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Cell Cycle Changes after Glioblastoma Stem Cell Irradiation: The Major Role of RAD51.

ABSTRACT: "Glioma Stem Cells" (GSCs) are known to play a role in glioblastoma (GBM) recurrence. Homologous recombination (HR) defects and cell cycle checkpoint abnormalities can contribute concurrently to the radioresistance of GSCs. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for HR and its inhibition has been shown to sensitize GSCs to irradiation. The aim of this study was to examine the consequences of ionizing radiation (IR) for cell cycle progression in GSCs. In addition, we intended to assess the potential effect of RAD51 inhibition on cell cycle progression. Five radiosensitive GSC lines and five GSC lines that were previously characterized as radioresistant were exposed to 4Gy IR, and cell cycle analysis was done by fluorescence-activated cell sorting (FACS) at 24, 48, 72, and 96 h with or without RAD51 inhibitor. Following 4Gy IR, all GSC lines presented a significant increase in G2 phase at 24 h, which was maintained over 72 h. In the presence of RAD51 inhibitor, radioresistant GSCs showed delayed G2 arrest post-irradiation for up to 48 h. This study demonstrates that all GSCs can promote G2 arrest in response to radiation-induced DNA damage. However, following RAD51 inhibition, the cell cycle checkpoint response differed. This study contributes to the characterization of the radioresistance mechanisms of GSCs, thereby supporting the rationale of targeting RAD51-dependent repair pathways in view of radiosensitizing GSCs.

SUBMITTER: Tachon G

PROVIDER: S-EPMC6213228 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Cell Cycle Changes after Glioblastoma Stem Cell Irradiation: The Major Role of RAD51.

Tachon Gaelle G Cortes Ulrich U Guichet Pierre-Olivier PO Rivet Pierre P Balbous Anais A Masliantsev Konstantin K Berger Antoine A Boissonnade Odile O Wager Michel M Karayan-Tapon Lucie L

International journal of molecular sciences 20181003 10

"Glioma Stem Cells" (GSCs) are known to play a role in glioblastoma (GBM) recurrence. Homologous recombination (HR) defects and cell cycle checkpoint abnormalities can contribute concurrently to the radioresistance of GSCs. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for HR and its inhibition has been shown to sensitize GSCs to irradiation. The aim of this study was to examine the consequences of ionizing radiation (IR) for cell cycle progression in GSCs. In addition, we inte ...[more]

PMID: 30282933

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Project description:BackgroundIonizing radiation exposure is a great threat to human health. MicroRNAs (miRNAs) have been shown to play an important role in radiation-induced biological effects. Here, we investigated plasma miRNA expression changes and differentially expressed miRNAs in radiotherapy patients exposed to cobalt-60 (60Co) gamma rays to provide an experimental basis for human plasma miRNAs as an estimation indicator for ionizing radiation injury.MethodsSix patients with acute lymphoblastic leukemia (ALL) received continuous 5 gray (Gy) total body irradiation (TBI) twice. At 12 hours after irradiation, miRNA microarray was applied to screen for differentially expressed miRNAs, with some miRNAs confirmed by real-time polymerase chain reaction (RT-PCR) assay. Bioinformatic analysis was carried out to identify the relevant target genes and biological function of the differentially expressed miRNAs.ResultsAfter radiotherapy patients were exposed to 5 Gy gamma radiation, the expression of 9 plasma miRNAs was significantly upregulated, and the expression of 2 miRNAs was downregulated. After irradiation with 10 Gy gamma radiation, the blood plasma of radiotherapy patients contained 18 differentially expressed miRNAs, of which 17 were upregulated and 1 was downregulated (P<0.05). The expression of miR-4532, miR-4634, miR-4655-5p, miR-4763-3p, miR-4785, miR-6087, miR-6850-5p, and miR-6869-5p were significantly upregulated in both the 5-Gy and 10-Gy dose groups, showing a certain dose-response relationship. The RT-PCR results were consistent with the findings of high-throughput sequencing. In addition, the target genes of the differentially expressed miRNAs were mainly involved in RNA transcription and DNA damage. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these miRNAs participated in phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), Ras, mitogen-activated protein kinase (MAPK), and other signaling pathways.ConclusionsThe expression of differential plasma miRNAs of radiotherapy patients was associated with irradiation injury and showed a certain dose-effect relationship. These differentially coexpressed plasma miRNAs could be used as an early indicator for estimating radiation injury.

