Abstract
A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1β in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1β complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1β foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses.
Highlights
Addition, inhibitors of signaling pathway proteins such as RAS9, ErbB receptor tyrosine kinase[10], and HER211,12 have been described as radiosensitizers
Together with the detection of interaction between DNMT3B and H2AX induced by ionizing radiation (IR), our current study suggested that DNMT3B regulates IR-induced HP1β foci-formation and H2AX accumulation, and consequent DNA damage responses thereby protecting cells from cell death
Previous studies showed that DNMT3B interacts with HP1 and the accumulation of HP1β at double strand break (DSB) sites has been reported as an early response to irradiation[31,32]; we examined whether knockdown of DNMT3B impaired the regulation of HP1β in HeLa cells after γ -irradiation
Summary
Addition, inhibitors of signaling pathway proteins such as RAS9, ErbB receptor tyrosine kinase[10], and HER211,12 have been described as radiosensitizers. Bevacizumab is a radiosensitizer that blocks vascular endothelial growth factor (VEGF) in the tumor micro-environment[13]. This study aims to comprehensively identify genes that promote the radiosensitization of p53-inactivated cancer cells, HeLa when downregulated. We demonstrated that DNMT3B regulates HP1β and H2AX to protect cells from ionizing radiation (IR). DNMT3B dysfunction impaired HP1β foci-formation and H2AX accumulation induced by IR. Together with the detection of interaction between DNMT3B and H2AX induced by IR, our current study suggested that DNMT3B regulates IR-induced HP1β foci-formation and H2AX accumulation, and consequent DNA damage responses thereby protecting cells from cell death. Comprehensive screening accompanied by cluster analysis might be useful for the identification and evaluation of radiosensitization targets
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