Carcinogenesis and Cancer Treatment Resistance - Assignment Example

0 Carcinogenesis and Cancer Treatment Resistance Cellular DNA was considered to be an inert substance. However, a recent research in the field along with our knowledge of molecules has changed this view. Now it is known that DNA is damaged and then repaired constantly by agents both inside and outside the cell. Some of those agents, among many other agents inducing mutations, are chemicals from the environment, coming either as pollutants from human industry and naturally from foods. As a result, DNA is often damaged and has to be repaired by the pathways of repair of DNA polymerase (Loeb, Loeb 2000). For instance of repair pathways are homologous recombination (HR), trans-lesion synthesis (TLS), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR) (Martin et al., 2010). When there are double-strand breaks in the DNA strands and the replication forks, HR and the NHEJ pathways are at work to repair it. In the case of modified or incorrect basis, which can happen in DNA synthesis, the BER path works to remove them. When there are chemically induced damages to DNA or UV damage, the NER pathway removes the damage. MMR is the pathway repairing deletion, mismatch or insertion mutations that happen in the DNA and TLS pathways makes the polymerases bypass modified bases which can disrupt the replication forks. In other cases, multiple protein pathways take part in maintaining the genetic wholeness in different cell cycles, stopping the aberrant or anomalous cells from dividing. Thus, the daughter cells inherit the correct genomic information (Martin et al. 2010). There are various mutations are happened at the same time, the repair process misses some of mutations and become part of DNA (Loeb, Loeb 2000). Currently, there are 346 genes, which are associated with the development of cancer and this number keeps increasing. In some cases these genes are mutated only in cancer cells, not in the normal cells. In other cases genes may show increased levels of deregulated. In both cases, the result can be a significant decrease in the effectiveness of the genes, which may lead to change pathways both in the cancer cell and the body itself (Huang, Wallqvist & Covell 2006). This suggests that mutations are very essential part of cancer formation. It is observed that when human cancer is first detected, there are already thousands of different mutations. Also, information that cancer can be passed from one generation to the next, means that some of those mutations are inherited in the genes (Loeb, Loeb 2000). Cancer is characterised by a high rate of somatic cells cloning themselves. These cells do not go by the normal growth regulation mechanisms acting in a cell. This is why these cells can multiply far more rapidly than a normal cell would. In a similar way, mechanisms controlling apoptosis are also not working normally, which means that the cancerous cell does not die when it should (Evan, Vousden 2001). Most tumours that develop in the human body are substantially heterogeneous. This suggests that many mutations occur in the cancer cells leading to the creation of tumours and to the change in function of normal cells compared to cancerous cells (Loeb Loeb & Anderson 2003. That is why cancer can be seen as a disease, or many diseases, which happen because of genetic abnormalities piling up in the cells (Huang, Wallqvist & Covell 2006). In each generation of cancer cells, significant mutation occurs within different cells, and those that develop mutations that facilitate cancer growth are selected for, resulting in an increase in the prevalence of these cells. Mutations may be subtle, such as changes in the sequence of nucleotides, or more substantial, involving changes in the chromosomes themselves (Wang et al, 2002). One important factor in the treatment of cancer is that therapeutic killing cancer cells, which make the cancer go into remission, can also make it more resistant to treatment in a similar manner to how some microorganisms can become more resistant after extended treatment with antibiotics. This can lead to increase in the tumour growth, which then has cells that cannot be targeted by the same treatment (Blagoskonny, 2005). Another form of resistance which can happen depends on the location of the cell in the division cycle. Cancer therapy is most successful on rapidly replicating cells. Also, resistance to treatment can often be stronger in some stages of the cell cycle than others (Schwartz, Shah 2000). Similarly, treatment can be made more effective by treating the cancer when the cells are at the most appropriate stage of cell division, (Schwartz, Shah 2005) Cancer cells have many different mutations which may cause the formation and spreading of tumours. Research shows that cancer cells are very unstable. Because of that there is a process of selection of the aspects of the cancer cells that help it avoid the usual forms of control, especially replication and apoptosis. This difference within individual cells leads to creating very different tumours. Furthermore, cancer cells seem unstable genetically, and easy to