Abstract
Abstract Radiation therapy is often associated with normal tissue injury as an undesired side effect. This is promoted by the depletion of normal stem cells and a decreased regenerative capacity of the affected organs. To reduce normal tissue injury during radiotherapy protective treatments have to be developed that improve stem cell survival without protecting tumor cells. In this study we investigate the acetylation and methylation of histone 3 at lysine 9 (H3K9), that is responsive to DNA damage and uniquely regulated in stem cells, as potential target for the development of radioprotective therapeutics. We analyzed stem cells in mouse tissue niches in vivo, murine embryonic stem cells in culture and neural stem cells freshly isolated from newborn mouse pups. We investigate repair factors, histone modifications and apoptosis pathways by immunofluorescence staining, western blotting and flow cytometry after X-ray irradiation or micro-irradiation of individual cells. H3K9 modifying enzymes are targeted with inhibitors, activators, RNA interference or overexpression by plasmid transfection and the effect on stem cells’ radiosensitivity evaluated. We show that stem cells are highly radio-sensitive and have reduced DNA repair with low activation of DNA damage factors like ATM. We screened for stem cell specific levels of histone modifications that are responsive to DNA damage and discovered that stem cells of adult tissue niches like brain and testis as well as murine embryonic and neuronal stem cells show very high levels of H3K9ac, while H3K9me3 levels are decreased. In tumor cells H3K9 acetylation (H3K9ac) was shown to be reduced after damage induction, while H3K9 tri-methylation (H3K9me3) is increased to fully activate the repair factor ATM. We hypothesize that in stem cells, an inept transition between H3K9 acetylation and tri-methylation prevents robust activation of ATM leading to high radiosensitivity. In agreement with this hypothesis, we find reduced down-regulation of H3K9ac in stem cells at the DNA damage sites. To transiently improve the H3K9ac/me3 switch in stem cells in order to enhance activation of DNA repair factors and stem cell survival after irradiation, we aim to transiently reduce H3K9 acetylation by targeting the responsible acetyltransferases and deacetylases. We show expression levels of H3K9 modifying enzymes in stem cells before and after irradiation as well as preliminary results of the effect of down- or up-regulation of H3K9 modifying enzymes on stem cell radiosensitivity. We conclude that elevated H3K9 acetylation and reduced H3K9 tri-methylation levels in stem cells and their responsible H3K9 modifying enzymes are potential targets to improve ATM activation and stem cell survival after irradiation. Citation Format: Barbara Meyer, Keith M. Jacobs, Suyash Raj, Cheri L. Zobel, Dennis E. Hallahan, Girdhar G. Sharma. The role of H3K9 acetylation and tri-methylation in stem cell radiosensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3299. doi:10.1158/1538-7445.AM2015-3299
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