GBM CSC’s display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation

GBM CSC’s display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation. Inhibition of ATM kinase by KU\55933 produced GDC-0980 (Apitolisib, RG7422) potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6C3.5) and effectively abrogated the enhanced DSB repair proficiency observed in GBM CSCs at 24?h post irradiation. and differentiated tumour cells were exposed to increasing concentrations of KU\55933 and incubated for 24?h prior to performing CellTiter\Glo assay (promega). Each data point represents mean plus SEM for 3 GDC-0980 (Apitolisib, RG7422) independent experiments. B, E2 and G7 CSC and differentiated tumour cells were exposed to increasing concentrations of KU\55933 for a period GDC-0980 (Apitolisib, RG7422) of 6 days prior to cell viability assay in order to assess toxicity of prolonged exposure. Data points represent mean plus SEM for 3 independent experiments. C, Demonstration of specificity of the Novus pATM s1981antibody. ATM crazy type and null mouse embryonic fibroblasts were treated with 5?Gy or sham irradiated, then lysed at 1?h following treatment and probed for manifestation of pATM s1981 by western blot. MOL2-9-192-s002.jpg (61K) GUID:?8F07A5F2-529A-4510-91A3-50EBDDAD0F48 Supplementary Figure?3 Cell cycle profiles and FACS gating for G2/M checkpoint analysis. A, Representative images of cell cycle profiles acquired in E2 and G7 CSC and differentiated tumour cell cultures following incubation with KU\55933 and irradiation at timepoints indicated. B, Representative images of gating used during analysis of phosphorylated histone H3 for G2/M checkpoint interrogation in E2 and G7 CSC and differentiated tumour cell cultures. MOL2-9-192-s003.jpg (53K) GUID:?C5C74E57-7C7A-41B1-90D4-163140BA25A7 Supplementary Figure?4 Images of gamma H2AX immunofluorescent staining of DNA DSBs (green foci) in E2 CSC and differentiated tumour cell cultures following irradiation with 1?Gy at timepoints indicated. Cells in G2 cell cycle phase are Vezf1 stained in reddish for the G2 marker CENPF, nuclei are counterstained with DAPI. MOL2-9-192-s004.jpg (62K) GUID:?E2046DB8-A1B1-4DEB-9FBD-7A1F1793FA26 Supplementary Figure?5 Images of gamma H2AX immunofluorescent staining of DNA DSBs (green foci) in E2 CSC and differentiated tumour cell cultures following treatment with 10?M KU\55933 or 0.01% DMSO and irradiation with 1?Gy at 1 and 24?h. Cells in G2 cell phase are stained in reddish for the G2 marker CENPF, nuclei are counterstained with DAPI. MOL2-9-192-s005.jpg (78K) GUID:?A8D647AE-4121-436E-9B9D-3A3C4DF181C7 Supplementary Table 1 Mean SF2Gy ideals for R10, E2 and G7 CSC and tumour bulk cultures derived from 9 indie experiments in the case of E2 and G7, and 3 indie experiments in the case of R10 each performed in triplicate. MOL2-9-192-s006.jpg (25K) GUID:?A4088B22-72FE-4273-A53D-995100A4E814 Abstract Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM malignancy stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs show upregulated DNA damage responses and are resistant to radiation but the current literature is definitely conflicting. We investigated radioresistance of main GBM cells produced in stem cell conditions (CSC) compared to combined GDC-0980 (Apitolisib, RG7422) differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU\55933. We statement that GBM CSCs are radioresistant compared to combined differentiated tumour cells as measured by clonogenic assay. GBM CSC’s display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and restoration DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation. Inhibition of ATM kinase by KU\55933 produced potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6C3.5) and effectively abrogated the enhanced DSB repair skills observed in GBM CSCs at 24?h post irradiation. G2/M checkpoint activation was reduced but not abolished by KU\55933 in GBM CSCs. ATM kinase inhibition overcomes radioresistance of GBM CSCs and, in combination with conventional therapy, offers potential to improve outcomes for individuals with GBM. following temozolomide treatment (Chen et?al., 2012). Reactions of GBM CSCs to radiotherapy have also been investigated, with conflicting results. Bao et?al. shown that CD133+ tumour cell populations were radioresistant compared to CD133? populations (Bao et?al., 2006), a phenotype that was mediated by upregulation of the DNA damage response (DDR). Enhanced phosphorylation of cell cycle checkpoint proteins was shown along with evidence of more efficient DNA repair, even though kinetics of DNA double strand break (DSB) restoration were not examined in detail. In contrast,.