRNA is synthesized and the ribosomes are "assembled") of cancer cells are increased because they have an increased transcriptional activity, representing predictive aspects of cell proliferation rate and prognosis of patients, which explains why the cancer cells have elevated expression ribosomal proteins, revealing higher content of ribosomes, hence the ribosome biogenesis and oncogenesis are intimately connected; 11- Because of its size (25 nm) the counting of free ribosomes can only be studied with the resources of nanotechnology; 12 - In the present study one demonstrates that it is possible to define some guiding principles about the value and significance of the increased number of free ribosomes above a certain concentration level established as the limit of normal; 13 - This significant progressive increase in the number of free ribosomes represents the clĂ­nicolaboratorial translation of the dynamics of biochemical process toward malignant cells in a tissue-specific cellular community, or organ; 14 -The verification in the field of electron microscopy observations show the existence of large amounts of free ribosomes in neoplastic tissues or premalignant. With flow cytometry it also verifies the same, but in a measurable way, after isolation, labeling with fluorescent dyes and quantification of free ribosomes, allowing comparison of values; 15 - The finding of such large amounts of free ribosomes can be translated into a graph curve that represents and sets high, over time, the propensity for malignant growth of tissue or cell community under certain conditions; 16 - This curve graph will have the designation of RIBOGRAMA. Has a mathematical basis (that may be reproducible in equivalent circumstances) which corresponds to a non-subjective language; 17 - The analysis of trends and changes found in the high curve will be a reliable indicator of the degree of development and propensity for malignancy; 18 - The RIBOGRAMA corresponds to a phenotype, which can provide quantitative information on levels of risk and significance, prior to the "opened", malignancy, with information on the risk profile, in the heredo-familiar profile, in the lifestyle profile, in the food and diet profile, in any tissue being studied about their proliferative dynamics.

19 - Compared to existing noninvasive tests in use (occult blood in the stool and analysis of DNA mutations in the colonocytes) the ribosome counting test has features that have major advantages over existing tests: 14

i. Knowing that it will be studied isolated colonocytes following a specific technique, the specificity of the results is 100% relatively to the count of the free ribosomes; ii. The different levels of the amounts of free ribosomes, on grounds of risk and significance, allows time for preventive and therapeutic attitudes against a tendency of malignancy, while the discovery of mutations in the test DNA in the stool is only positive/negative response for certain mutations, do not allowing a quantification by levels or degrees with a signification risk; iii. The test for DNA mutations in the stool have false positives and false negatives, which vary broadly, according to the authors; iv . The positivity test for fecal occult blood has a high percentage of false positives and false negatives are not negligible v . When a tumor is bleeding, this means that the mucosal barrier has already been destroyed by erosion/invasion of the submucosa, with their blood and lymph vessels, ensuring no certainty as to the metastization and lymph node involvement, meaning this that the tumor is not a Tis/0; vi. Mutations in the DNA tests in the stool can be from other organs (respiratory tract, gastrointestinal tract, upstream of colorecto, etc.), not allowing the warranty about the location of a possible tumor; vii . In addition, mutations in DNA testing in the stool can be no mutant phenotypes, which contributes to the many false positives; viii . In the counting test of free ribosomes, to which correspond levels significant of its increase, the number of free ribosomes in colonocytes isolated from feces represents the existence of a real uncontrolled increase in the synthesis of proteins, and cannot therefore to exist false positives because a tumor depends crucially on the excessive production of proteins in the growth process, and this in the cell can only be done by free ribosomes;

POTENTIAL CLINICAL USE OF RIBOGRAMA IN SCREENING, DIAGNOSIS AND "FOLLOW UP" OF COLORECTAL CANCER: 20 - In a scenario of utilization of the RIBOGRAMA method as an expression of phenotypic behavior of coloncytes (free ribosome density expressed in units of volume, over time), it will open the doors to many fields of clinical and laboratory research. It will enable the screening and the very early diagnosis of colorectal cancer in conditions conducive to convince family members also to undergo a noninvasive test that does not force anyone to manipulate their feces and convincing, if there are high RIBOGRAMA curves, to submit them, quite 15

justifiably, to a diagnostic and therapeutic colonoscopy, once does not exists any clinical signs; 21 - Through RIBOGRAMA of desquamated cells of colorectal mucosa, conveyed in feces and collected in a suitable and specific container, without manipulating of their feces, with a design created and appropriate for this purpose, people would have a easy way to access to this diagnostic and generalized screening and could be oriented towards an endoscopic examination and be routed to a risk group and be observed and later to be studied graphic profiles of the numerical values of quantities of free ribosomes (RIBOGRAMA) compared to a standard curve-middle-drawn with the study of large populations.