mutate. These differences in the tumours and the instability have another very important role. They help the cancer cell to become resistant to treatment. This means that one treatment may not be effective for all the cells in a tumour and the cells that are not destroyed by the treatment are resistant to it. This leads to selection for the elements that make a cancer cell resistant. The genetic instability also allows cells that don’t have the mutation for resistance and still survive by chance, to develop this resistance before the next treatment happens (Loeb, Loeb 2000). All types of cancer treatment have the risk of causing resistance in cancer cells. This means that the most successful method of dealing with cancer would be to target all abnormal cells at the same time and to attack lesions specific to tumours at the individual level. However, this method is still in development and there are many challenges which prevent it from resembling an effective treatment at this time (Evan, Vousden 2001). Therefore, the research needs to focus on molecular mechanisms of cancer cells and find treatments that do not cause resistance. An interesting thing is that carcinogenesis has an important role in both cancer and cancer therapy. Sometimes cytotoxic agents cause cancer and those agents are used for its treatment. This is based on the fact that genetic instability makes the tumour spreads faster and predisposes an individual to cancer. Genetic instability can be caused by a number of factors, and the most important of them is the action of carcinogenic agents or errors in DNA repair. However, DNA repair is also used to treat cancer (Loeb Loeb 2000). In a similar way, errors in DNA replication are the reason for mutations that cause cancer, but once the cancer is formed, high fidelity DNA replication helps the mutant cells to continue growing and dividing. This is something that needs to be taken into account when looking at the resistance to cancer treatment, because it demonstrates that the most logical things are not always the right ones. Another element of carcinogenesis and cancer treatment which needs to be taken into account when deciding upon a course of treatment is that the cells originated within the host and therefore share many similarities genetically (Bunn 1999). This means that the cells cannot be targeted as a bacterium or virus would be, as treatments aimed at cancer cell apoptosis can cause trauma to the individual’s healthy cells. We see this manifest in many of the current cancer treatments such as traditional chemotherapy and radiotherapy, which rely on the fast-dividing nature of cancer cells to differentiate them from normal host cells (Blagoskonny 2005). In these cases, side-effects and collateral damage are common because of the very nature of the disease (Blagoskonny 2005). Many forms of cancer rely on this disguise mechanism to become indistinguishable and flourish within the body, in many cases going undetected for years. This is perhaps the biggest challenge for those researching cancer cell treatment and the causes of resistance, as many treatment options that target all cancerous cells (thus leaving no room for the development of resistance) would be extremely harmful to healthy cells. 2.0 Mechanisms in Cancer Cells Finding the way in which cancer happens at the molecular level has been promoted as a method for developing better and more effective cancer therapies. It is often believed that cancers are very different from each other and that the ‘cancer’ to describe the disease is a misnomer, as the treatment requires very different attitudes depending on several different factors, including originating tissue and division speed, which effectively makes it several different diseases. From this point of view, different cancers need different treatments and this is why many different treatments are developed for cancer (Evan, Vousden 2001). Normal cells in the body are regulated by mitogenic signals which control the rate of their division. Besides, cells can only proliferate at the end of the G1 component of cell cycle. On top of that, mitogenic signalling can be performed successfully under some circumstances (Evan, Vousden 2001). This is why limitations on the division of cells are very important in normal conditions. In normal cells, apoptosis, which is the cell’s way to kill itself, is signalled through two different pathways. The first one is the extrinsic (outer) pathway, which uses the death receptors located on the surface of the cell. The second one is the intrinsic (inner) pathway, which is controlled by the mitochondria in the cell (Igney, Krammer 2002). In the healthy cell, these pathways activate themselves when a cell becomes unviable or damaged beyond repair. In cancerous cells, the pathways that prevent the cell division are abnormal. One such abnormality is the mechanism that needs cells to receive mitogenic signals to proliferate. The mitogen activated protein (MAP) kinases fall into three major groupings, one of which is the extracellular-signal regulated kinase (ERK). ERK1 and ERK2 are transducers of proliferation signals and are typically active