Recent studies indicate that significant inter-individual variation exists at the amino acid sequence level of BER proteins, and that variability presumably exists in the BER capacity of the general population. We have taken on efforts to delineate the impact of amino acid variants found in BER proteins within the population and to develop assays to determine the extent of inter-individual variation at specific steps of BER. We have recently completed work in which we describe the functional capacity of XRCC1 proteins that harbor a site-specific amino acid substitution found within the human population. Such studies represent an important step towards understanding the molecular activities of variant proteins that may be susceptibility factors. In addition, using established biochemical assays, we are evaluating whether inter-individual variation in BER associates with disease susceptibility and/or clinical agent responsiveness;we are also assessing for age-dependent or gender-specific variation, within the limits of the sample set in hand. The establishment of such techniques, and ultimately a high throughput BER pathway assay presently in design, will be necessary to evaluate the relationship of BER capacity to disease susceptibility among the Baltimore Longitudinal Study of Aging (BLSA) population. Current strategies to eradicate cancer cells rely on the fact that they divide rapidly. Thus, to induce cell death, many anti-cancer agents interact with DNA to block replication and prevent duplication. Not surprisingly, cells with efficient repair of the cytotoxic DNA lesions generated by anti-cancer agents are more resistant to cell killing. Hence, a goal has been to regulate strategically the repair capacity of cancer and/or normal cells to improve the efficacy of specific therapeutic paradigms. In particular, inhibiting the DNA repair capacity of cancerous cells has been an area of promising interest. Using a catalytically-inactive, dominant negative form of the major human abasic endonuclease (APE1), which binds with high affinity to substrate DNA and blocks subsequent repair steps, we have assessed the role of BER in mediating cellular resistance to a broad range of clinically relevant DNA-interactive drugs. Our results indicate that expression of the dominant-negative protein, termed ED, increases cellular sensitivity to clinical alkylators, particularly streptozotocin and temozolomide;to the chain-terminating nucleoside analog troxacitabine, but not gemcitabine;and most impressively, to 5-fluorouracil and 5-fluorodeoxyuridine. These data suggest that APE1, and BER more generally, is a reasonable target for inactivation in anti-cancer treatment paradigms that involve select alkylating drugs and antimetabolites. We are currently designing adenoviral-based gene delivery systems to determine the effects of ED on modulating human cancer cell survival in culture and in mouse models. In addition, we are screening for small molecule, non-covalent chemical inhibitors of the major human abasic endonuclease APE1, with the long term goal of creating high affinity inhibitors with therapeutic value. Along these lines, we have recently described (i) the development of a panel of complementary and improved miniaturized high-throughput screening and profiling assays, which will have a broad appeal to other research investigators, particularly those in the field of DNA repair, and (ii) the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals. The establishment of adenoviral and complementary techniques, including small molecule inhibitors, will provide a platform for more extensive investigations on the therapeutic benefits of regulating cellular DNA repair capacity.
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