Abstract 3659: NF-κB1-p50 is a novel mediator of methylation-induced apoptosis

SN1-methylating agents form O6-methylguanine (O6-MeG) lesions that induce apoptosis following mis-pairing with thymine (T) and activation of the mismatch repair (MMR) system. Previous work has shown that O6-MeG inhibits the activity of the transcription factor, nuclear factor-κB (NF-κB), by a mechanism involving MMR-dependent inhibition of NF-κB DNA-binding. In the current study, we set out to determine the mechanism by which methylating agents block NF-κB as a prerequisite to understanding the role of NF-κB in the response to O6-MeG. We hypothesize that a single NF-κB subunit is the target that is essential for mediating both inhibition of NF-κB and cytotoxicity in response to methylation damage. Methods: Temozolomide and methyl-nitrosourea were used as model methylators and both human glioma cells and mouse embryonic fibroblasts were used. Loss of function was performed with siRNA-mediated NF-κB knockdown and targeted NF-κB subunit knockout. Techniques used include EMSA, ChIP assay, NF-κB- and Gal4-dependent luciferase reporter assays, quantitative real time-PCR, single cell gel electrophoresis (comet) assay, flow cytometric analysis of annexin V binding, MTS short term cell killing assay and long-term colony formation assay. Results: The NF-κB subunits affected by SN1-methylators include p65 and p50 suggesting that one or both of these subunits mediates the response to SN1-methylation. Studies demonstrate that while p65 is dispensable for methylation-induced inhibition of NF-κB, p50 is required for inhibition of NF-κB. Furthermore, p50 is necessary for methylation-induced inhibition of endogenous NF-κB-dependent anti-apoptotic gene expression. The specific role of O6-MeG: T mismatches to inhibition of NF-κB was confirmed using oligonucleotide duplexes bearing a central O6-MeG: T, G: T, O6-MeG: C, or G: C base pair and observing that only O6-MeG: T mismatches inhibit NF-κB, in a p50-dependent manner. Because of the importance O6-MeG and MMR to inhibition of NF-κB, the requirement of p50 for methylator-induced cytotoxicity was examined. p50-deficient cells are resistant to methylation-induced apoptosis and also show increased clonal viability following methylator treatment compared to wild-type (wt) cells. The resistance to killing is specific to MMR-mediated cytotoxicity as p50 deficient cells are as sensitive as wt cells to ionizing radiation and doxorubicin. Importantly, there is equivalent damage induction following methylator treatment in p50 deficient cells relative to wt cells as assessed by comet assay. The specificity of p50 to methylation-induced killing was further confirmed by demonstrating that deletion of the p65 subunit increases methylation-induced cytotoxicity. Conclusion: The p50 NF-κB subunit acts downstream of MMR to enable apoptosis in response to SN1-methylation damage. This study sets the groundwork for evaluation of a novel intermediate, p50, in the MMR-directed cytotoxic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3659.