References: 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Darzynkiewicz Z. (1998) Cellular ARN content, a feature correlated with cell kinetics and tumour prognosis. Leukemia. 2, 777 – 787.

Ruggero D. and Pandolfi P.P. (2003) Does the ribosome translate cancer? Nature Reviews Cancer, 3, 179 – 192.

Derenzini M., Sirri V. and Treré D. (1994). Nucleolar organizer regions in tumour cells. Cancer Journal. 7, 71 – 77.

Pomeroy S.L. et al. (2002) Prediction of central nervous system embryonic tumour outcome based on gene expression. Nature. 415, 436 – 442.

Derenzini M., Treré D., Pession A., Montanaro L., Sirri V. and Ochs R.L. (1998) Nucleolar function and size in cancer cells. American Journal of Pathology. 152, 1291 – 1297.

Stanners C.P., Adams M.E., Harkins J.L. and Pollard J.W. (1979).Transformed Cells Have Lost Control of Ribosome Number through their growth Cycle. J. Cell Physiol. 100, 127 – 138.

Sakiyama, H., S. K. Gross and P. W. Robbins (1972). Glycolipid synthesis in normal and virustransformed hamster cell lines. Proc. Nat. Acad. Sci. (U.S.A). 69, 872 –876.

Bose S. K. and B. J. Zlotnick (1973). Growth - and density – dependent inhibition of deoxyglucose transport in Balb 3T3 cells and its absence in cells transformed by murine sarcoma virus. Proc. Nat. Acad. Sci. (U.S.A). 70, 2374 – 2778.

Hirschberg C. B., B. A. Wolfund and P.R. Robbins (1975). Synthesis of glycolipids and phospholipids in hamster cells: dependence on cell density and the cell cycle. J. Cell. Physiol. 85, 31 – 40.

Chou I. N., O´Donnell, P. H. Black and R.O. Robbin. (1977) Cell density-dependent secretion of plasminogen activator by 3T3 cells. J. Cell. Physiol. 91, 31 – 32.

Hanahan D., Weinberg R.A. (2000) The hallmarks of cancer. Cell. 100, 57 – 70.

De Young L.M., Argyris T.S. and Gordon G.B. (1977) Epidermal ribosome accumulation during two-stage skin tumorigenesis. Cancer Research. 37, 388 – 393.

Chu T.Y., Hwang K.S., Yu M.H., Lee H.S., Lai H.C. and Liu J.Y. (2002) A research-based tumour tissue bank of gynaecologic oncology: characteristics of nucleic acids extracted from normal and tumour tissues from different sites. International Journal of Gynaecological Cancer. 12, 171 – 176.

Boon K. et al. (2001) N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis. EMBO Journal. 20, 1383 – 1393.

Linaggar A.K., Kemp B.L., Sneige N., Hurr K.G., Steck K., Tu Z.N., Pritsche H.A., Singletary S.E. and Balch C.M. (1996) Bivariate ARN and ADN content analysis in breast carcinoma: biological significance of ARN content. Clinical Cancer Research. 2, 419 – 426.

Lourenco G., Embury S., Schrier S.L. and Kedes L.H. (1978). Decreased ribosomal ARN content and in vitro ARN synthesis in purified bone marrow erythoblasts of patiens with idiopathic ineffective erythropoiesis and DiGuglielmo disease. America Journal of Hematology. 5, 169 – 182.

16

17. Johnson L.F., Abelson H.T., Green H. and Penman S. (1974) Changes in ARN relation to growth of the fibroblast I. Amounts of mARN, rARN, and tARN in resting and growing cells. Cell. 1, 95 – 100.

18. Loeb J.N. and Yeung L.L. (1975) Synthesis and degradation of ribosomal ARN in regenerating liver. J Exptl Med. 142, 575 – 587.

19. Malt R.A. and LeMaitre D.A. (1968) Turnover of ARN in the renoprival kidney. Am J Physiol. 214, 1041 – 1047.

20. Tata J.H. (1967) .The formation and distribution of ribosomes during hormone-induced growth and development. Biochem J. 104, 1-16.

21. Levin D.L., Devessa S.S., Godwin J.D. and Silverman D.T. (1974) Cancer rates and risks.

Washington D.C: United States Department of Health Education and Welfare.

