Abstract 262: O6-methylguanine:Deoxythymidine mismatches sensitize cells to DNA strand break-induced cytotoxicity

Background: O6-methylguanine (O6-MeG) is the primary cytotoxic lesion formed by SN1-methylating agents such as temozolomide. Cytotoxicity is induced following the mispairing of O6-MeG with deoxythymidine (T) residues and the subsequent activation of a mismatch repair (MMR)-mediated process. While it is generally recognized that MMR-induced DNA double strand breaks (DSBs) are central to O6-MeG-induced cell death, our previous work demonstrates that signaling via the p50 subunit of nuclear factor-κB (NF-κB) is also necessary for efficient SN1-methylation-induced cytotoxicity. In the current study, we isolated O6-MeG:T mismatches using a series of oligonucleotide duplexes and examined the hypothesis that O6-MeG:T mismatches induce a p50-dependent signaling pathway that sensitizes cells to DNA strand break-mediated cell death. Methods: A series of 40bp oligonucleotide duplex substrates bearing a unique central O6-MeG:T, G:T, O6-MeG:C, or G:C base pair were used. Cell lines included both human glioma cells and mouse embryonic fibroblasts. Loss of function was performed with shRNA-mediated p50 knockdown and targeted p50 knockout. Techniques used include EMSA, NF-κB-dependent luciferase reporter assays, in-vitro kinase assay, indirect immunofluorescence, annexin V binding and colony formation assays. Results: When administered to nuclear extracts, O6-MeG:T substrate inhibits NF-κB DNA binding substantially more than control duplex, and, when used in live cells, inhibition of NF-kB-dependent activity is seen only in response to O6-MeG:T substrate. The inhibition of NF-kB by O6-MeG:T substrate is noted only in p50-proficient cells. O6-MeG:T, but not control substrate, induces Chk1-mediated phosphorylation of p50 at a novel serine residue, a pathway that is shown to be necessary for inhibition of NF-kB DNA binding. O6-MeG:T substrate, used at a concentration sufficient to induce MMR- and p50-mediated signaling, does not increase apoptosis or decrease clonogenic survival relative to control duplexes. However, O6-MeG:T, but not control duplex, sensitizes p50-proficient, but not p50-deficent, cells to non-cytotoxic doses of ionizing radiation. O6-MeG:T-induced sensitization to cytotoxicity occurs without changing the level of IR-induced DSB and is only seen when duplex substrate is given prior to, but not after, IR. Conclusion: While an O6-MeG-induced signaling pathway has been previously described, the role of such a pathway to the damage response is disputed. The above findings suggest that O6-MeG:T-induced, p50-dependent signaling sensitizes cells to cytotoxicity that is ultimately induced by DNA DSBs. Such a model is temporally consistent with the response to O6-MeG in that MMR-induced signaling is induced after the first S-phase following O6-MeG formation while DSBs are formed after the second S-phase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 262. doi:1538-7445.AM2012-262