by mitogenic signals (Evan, Vousden 2001). Excess levels of certain activating agents, such as epidermal-derived growth factor (EGF) can promote the proliferation of cancerous cells beyond normal and appropriate levels, leading to tumorous growth (Igney, Krammer 2002). The typical mechanisms for apoptosis in the cancer cells are also absent in cancerous growth (Evan, Vousden 2001). This happens because of the expression of proteins which stop apoptosis (anti-apoptosis proteins) or because of mutation or down regulation of the proteins responsible for apoptosis (Igney, Krammer 2002). Many cells proliferating and few cells dying by limited apoptosis lead to the fast cell growth typical of cancer. Research shows that controlling the rate at which cells grow, their proliferation and apoptosis are very connected with each other. Also, apoptosis has a very important role in developing cancer and its maintenance. Cells which replicate a lot but have no disturbance in apoptosis, do not become too many in the human body because the mechanisms of cell death function normally and many of the cells die (Igney, Krammer 2002). These mechanisms are dominant in the cancer cells and this is why cancer therapy deals with them. This way, many cancer treatments are target the cell cycle itself. Research has shown that arresting the cell cycle is an effective method of inducing apoptosis and this plays a significant role in cancer treatment (Schwartz, Shah 2000), due to the fact that this mechanism is in place to prevent the proliferation of cells that have not followed the typical growth cycle and thus may contain errors in mutation. Without this, the mutated cancer cells may continue to proliferate. 3.0 Nucleoside Analogues Most of the drugs used in treating cancer interfere with the mitotic cycle, but they cannot do it in a very precise way. Instead, treatments for cancer work by targeting the cell division and DNA replication processes (Evan, Vousden 2001). Inhibiting mitosis is important, as without cell division the cells are evidently not able to replicate, grow and metastasize. This mechanism relies on the fact that many cancerous cells go through mitosis at a much faster rate than healthy cells (Blagoskonny, 2005). Without this factor, arresting mitosis will have the effect of stopping healthy cell growth throughout the body, which causes many of the side effects typically seen in some cancer treatments relying on arresting mitosis (Evan, Vousden 2011). One sort of drugs that is becoming efficient in cancer treatment is nucleoside analogues (NAs). They can stop transcription and replication of DNA in the cancer cells (Milas et al, 2003). Nucleoside analogues (NAs) are type of antimetabolites used in cancer treatment and solid tumours. These molecules include analogues for both purine and pyrimidine DNA particles. The analogues are actively transported in the cells with membrane transporters due to their innate similarities to typical nucleosides found within the body (Galmarini, Mackey & Dumontet 2001). Once inside the cell they interfere with a number of cellular processes such as cytotoxity (Milas et al, 2003). Nucleoside analogues rely on their similarity to nucleotides, as this characteristic allows them to be incorporated into DNA. However, after being incorporated into DNA strands in the typical way, their analogous nature means that they function effectively as chain terminators and by inhibiting the action of DNA polymerase (Galmarini, Mackey & Dumontet 2001), an enzyme which is vital for replication of cellular information and thus proliferation of cells. Different NAs are used for different types of cancer. For example, fluorophyrimidine 5-fluorouracil (5-FU), a pyramidine analogue, is used for many different cancer types by irreversible inhibition of the enzyme thymidylate synthase which is implicated in mechanisms of DNA synthesis and repair (Obata et al, 2003). NAs were some of the first methods for cancer treatment. Nowadays, they are still commonly used for treating many different forms of cancer. Understanding how each nucleoside analogue works specifically to affect processes in the cells is very important for making them more specific and efficient in the treatment of the disease (Galmarini, Mackey & Dumontet 2002). Two NAs that have been shown to be particularly important in cancer treatment are fludarabine and gemcitabine, purine analogues. Both of these are very important in making the tumours more responsive to radiography by making the cells more susceptible to the cytotoxicity of radiation. The explanation for this may be that they do not let the cell repair itself after damage from radiation as they interfere with the enzymes DNA polymerase and ribonucleotide reductase, thus making repair mechanisms ineffectual (Obata et al, 2003). In this way, fludarabine and gemcitabine can be used to make the cancer cell fit for radiation treatment by making radiation more effective on solid tumours (Milas et al, 2003), which are notoriously difficult to treat because of their depth, size and the fact that they contain an extremely large number of cancerous cells, meaning that there is more