| Summary: | We are interested in studying potential drugs that have the ability to make tumors more sensitive to radiation. There has been much interest in examining the effect of modulation to chromatin structure on the DNA damage response (DDR). We therefore examined histone deacetylase (HDAC) inhibitors, small molecules that modify the structural features of chromatin involved in the packaging of DNA. HDAC inhibitors are considered to be potential drug candidates to complement radiation therapy, yet the mechanisms underlying their effects have been elusive.
To study the DDR signaling effects of HDAC inhibitors, we developed a high-throughput automated microscopy assay to assess effects on elements of DNA damage-mediated signaling through quantification of H2AX phosphorylation, cell cycle profiling, and cell survival following irradiation. We then applied this assay to a library of diverse HDAC inhibitors as well as a collection of shRNAs targeting the individual HDACs. We next assessed DSB induction and repair kinetics on a selection of active HDAC inhibitors with CometChip, a novel high-throughput adaptation of the single-cell DSB comet assay. Finally, with the recent discovery of the role of the bromodomain chromatin reader protein Brd4 in the insulation of chromatin from the DDR, we explored combinatorial use of HDACi and JQ1 to achieve altered DDR through simultaneous alteration of chromatin structure and acetyl-lysine reading.
Our data suggest that class I HDAC inhibitors potently elevate H2AX phosphorylation, but without strong effects on DSB induction or repair kinetics. We find that many HDAC inhibitors also enhance G2/M cell cycle arrest and decrease proliferation, even in the absence of irradiation. These data suggest that HDAC inhibitors influence radiation effects primarily through altering DNA damage-induced signaling events. We have also found that overexpression of specific Brd4 isoforms can abrogate the elevated H2AX phosphorylation induced by HDAC inhibition and lead to preferential cell death. This may have important implications for the clinical use of these agents.
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