Direct and indirect modulation of inflammation-induced DNA damage

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 164-183).Cancer causes 13% of all deaths worldwide. Inflammation-mediated cancer accounts for ~15% of all malignancies, strongly necessitating investigation of the molecular interactions at play. Inflammatory reactive oxygen and nitrogen species (RONs), including peroxynitrite and nitric oxide (NO'), may potentiate malignancy. We hypothesize that the base excision repair (BER) pathway modulates susceptibility to malignancy, by modulating the BER-intermediate levels, large scale genomic rearrangements and toxicity following exposure to RONs. We further hypothesize that DNA methyltransferases are responsible for the memory of genotoxic insult, and the epigenetic propagation of genomic instability, following exposure to genotoxins. Here, we exploited cell lines engineered to carry deficiencies in BER to study repair of DNA damage induced by RONs. Toxicity and BER-intermediate levels were evaluated in XRCC1 proficient and deficient cells, following exposure to the peroxynitrite donor, SIN-1 and to NO*. Using the alkaline comet assay, we find that while XRCC1 proficient and deficient CHO cells incur equivalent levels of SIN-1 induced BER-intermediates, the XRCC1 null cells are more sensitive to killing by SIN-1, as assessed by clonogenic survival. Furthermore, using bioreactors to expose CHO cells to NO', we found that the BER-intermediate levels measured in XRCC1 null cells were lower than in WI cells. We found that while XRCC1 can facilitate AAG-mediated excision of the inflammation-associated base lesions ethenoadenine and hypoxanthine, in vitro; XRCC1 deficient human cells were no more susceptible to NO' than WT cells. However, in live glioblastoma cells, XRCC1 is acting predominantly downstream of AAG glycosylase. This work is some of the first to assess the functional role of XRCC1, in response to RONs and suggests complexities in the role of XRCC1. We also demonstrate that the underlying basis for the memory of a genotoxic insult and the subsequent propagation of genomic instability is dependent on the DNA methyltransferases, Dnmtl and Dnmt3a. We found that a single exposure led to long-term genome destabilizing effects that spread from cell to cell, and therefore provided a molecular mechanism for these persistent bystander effects. Collectively, our findings impact current understanding of cancer risk and suggest mechanisms for suppressing genomic instability, following exposure to inflammatory genotoxins.by James T. Mutamba.Ph.D

XRCC1 and base excision repair balance in response to nitric oxide

Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO−) and nitric oxide (NOradical dot), create base lesions that potentially play a role in the toxicity and large genomic rearrangements associated with many malignancies. Little is known about the role of base excision repair (BER) in removing these endogenous DNA lesions. Here, we explore the role of X-ray repair cross-complementing group 1 (XRCC1) in attenuating RONs-induced genotoxicity. XRCC1 is a scaffold protein critical for BER for which polymorphisms modulate the risk of cancer. We exploited CHO and human glioblastoma cell lines engineered to express varied levels of BER proteins to study XRCC1. Cytotoxicity and the levels of DNA repair intermediates (single-strand breaks; SSB) were evaluated following exposure of the cells to the ONOO− donor, SIN-1, and to gaseous NOradical dot. XRCC1 null cells were slightly more sensitive to SIN-1 than wild-type cells. We used small-scale bioreactors to expose cells to NOradical dot and found that XRCC1-deficient CHO cells were not sensitive. However, using a molecular beacon assay to test lesion removal in vitro, we found that XRCC1 facilitates AAG-initiated excision of two key NOradical dot-induced DNA lesions: 1,N[superscript 6]-ethenoadenine and hypoxanthine. Furthermore, overexpression of AAG rendered XRCC1-deficient cells sensitive to NOradical dot-induced DNA damage. These results show that AAG is a key glycosylase for BER of NOradical dot-induced DNA damage and that XRCC1's role in modulating sensitivity to RONs is dependent upon the cellular level of AAG. This demonstrates the importance of considering the expression of other components of the BER pathway when evaluating the impact of XRCC1 polymorphisms on cancer risk.Massachusetts Institute of Technology. Center for Environmental Health Sciences (NIEHS P30-ES002109)National Institutes of Health (U.S.) (NIH grant P01-CA026731)National Institutes of Health (U.S.) (NIH grant 2-R01-CA079827-05A1)National Institutes of Health (U.S.) (NIH Grant U01-ES016045)National Institutes of Health (U.S.) (NIH Grant GM087798)National Institutes of Health (U.S.) (NIH Grant CA148629)National Institutes of Health (U.S.) (NIH Grant ES019498)National Institutes of Health (U.S.) (Cancer Center Support Grant P30 CA047904