opportunity for the treatment to be ineffectual on some and thus lead to treatment resistance. There are three general mechanisms that have been identified that result in the cell being resistant to the NAs. The first one is where there is a low concentration of NA triphosphates. This low concentration can be caused by different things, for example high levels of NA degradation, a reduction in how much the enzymes are active or increase in the size of dNTP pools. The second way is when apoptosis of the cells is done badly and the NAs cannot be taken up efficiently enough to disrupt normal mechanisms of cell replication. The third way is that NAs cannot make the necessary changes in the strands of DNA or the pools of dNTP (Galmarini, Mackey & Dumontet 2001). The mechanism that NAs rely upon varies depending on the individual NA that is being used. Some NAs prevent the synthesis of typical nucleotides, other NAs depend on being integrated in DNA during its synthesis. In many cases, wen NAs are integrated in DNA, they prevent the normal addition of nucleotides to the DNA strand being synthesized due to their different structure, as mentioned above. This happens because of the phosphorylation of the compound when it enters the cell, which makes it active. To continue replicating, the cell must remove the NA from the DNA chain. This happens with a repair mechanism identifies NA as an abnormal part of the chain and removes it from DNA, which may not always be possible (Zhu et al, 1998, although it is a major challenge for using nucleoside analogues in cancer treatment. The use of NAs as a form of treatment is effective because they so similar to the pyrimidines and purines that typical, healthy cells take up and incorporate in their DNA in cell division mechanisms. About 20% of all cancer treatment drugs are NAs of either purines or pyrimidines. Their development as drugs for cancer treatment continues and researchers are trying to find new and better ways of stopping the replication of cancer cells by using analogues to elements typically required for mitotic cell division and thus proliferation (Parker 2009. 3.1 Purine nucleobases and purine nucleoside analogues The use of purine nucleobases is crucial in cancer treatment, and is one of the most important types of cancer treatment available, having been involved in cancer treatment for the past 50 years. Development and creation of new purine NAs continues to this day, as continual improvement and modification can help to fine-tune the way that they work to combat cancer in the body and help to increase their specificity for cancer cells over normally functioning healthy cells. They can be the way to creating a more effective way of cancer treatment and there is still more to be explored within the compound to creating a new anticancer drugs (Parker, Secrist & Waud 2004). Many different types of purine NAs are used and developed for this purpose, including nelpanocin A, tiasofurin and 3-deazaguanosine. Purine NAs can differ very much between themselves in the way they act and in the things they need to become biologically activated and active (Plunkett, Saunders 1991). Nelpanocin A, for example, acts as a tight binding inhibitor by exhibiting stoichiometry between one molecule of inhibitor and one tetramer of a typical enzyme, and works by inhibiting S-adenosylhomocysteine hydrolase, as does the nucleoside analogue 3-deazaguanosine (Plunkett, Saunders, 1991. Tiasofurin, on the other hand, works as an inhibitor of IMP dehydrogenase, thus preventing normal de novo DTP biosynthesis and thus effecting cell proliferation. This is a treatment approach is the best for actively rapidly growing cells (which includes cancerous cells and healthy fast-replicating cells such as white blood cells alike). However, it can also have an effect on cells that are not doing so (Robak et al, 2005), meaning that developing these drugs to become more appropriate for cancer treatment is of a high priority. 3.2 Deoxyadenosine derivatives The chemical structure of purine NAs looks like the structure of adenosine or deoxyadenosine. Their derivatives frequently use substitutions or adding a chemical group. For instance, when the hydrogen in deoxydenosine is changed with chlorine, the result is cladribine. Deoxyadenosine derivatives have many characteristics in common, despite any differences in their chemical structures – for example, the and dephysphorylation by a 5’-nucleotidase and also the way of transport in the cell (Robak et al, 2005). This group of compounds are a frequently used in cancer therapy and many of the other commonly used drugs have the same form (Kantarijian et al. 2003) One of the most commonly used deoxyadenosine derivatives is 3’deoxyadenosine N1-oxide, which exhibits a synergistic effect. Studies have shown that ‘3'-dANO resistant Ehrlich cells initiate the metabolism of 3'-dANO by a reduction to 3'-deoxyadenosine, which is converted primarily to 3'-deoxyinosine by adenosine deaminase and, to a small extent, phosphorylated to the cell toxic agent 3'-dATP’ (Svendson, Overgaard-Hansen & Frederiksen 1988) thus changing the activities of enzymes reliant on this pathway. This subsequent change in enzyme activity, primarily of the reductase, adenosine kinase and adenosine deaminases (Svendson, Overgaard-Hansen & Frederiksen 1988) prevented the normal methylation actions required within normal cell proliferation and therefore can be very effective in preventing cancerous growth. 