Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

Abstract

Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA damage signalling in low-and highgrade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n ¼ 41) and grade IV GBM (n ¼ 60), we conclude that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (cH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high-or lowoxygen culture conditions and in clinical specimens of both low-and high-grade tumors. The observed global checkpoint signaling, in contrast to only focal areas of overabundant p53 (indicative of p53 mutation) in grade II astrocytomas, are consistent with DDR activation being an early event in gliomagenesis, initially limiting cell proliferation (low Ki-67 index) and selecting for mutations of p53 and likely other genes that allow escape (higher Ki-67 index) from the checkpoint and facilitate tumor progression. Overall, these results support the potential role of the DDR machinery as a barrier to gliomagenesis and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.

Therapeutic targeting of constitutive PARP activation compromises stem cell phenotype and survival of glioblastoma-initiating cells

“…Although we have previously shown high and variable levels of oxidative DNA lesions in biopsies of human glioblastomas, 34 there have been no such studies in GICs versus non-GIC. We first evaluated the baseline ROS levels in low-passage GICs derived from human glioblastoma specimens previously validated to fulfill functional criteria of GICs: self-renewal, sustained proliferation, stem cell marker expression, capacity for lineage commitment, and tumor propagation.…”

“…We next evaluated the homeostatic levels of single-strand DNA (ssDNA) in matched GICs and non-GICs as assessed by BrDU incorporation under non-denaturing conditions and detected enhanced ssDNA in GIC populations (Supplementary Figure 3b). 34,40,41 We also used the alkaline comet assay to measure DNA strand breaks. GICs had significantly longer tails and higher comet tail DNA content as compared with the non-GICs, indicating the extent of fragmented DNA at baseline was greater in the GICs ( Supplementary Figure 3c-e).…”

“…We previously demonstrated higher basal activation of the DDR in GBM and specifically the GIC population. 2,34 We have now expanded these findings to highlight a requirement for the SSBR protein, PARP1, in GICs. Higher ROS and DNA damage within GICs likely contributes to constitutive activation of PARP1 within this population.…”

“…Detection of gH2AX (1 : 500; Abcam, Cambridge, UK) was performed as described previously. 34,40 GICs and non-GICs were grown on GelTrex (Gibco, Invitrogen, Carlsbad, CA, USA)-coated cover slips and treated with DMSO or 10 mM olaparib and left unirradiated or irradiated with 3 Gy and fixed 1, 6, or 24 h after irradiation. Cells were then immunostained for gH2AX.…”

Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells: prognostic significance and biological impact

“…21,22 Four cellular phenotypes have been identified: proneural, neural, mesenchymal, and classical. 23 Each phenotype has a slightly different prognosis, with the mesenchymal subtype being the most aggressive and leading to the shortest life expectancy.…”