Wednesday, 25 January 2017

CONTRIBUTION OF DEFECTIVE DNA REPAIR IN CANCER DEVELOPMENT AND TREATMENT

DNA is the most important component of a cell to maintain its integrity. The process of replication and transcription has to be accurate to stabilize the genome. However, Without the enzyme polymerase (with both exonuclease and proofreading activity) error rate in DNA replication is found to be 10-6 to 10-8 (Thomas A Kunkel, 2004). Along with the enzyme polymerase, there are other types of machinery to repair damaged DNA during replication and other biochemical activities. The bacterial transforming DNA was inactivated by creating a lesion in presence of UV light of lower wavelength. Incorporation of the enzyme-like agent from the yeast on photoactivation restored the activity of bacterial DNA (Claud S Rupert, 1962). The experimental result was very much evident and suggested the implication of a machinery for DNA repair. UV radiations are known to cause thymidine dimers; cellular uptake of acridine dye is inversely proportional to thymidine dimers present in a cell. Three cell lines (desquamated buccal cells, cells from asymptotic smokes and cancer cells from oral cavity) were exposed to UV radiation and treated with enzyme-like complex from yeast in a medium containing acridine. Cancer cells distinctly expressed the deficit of repair response for damaged DNA and were analyzed by least cellular uptake of acridine (Daniel Roth, 1969). This was a landmark experiment to describe the role of defective DNA repair in cancerization.
Both external and internal factors can result in DNA damage. Several signalling pathways come into the picture as a response to the damaged DNA. The outcome of signalling pathways decide the fate of a cell and is categorised into three sections, namely; DNA repair, damage tolerance, and apoptosis. Several mutagenic responses are directly linked to defect in DNA repair machinery and mutations in sensors which recognizes DNA damage (Wynand P Roos, 2016).

Significant DNA damages like single strand break (SSB), double-strand break (DSB) and stalled replication fork activates the damage response elements. Functioning of DDR cascade comprises of sensory molecules like DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 Related (ATR) and PI3K related kinases (PIKK). Mutations in ATM or ATR cannot activate the
tumour suppressing protein, p53 and apoptotic initiator protein, Caspase 2; as a result, cell proliferation continues without the checkpoint barriers in the cell cycle (Figure 2). Laps of DDR elements explains the intricacies leading to uncontrolled cell proliferation (Wynand P Roos, 2016). Apart from sensors which detect DNA damage, repair system itself would be a deficit to rectify the damaged DNA.
Several examples are quoted to explain the cause of cancer on the basis of a defect in DDR or defect in DNA repair.

Xeroderma Pigmentosum (XP):
Person having XP explicates a 1000 fold more chances to get skin cancer (Cleaver, 1969). The cells from XP patient showed that there was a defect in repairing the DNA damage caused by UV radiation. Subsequent investigation of the cells from XP patient showed the mutation in the component of Nucleotide Excision Repair (NER) system which blocked repair mechanism (Lehman, 2011). Due to the defective repair system the dimers (thymidine dimer) formed by the UV light cannot be eliminated; resulting in the specific skin cancer predisposition in XP patients.

Lynch Syndrome or Hereditary Non-Polyposis Colorectal Cancer (HNPCC):
Instability of repeats of microsatellite causes a familial pattern of colorectal cancer characterized by mutations in the homologues of mismatch repair proteins, MutS and MutL (Leach, 1993). Lynch syndrome significantly increased the risk of other cancer types like endometrial cancer, ovarian cancer, pancreatic cancer, kidney cancer, urinary tract cancer (Cancer.net, 2014).

Werner Syndrome (WS):
WRN is a protein known to have both exonuclease and helicase activity. WRN protein is a key protein in DSB repair by Non-Homologous end joining (NHEJ) or homologous recombination (HR). Mutations in WNR gene can cause predisposition to cancer and early aging (Gray, M. D., 1997).

Fanconi Anemia (FA):
FA pathways includes three complex protein systems involved in DNA repair mechanism. HR, NER, and mutagenic translesion synthesis are dependent on FA pathway which employs nuclear protein complexes to ubiquitinate FANCD2 and FANCI, which in turn results in the formation of repair complex. The absence of ubiquitination of FANCD2 and FANCI unambiguously leads to collapse in the repair system causes uncontrolled cell proliferation (Moldovan, G.L, 2009).

Breast and Ovarian cancer:
BRCA1 is a vital component involved in DNA repair mechanism and is found to be in association with RAD51, protein functions in DSB repair system by homologous recombination. Mutant BRCA1 evidently altered homologous and non-homologous DNA integration and DSB repair. Maintenance of genomic integrity is chiefly regulated by BRCA1; thus, mutation of which results in the predisposition to cancer (Moynahan, M. E, 1999).

These instances evidently explain the consequences of faulty DNA damage repair. However, DNA repair mechanism itself act as a barrier in therapeutics of many cancer types. Anti-cancer agents target the DNA and create breaks in them so as to draw the cancer cells to apoptotic pathway. Cancer caused by other means (with unaltered repair machinery) would bring out efficient repair system to neutralize the activity of the anti-cancer drug. In such cases, it is obvious to use the supportive drug to inactivate the repair process. Functioning of supportive drugs is based on the pattern of damage caused by the initial drug used to damage the DNA. Thus it is a big challenge to reduce the risk that would be caused by the usage of the secondary drug molecule. This strategy cannot be employed to cancer caused by faulty DNA damage repair system. Cancer caused by defective DNA repair must be treated with a drug molecule which could either create cell senescence or cell death (apoptosis, necrosis or autophagy).
Any repair mechanism could be specifically targeted to get a better result for a cancer type with DNA damaging agent.

Targeting BER:
Anti-cancerous alkylating agents results in alkylated and oxidized bases which can be removed by Base excision repair mechanism. Apurinic or Apyrimidinic endonucleases play a vital role in BER. Inhibition of APE1 blocks the BER pathway and thereby alkylating agent could damage the DNA molecules go arrest the cell cycle. AP site can also be targeted to modify it by the virtue of which APE1 fails to bind to it. Methoxyamine (MX) is a tiny molecule with lesser Km value to APE1. Hence MX strongly binds with AP site and blocks APE1 to proceed with the repair process. APE1 is a multifunctional enzyme and is better to target APE1 to block all its functions. Thus, the combination of Temozolomide (Alkylating agent), E3330 (blocks redox activity of APE1) and MX can increase the efficiency of therapeutic activity and promising results are obtained.

Targeting HR and NHEJ:
Exposure to IR for a prolonged duration, ROS is a highly potential agent to create DSB. Meanwhile, these therapeutics cannot be efficient due to DDR. DDR will bring out the activation of HR or NHEJ repair systems which neutralizes the therapeutic activity of IR and ROS. DDR is a function mediated by ATM, ATR, and DNA-PK which transduces the signals to activate repair pathway. Inhibition of ATM and ATR were not significance due to the side effects and sensitivity to switching over to other cancer types (Collis SJ, 2005). Thus targeting DNA-PK looks promising to increases the therapeutic activity with fewer side effects. DNA-PK inhibitors like vanillin, NU7026 are very effective and efficiently blocked the repair system. Some of the
DNA damaging agents can even inhibit DNA-PK thereby performs the dual function and reduces the risk of side effect. For instance, inhibitors of Topoisomerase 2 even blocks NHEJ. Highly advanced and accurate technique is the use of siRNA and it is found to reduce the activity of ATM, ATR and DNA-PK by 90% and was significantly higher than wortmannin (Collis SJ, 2003).

Targeting direct repair:
O6 Methyl Guanine-DNA-Methyl Transferase (MGMT) results in resistance to many anticancer drugs, like Temozolomide, dacarbazine, BCNU by transferring the adduct to its own cysteine residue. Thus MGMT gene is targeted and subjected for methylation of its gene. Analogue of guanine is used to suppress the enzymatic activity of MGMT (Yongjian Zhu, 2009).
Therapeutic actions are very crucial and have to be checked for side effects and other possible consequences. Personalised medicine would possibly give a better result; along with which whole genome sequencing and exome sequencing techniques should be employed for cancer therapy. Each cancer patterns have to be sequestered with genome sequencing through which medication could be done at the molecular level. Cancer with a family history should be treated with suitable prognostic methods.





References:
1. Thomas A Kunkel, JBC – Apr 23, 2004
2. Claud S Rupert, The journal of General Physiology, Vol 45,1962
3. Daniel Roth and Harold H Sage, Cancer, Sept 1969.
4. Cleaver, J. E. Proc. Natl Acad. Sci. USA 63, 1969
5. Lehmann, A. R., McGibbon, D. & Stefanini, 6, 70 (2011).
6. Leach, F. S. et al. Cell 75, (1993)
7. Cancer.net, Editorial board, 12/2014
8. Gray, M. D. et al. Nat. Genet. 17, (1997).
9. Moldovan, G. L. & D’Andrea, A. D. Annu. Rev. Genet. 43, (2009).
10. Moynahan, M. E., Chiu, J. W., Koller, B. H. & Jasin, M. 1999
11. Yongjian Zhu a,d,e, Jue Hu b,d,f, Yiduo Hu c,g, Weiguo Liu a
12. Collis SJ, DeWeese TL, Jeggo PA, Parker AR. Oncogene 2005.

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The article is written by using the data from several research articles and are subjected for copyright. Hence, they are referenced accordingly.

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