3.3 Deoxycytidine analogues Another drug commonly used in cancer treatment is Gemcitabine, which is a deoxycitidine analogue, different only because two hydrogen atoms on the 2’ carbon of deoxycitidine are replaced by fluorine atoms (Bunn 1999). It is often used for treating non-small cell lung cancer. It is not very toxic and because of that it can be used with variety of patients, including elderly people with weakened systems (Shepherd et al. 1997). Also, this low amount of toxicity enables the effective use of the drug in other forms of treating of cancer (Bunn 1999). In this drug, the deoxycytidine analogue is substituted with fluorine. Once it becomes active in the cell, it can be incorporated in the DNA chain, thus affecting the DNA polymerase. This makes the elongation of the chain impossible, along with the repair and synthesis of DNA. Despite all this, patients with cancer can still become resistant to the drug (Kang, Saif, 2008) The molecular target of this group, as with the others, tends to vary slightly. However, Gemcitabine and the similar antitumour drug Ara-C have a similar mechanism. Like other nucleoside analogues, they are transported into the cell via nucleoside transporters and then phosphorylated, in this case towards each specific monophosphate form by a kinase known as dCyd kinase (Obata et al, 2003). This leads to the enzymatic production of tri-phosphate forms, which effectively inhibits normal DNA synthesis mechanisms. dCyd kinase is important in itself because it is an activator of antitumour analogues of itself. Whilst this can be beneficial in the early rounds of cancer treatment, exposure to 2’dCyd kinase can cause cancer cells to become severely resistant to subsequent treatments relying on this mechanism (Obata et al, 2003). In this way, this molecular group hold several parallels to the others, in that resistance and specificity pose the biggest challenges to their effective use in cancer medicine. 4.0 Topoisomerases Topoisomerases are a type of enzymes which take part in regulating the winding of DNA, a precise mechanism which arose as a solution to the problem of the precise intertwined structure of double helical DNA. When replication or transcription occurs, the DNA becomes overwound in front of the site of replication. This can result in an inability for the processes to continue due to the heightened tension of the strands in the structure. This is why topoisomerases make small cuts along the DNA, helping it to unwind by making small nicks in the phosophodiester backbone of the double helix, thereby reducing tension in the structure. The cut gets sealed later by other enzymes involved in this process. So, if there were no topoisomerases, DNA replication would not be possible and cells would not be able to proliferate. This is why topoisomerases are a very important aspect of treating cancer, because changing what they do can make cell proliferation impossible. Also, stopping the cell proliferation in such way, results in cell apoptosis. This is why many drugs for cancer treatment focus on these important enzymes (Pommier et al. 2010). Due to their role as described above, many efforts in chemotherapy have been aimed at developing so-called topoisomerase inhibitors, usually preventing the ligation of the phospodiester backbone needed for normal DNA structure (Bunn 1999). Introduction of such breaks is detected by normal cell mechanisms and therefore leads to apoptosis, preventing proliferation. Another element crucial to their success is that cancer cells are often extremely sensitive to such breaks in their DNA, increasing their specificity for cancer cells over healthy cells. One important type of drug of this class is the campothecins, which targets type I topoisomerase and stabilizes cleavable complexes and prevents the annealing of such breaks by its action (Bunn 1999). Whilst campothecins have their limitations, such as the destruction of white blood cells, they have become increasingly important in cancer treatment today. Another such drug is doxorubicin, which is commonly involved in breast cancer treatment. It works by inhibiting DNA topoisomerase II-alpha. Research suggests that the drug is effective mostly when topoisomerases II-alpha expression is high and when the expression is low, resistance to the drug can be developed. This is what makes using the drug very specific – topoisomerases II-alpha levels are high when the cell is replicating and as a result there is much of it in cancer cells which replicate very fast (Lynch, Guinee & Holden 1997). DNA topoisomerase II is important for a huge variety of cancer drugs (Glisson, Ross, 1987). Topoisomerase II has a very important role in DNA replication and this is why research is targeting it. This enzyme is homodimer and its two isoforms are topoisomerase II-alpha and