Thesis (M.Sc.)--Chulalongkorn University, 2011

Inhibition of DNA-PKcs-mediated non-homologous end joining (NHEJs) under drug- induced histone acetylation reduces cell survival. However the mechanism is not well understood. Recently, we developed a new PCR-based technique to measure the amounts and types of epigenetic modification of endogenous DNA double strand breaks (EDSBs). We found that EDSBs in cells at G0 stage, known as replication-independent EDSBs (RIND-EDSBs) exist in two classes, namely, those present at a given level in all cell types under normal physiological conditions (named NRIND-EDSBs), which are hypermethylated, retained in facultative heterochromatin, devoid of γ-H2AX phosphorylation, and are repaired by a slow but more precise ATM-mediated NHEJs; and those (known as PRIND-EDSBs) that are hypomethylated and bound by γ-H2AX are only detected when DSB repair is inhibited. As NRIND-EDSBs are found in all cell types, we propose that they may possess normal physiological function. On the other hand, cells recognize PRIND-EDSBs as equivalent to radiation-induced DSBs and may be pathogenic. NRIND-EDSBs were reduced when chromatin became hyperacetylated by treatment with 100 ng/ml trichostatin A (TSA) (histone deacetylase inhibitor), but were increased when cells were treated with a combination of 100 ng/ml TSA, 2.5 mM vanillin (DNA-PKcs inhibitor) and 5 mM caffeine (ATM inhibitor). Therefore, we hypothesize that NRIND-EDSBs prevent PRIND-EDSB production, and that reduction of NRIND-EDSBs should increase PRIND-EDSBs, leading to cell death when repair of EDSBs are inhibited. We observed a reduction in NRIND-EDSBs under three situations: DNA hypomethylation, histone hyperacetylation and down regulation of high-mobility group protein B1 (HMGB1). We also found variations in NRIND-EDSB levels under normal culture conditions. When TSA-treated cells or those containing reduced levels of HMGB1 were exposed to vanillin, there was an increase in PRIND-EDSBs after 2 hours. Consequently, lower survivals were observed in both types of test cells. There is also a direct correlation between NRIND-EDSB level and resistance to vanillin treatment. In summary, NRIND-EDSBs are maintained in normal cells at a given level by DNA methylation, histone acetylation and an appropriate amount of HMGB1. Reduction in NRIND-EDSBs results in an increase in PRIND-EDSBs, thereby causing cell death when cells are treated with inhibitor of DSB repair (viz. vanillin). We postulate that in the absence of DNA repair inhibitors, PRIND-EDSBs should lead to DSB repair errors. Thus, a reduction of NRIND-EDSBs as a result of global hypomethylation, histone hyperacetylation or reduction of HMGB1 may underlie genomic instability.