22. Blobel G. and Potter V.R. (1966) Nuclei from rat liver: isolation method that combines purity with high yield. Science. 154, 1662.

23. Blobel G. and Potter V.R. (1967) Studies on free and membrane-bound ribosomes in rat liver. J Mol Biol. 26, 279.

24. Raick A.N. (1974) Cell diferentiation and tumour-promotor action in skin carcinogenesis. Cancer Research. 34, 2915 – 2925.

25. De Young, Lawrence M., Argyris, Thomas S. and Gordon, Gerald B. (1977) Epidermal ribosome accumulation during two-stage skin tumorigenesis, Cancer Research 37, 388-393.

26. Ruggero D. et al. (2003) Dyskeratosis congenita and cancer in mice deficient in ribosomal ARN modification. Science. 299, 259-262.

27. Draptchinskaia N. et al. (1999)The gene encoding ribosomal protein S19 is mutated in DiamondBlackfan anaemia. Nature Genet. 21, 169-175.

28. Pardee A. B. (1989) G1 events and regulation of cell proliferation, Science. 246, 603-608.

29. Pyronnet and S. & Sonenberg N. (2001) Cell-cycle-dependent translational control, Curr. Opin.

Genet. Dev. 11, 13-18.

30. Grummt I. (1999) Regulation of mammalian ribosomal gene transcription by ARN polymerase I. Prog. Nucleic Acid Res. Mol. Biol. 62, 109-154.

31. Grummt I., Smith V.A. & Grummt, F. (1976) Amino acid starvation affects the initiation frequency of nuclear ARN polymerase. Cell. 7, 439-445.

32. Kief D. R. & Warmer J.R. (1981) Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae. Mol. Cell. Biol.

1, 1007-1015.

33. Klein J. & Grummt I. (1999) Cell cycle-dependent regulation of ARN polymerase I transcription: the nucleolar transcription factor UBF is inactive in mitosis and early G1. Proc. Natl. Acad. Sci. USA 96, 6096-6101.

34. Cavanaugh A.H. et al. (1995) Activity of ARN polymerase I transcription factor UBF blocked by gene product. Nature. 9,177-180.

35. Beckmann H., Chen J.L., O’Brien T. &Tjian R. (1995) Coactivator and promoter-selective properties of ARN polymerase I TAFs. Science. 270, 1506-1509.

36. Brandenburguer Y., Jenkins A., Autelitano D. J. & Hannan R. D. (2001) Increased expression of UBF is a critical determinant for rARN synthesis and hypertrophic growth of cardiac myocytes Faseb J. 15, 2051-2053.

37. Voit R., Hoffmann M. & Grummt I. (1999) Phosphorylation by G1-specific cdk-cyclin complexes activates the nucleolar transcription factor UBF. Embo J. 18, 1891-1899.

38. Voit R. & Grummt I. (2001) Phosphorylation of UBF at serine 388 is required for interaction with ARN polymerase I and activation of rADN transcription. Proc. Natl. Acad. Sci. USA. 98, 1363113636.

39. Voit R. et al. (1992) The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation. Embo J. 11, 2211-2218.

40. Johnson L. F., Williams J. G., Abelson H. T., Green H. & Penman S. (1975) Changes in ARN in relation to growth of the fibroblast. III. Posttranscriptional regulation of mARN formation in resting and growing cells. Cell. 4, 69-75.

41. Baxter G. C. & Stanners C.P. T (1978) he effect of protein degradation on cellular growth characteristic. J. Cell. Physiol. 96, 139-145.

42. Jefferies H. B. et al. (1997) Rapamycin suppresses 5’TOP mARN translation through inhibition of p70s6k. Embr J. 16, 3693-3704.

17

43. Kawasome H. et al. (1998) Targeted disruption of p70 (s6k) defines its role in protein synthesis and rapamycin sensitivity. Proc. Natl Acad. Sci. USA 95, 5033-5038.

44. Orfao A., Ciudad J., López A., López-Berges M.C., Vidriales B., Macedo A. et al. (1993) La citometria de flujo en el diagnóstico clínico. Servicio General de Citometria. Universidad de Salamanca.

45. Orfao A., González M., Ciudad J., López-Berges M.C., López A., San Miguel J.F. et al. (1992) Aplicaciones de la citometría de flujo en el diagnóstico hematológico. Biol Clin Hematol 1992; 13: 456-523.

18