INTERVIEW BIOCHEMISTRY
One faulty cell An interview with Dr Anscombe Research is undoubtedly one of the most arduous yet rewarding careers in science. From trials and errors to meticulously analysing your findings, science research has the potential to shape the future of the world. Vanessa Y, Lily and Emma Tse sat down with Dr Anscombe to discuss her journey through a career in cancer therapy research. We’d like to thank Dr Anscombe for providing such great insight into the world of biochemical research - we hope you enjoy learning about her experiences as much as we did!
Background knowledge The cell cycle is regulated by cyclin-dependent kinases (CDKs). Kinases are enzymes that add phosphate groups to other molecules, a process called phosphorylation. Throughout the cell cycle, proteins called cyclins bind to CDKs, which allows CDKs to phosphorylate proteins such as pRb. pRb, short for retinoblastoma protein, is a tumour suppressor protein, which means it acts to inhibit cell proliferation through slowing mitosis or inducing apoptosis. Once pRb is phosphorylated, the cell is given the green light to continue to the next phase of the cell cycle; if not, the cell cannot progress further. When it comes to proteins themselves, understanding their 3D structure is imperative, because structure equates to function. X-ray crystallography is a widespread technique that can be used to find a protein's structure, which involves bouncing Xrays off a protein crystal and measuring the diffraction angles (in simple terms). Once the X-ray crystallography data is obtained, it's processed through a computer programme that executes a Fourier Transform, which is an image-processing tool that breaks down an image into cycles more specifically, their sine and cosine components. A simple analogy is to think of being given a smoothie and finding its recipe!
Could you summarise the research that you did for your PhD? There are four phases in a normal cell cycle, G1, S, G2 and M (mitosis), and within M you’ve got subphases. The protein I looked at was CDK2 (cyclin-dependent kinase 2) and that regulates the transition between G1, where the cell is growing fairly normally, and S, where it starts to replicate its DNA. Between each stage of the cell cycle, there are checkpoints that check, for instance, that there’s no damage to the DNA before replication, whether you’ve got the right number of copies of chromosomes, or that the cell is generally ready to enter the next phase of the cell cycle. We were looking at one particular cell cycle transition and the kinase involved in regulating it. We were essentially trying to halt that process because in cancer cells, the processes at these checkpoints become disrupted, so cancer cells escape the normal checks and balances a cell goes through. These checks keep cells functioning properly, avoiding things like DNA damage by UV or reactive oxygen species being passed down to the next generation of cells. Cancer cells usually have mutations that allow them to escape this G1-S checkpoint, so they don’t respond to pRb, for instance. So you want to switch off the CDK2 and upregulate the pRb activity. The idea we were looking at was whether we could reactivate those checkpoints and therefore stop cancer cells from dividing further. If we could, that would imply it could be used as cancer therapy! Essentially, the CDK2 phosphorylates another protein, and in order to do that, it has to bind ATP (the energy currency of the cell). In the binding site where the ATP binds to the kinase, we looked at developing inhibitors that bound there to stop ATP from working, and therefore stop the kinase from working. My PhD involved my team and I getting sent molecules, which were the potential candidates we would test. I was based at Oxford, and a lab in Newcastle would send us possible drug molecules that might potentially bind to this protein. We would see if they did bind and 27
if we could determine the structure of the protein, in addition to looking at the structure with the drug molecule it bound to, how closely it bound and whether it was a stable interaction or not.
So you’re basically trying to inhibit the CDK2, which is involved in the G1-S checkpoint, in order to reactivate that checkpoint. How exactly does that work?
Here is a simplified diagram of the cell cycle. This protein, pRb, which stands for the retinoblastoma protein, is a tumour suppressor. The function of this tumour suppressor protein, which determines whether DNA synthesis can start, is determined by CDK2. There are lots of cyclins in the cell cycle - CDK2 phosphorylates cyclin E, ATP has to bind to CDK2 in order to phosphorylate the protein, and if we can stop the ATP from binding to the CDK2, phosphorylation can’t take place.
In the process of your research, what did you do? The day-to-day process was that we would get sent a new set of chemicals to test - they’ve done all the basic chemistry and replicated all these potential drug molecules. They would send us a big batch to test, and we firstly did various assays to see if they-
