Dataset Information

Replication stress, DNA damage signalling, and cytomegalovirus infection in human medulloblastomas.

ABSTRACT: Medulloblastomas are the most common, and often fatal, paediatric brain tumours that feature high genomic instability, frequent infection by human cytomegalovirus (HCMV) and resistance to radiation and chemotherapy. The causes of the pronounced chromosomal instability and its potential links with HCMV infection and/or resistance to genotoxic therapies remain largely unknown. To address these issues, here we have combined immunohistochemical analysis of a series of 25 paediatric medulloblastomas, complemented by medulloblastoma cell culture models including experimental HCMV infection. Using eight established immunohistochemical markers to assess the status of the DDR machinery, we found pronounced endogenous DNA damage signalling (γH2AX marker) and robust constitutive activation of both the ATM-Chk2 and ATR-Chk1 DNA damage checkpoint kinase cascades, yet unexpectedly modest p53 tumour suppressor activation, across our medulloblastoma cohort. Most tumours showed high proliferation (Ki67 marker), variable oxidative DNA damage (8-oxoguanine lesions) and formation of 53BP1 nuclear 'bodies', the latter indicating (along with ATR-Chk1 signalling) endogenous replication stress. The bulk of the clinical specimens also showed expression of HCMV immediate early and late proteins, in comparative analyses using three immunohistochemical protocols. Cell culture experiments validated the chronic endogenous replication stress in medulloblastoma cell lines and showed sharply differential, intriguing responses of normal cells and medulloblastoma cells to HCMV infection, including differential subcellular mislocalization and enhancement of replication stress-related 53BP1 body formation, the latter in cell-non-autonomous manner. Overall, our results strongly indicate that in human medulloblastomas, the DDR checkpoint barrier is widely activated, at least in part due to replication stress. Furthermore, we propose that unorthodox p53 defects other than mutations may allow high proliferation despite the ongoing checkpoint signalling and that the highly prevalent HCMV may impact the medulloblastoma host cell replication stress and DNA repair. Collectively, the scenario we report here likely fuels genomic instability and evolution of medulloblastoma resistance to standard-of-care genotoxic treatments.

SUBMITTER: Bartek J

PROVIDER: S-EPMC5537913 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Despite recent progress in research on brain tumors, including identification of cancer stem-like cells (CSCs), little is known about the interplay of stemness with the commonly observed infection by the human cytomegalovirus (HCMV) and the widespread features of replication stress in these malignancies. To shed more light on these outstanding issues, here we combine immunohistochemical analysis of archival clinical specimens from a cohort of 25 human pediatric medulloblastomas, complemented by functional experiments and analytical approaches to examine three medulloblastoma cell lines. In the clinical samples, we find consistent, yet individually variable subsets of CSCs expressing the stem-cell markers CD133 and CD15, and a candidate marker VEGFR2, across the spectrum of endogenous DNA damage (γH2AX), expression of HCMV immediate early and late proteins, proliferation rate (Ki67) or molecular class of MB. Contrary to MB cell lines DAOY and D324, the D283 cells showed pronounced phenotypic features of stemness, associated with enhanced endogenous DNA damage, exceptionally high susceptibility to infection with HCMV, unorthodox signaling pathway response to ionizing radiation and hyperactive response to hydroxyurea-induced replication stress. Notably, single-molecule DNA fiber analysis revealed aberrantly slow replication fork progression, pronounced fork asymmetry and inability to timely recover from drug-induced fork stalling in stem-like D283 cells, all hallmarks of pronounced chronic replication stress and propensity to genomic instability. These findings provide insights into human medulloblastoma stemness phenotypes, with various susceptibilities to infection by HCMV and impact on replication fork (mal)function, with implications for better understanding pathogenesis and responses to treatment in pediatric brain malignancies.Abbreviations: CSC: cancer stem-like cells; FBS: fetal bovine serum; HCMV: human cytomegalovirus; MB: medulloblastoma; MBSC: medulloblastoma stem cells; MOI: multiplicity of infection; PBS: phosphate-buffered saline; RPA: replication protein A; RS: replication stress; SHH: sonic hedgehog; VEGFR2: vascular endothelia growth factor receptor 2.

Project description:UNLABELLED:BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability. IMPORTANCE:Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR activation is viral replication. Furthermore, we show that the virus is able to cause host DNA damage in the absence of viral replication and DDR activation. These results suggest an intricate relationship between viral replication, DDR activation, and host genome instability.

Dataset Information

Replication stress, DNA damage signalling, and cytomegalovirus infection in human medulloblastomas.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets