Radiation-Induced Hippocampal Neurogenesis Impairment and Cognitive Deficits Is Associated With Inhibition of BDNF-Trk-B Signaling

Abstract

Purpose/Objective(s): Histone deacetylase (HDAC) inhibitors are potential drug candidates to complement radiation therapy, and have shown a radiosensitizing effect in preclinical models of solid tumor malignancies. The molecular mechanisms underlying HDAC inhibitor effects on radiosensitivity are elusive, however. In order to investigate the mechanism of HDAC inhibitor-induced radiosensitization, we investigated the effects of multiple HDAC inhibitors on DNA double strand break (DSB) formation, DSB repair, and DNA damage-induced signaling events via high-throughput screening techniques. Materials/Methods: 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 IR-induced DSBs in U2OS osteosarcoma cells. 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 that facilitates automated imaging and analysis of DSB content by achieving a uniform single-cell spacing with a single focal plane. Results: Data from our screening platform suggest that class I HDAC inhibitors potently elevate H2AX phosphorylation and have mild cell cycle effects. Effects on cell proliferation indicate that in these cells there is not a robust enhancement of radiation-induced cell death. Our investigation using CometChip demonstrated that HDAC inhibition did not achieve strong effects on DSB induction or repair kinetics. Using both FACS analysis and live cell imaging, we find that some HDAC inhibitors also enhance G2/M cell cycle arrest and decrease cell proliferation, even in the absence of irradiation. Conclusions: Our data suggest that HDAC inhibitors influence radiation effects primarily through altering IR-induced signaling events, rather than through an increased frequency of DNA DSBs or a repression of DSB repair. This may have important implications for the clinical use of these agents. The two assay techniques we have developed can readily be extended to many tumor cell lines as a screening platform to identify candidate drugs to advance to preclinical and clinical studies. Author Disclosure: B. Greenberger: None. M.E. Pacold: None. J.J. Tay: None. R. Alter: None. Q. Huang: None. J. Ge: None. J.E. Bradner: None. B.P. Engelward: None. M.B. Yaffe: None. S.R. Floyd: None.