topoisomerase II-beta. It is still not clear how the latter functions, which is why most therapeutic approaches focus on the alpha isoform. Many drugs of this class, including doxorubicin, work by intercalating DNA. In this process, a molecule or group of molecules is reversibly included into a structure between two other molecules, such as within a chain of DNA (Lynch, Guinee & Holden 1997). This can induce the presence of structural distortions within the chain, causing many of the functions of the molecule and its derivatives that rely on the morphology to work correctly to become disrupted or inactive (Glisson, Ross, 1987). By doing this, normal DNA replication mechanisms are affected, which can halt their proliferation. However, as with many chemotherapies, this does have an effect on other cells which are rapidly proliferating such as hair cells and white blood cells because of their low specificity in targeting cancerous cells in particular. One of the most dangerous aspects of using a topoisomerase in chemotherapy is that its potency in killing cancerous cells can lead to the production of secondary neoplasms within the patient (Lynch, Guinee & Holden 1997). 5.0 The MRN complex Three components form the protein MRN complex: Mre11, Rad50 and Nbs1. This complex is very important in the repair of DNA not only in humans, but also in some mammals. Studies showed that it is when the double strand breaks, the MRN complex is one of the first things that get activated. It can recognize damage in DNA, including that which is produced by cancer treatment, and it can repair the DNA making the treatment ineffective. As previously mentioned, the cells involved in cancer originated within the host, so these mechanisms are available to healthy and cancerous cell alike. It also takes part in the cell-cycle signalling cascades (Williams Williams & Tainer 2007. It can also prevent the effective treatments, because many types of treatments depend on arresting the cell in a certain state and causing apoptosis, (Schwartz, Shah 2000), rather than the damage getting repaired by mechanisms such as the MRN complex. When the double strand of DNA breaks, the MRN complex registers and reacts to the break, activating specific molecules to go repair the damaged DNA (Lee, Paull 2005). This is why the functioning of MRN complex is necessary for the successful repair of DNA. If the MRN complex is not there, the ATM protein kinase cannot be activated and as a result, a repair is impossible (Uziel et al. 2003). Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two ways that cells use to repair DSB. If one of those mechanisms stops working properly, changes in the chromosomes can be seen, such as deletions, inversions, rearrangements and translocations. This leads to impaired division of the cell and its consequent death (Van Den Bosch, Bree & Lowndes 2003). Research has demonstrated that MRN has a role in many processes in the DNA metabolism, including DSBs, and when the MRN complex is mutated in mice, they die. This is why MRN is a necessary component for the successful repair of both endogenous and exogenous types of DNA damage (Van Den Bosch, Bree & Lowndes 2003). The MRN complex binds to double stranded breaks in the DNA and has actions in tethering broken ends prior to repair by non-homologous end joining or to initiate resection prior to repair by homologous recombination (Van Den Bosch, Bree & Lowndes 2003). Recent research has promoted the fact that these complexes are important for the toxicity of the cell, which is the result of breaks in the chromosomes. This means that they have a role in protecting the body from tumors and malignancy (Van Den Bosch, Bree & Lowndes 2003). 6.0 Aims and objectives The objective is to study the mechanisms used to minimize the effects of nucleoside analogues on cancer cells, due to their importance in cancer treatments as outlined in the literature review. This will be done by screening the S.pombe genome wide deletion library in conjunction with a RAD32 nucleus to determine the effect of nucleoside analogue removal or repair. The screening will identify and analyse the mRNA dependent nucleoside analogue resistant mechanism. Furthermore, a kinase and transporter will be introduced into the library as well as a RAD32 mutant and the sensitivity of these mutants to Gemcitabine (GemC) will be explored. 7.0 Bibliography Blagosklonny, M. 2005, "Carcinogenesis, cancer therapy and chemoprevention", Cell Death & Differentiation, vol. 12, no. 6, pp. 592- 602. Bunn, P.A.,Jr 1999, "Triplet chemotherapy with gemcitabine, a platinum, and a third agent in the treatment of advanced non-small cell lung cancer", Seminars in oncology, vol. 26, no. 1 Suppl 4, pp. 25-30. Deshpande, G.P., Hayles, J., Hoe, K.L., Kim, D.U., Park, H.O. & Hartsuiker, E. 2009, "Screening a genome-wide S. pombe deletion library identifies?novel genes and pathways involved in genome stability maintenance", DNA repair, vol. 8, no. 5, pp. 672-679. Evan, G.I. & Vousden, K.H. 2001, "Proliferation, cell cycle and apoptosis in cancer", Nature, vol. 411, no. 6835, pp. 342-348. 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