Project description:BackgroundClinical success of poly(ADP-ribose) polymerase inhibitors (PARP i ) has been limited to repair-deficient cancers and by resistance. Oncolytic herpes simplex viruses (oHSVs) selectively kill cancer cells, irrespective of mutation, and manipulate DNA damage responses (DDR). Here, we explore potential synthetic lethal-like interactions between oHSV and PARP i .MethodsThe efficacy of combining PARP i , oHSV MG18L, and G47Δ in killing patient-derived glioblastoma stem cells (GSCs) was assessed using cell viability assays and Chou-Talalay synergy analysis. Effects on DDR pathways, apoptosis, and cell cycle after manipulation with pharmacological inhibitors and lentivirus-mediated knockdown or overexpression were examined by immunoblotting and FACS. In vivo efficacy was evaluated in two GSC-derived orthotopic xenograft models (n = 7-8 per group). All statistical tests were two-sided.ResultsGSCs are differentially sensitive to PARP i despite uniform inhibition of PARP activity. oHSV sensitized GSCs to PARP i , irrespective of their PARP i sensitivity through selective proteasomal degradation of key DDR proteins; Rad51, mediating the combination effects; and Chk1. Rad51 degradation required HSV DNA replication. This synthetic lethal-like interaction increased DNA damage, apoptosis, and cell death in vitro and in vivo. Combined treatment of mice bearing PARP i -sensitive or -resistant GSC-derived brain tumors greatly extended median survival compared to either agent alone (vs olaparib: P ≤.001; vs MG18L: P = .005; median survival for sensitive of 83 [95% CI = 77 to 86], 94 [95% CI = 75 to 107], 102 [95% CI = 85 to 110], and 131 [95% CI = 108 to 170] days and for resistant of 54 [95% CI = 52 to 58], 56 [95% CI = 52 to 61], 62 [95% CI = 56 to 72], and 75 [95% CI = 64 to 90] days for mock, PARPi, oHSV, and combination, respectively).ConclusionsThe unique oHSV property to target multiple components of DDR generates cancer selective sensitivity to PARP i . This combination of oHSV with PARP i is a new anticancer strategy that overcomes the clinical barriers of PARP i resistance and DNA repair proficiency and is applicable not only to glioblastoma, an invariably lethal tumor, but also to other tumor types.

Project description:BACKGROUND:The transcription factor PAX6 is expressed in various cancers. In anaplastic astrocytic glioma, PAX6 expression is inversely related to tumor grade, resulting in low PAX6 expression in Glioblastoma, the highest-grade astrocytic glioma. The aim of the present study was to develop a PAX6 knock out cell line as a tool for molecular studies of the roles PAX6 have in attenuating glioblastoma tumor progression. METHODS:The CRISPR-Cas9 technique was used to knock out PAX6 in U251 N cells. Viral transduction of a doxycycline inducible EGFP-PAX6 expression vector was used to re-introduce (rescue) PAX6 expression in the PAX6 knock out cells. The knock out and rescued cells were rigorously characterized by analyzing morphology, proliferation, colony forming abilities and responses to oxidative stress and chemotherapeutic agents. RESULTS:The knock out cells had increased proliferation and colony forming abilities compared to wild type cells, consistent with clinical observations indicating that PAX6 functions as a tumor-suppressor. Cell cycle distribution and sensitivity to H2O2 induced oxidative stress were further studied, as well as the effect of different chemotherapeutic agents. For the PAX6 knock out cells, the percentage of cells in G2/M phase increased compared to PAX6 control cells, indicating that PAX6 keeps U251 N cells in the G1 phase of the cell cycle. Interestingly, PAX6 knock out cells were more resilient to H2O2 induced oxidative stress than wild type cells. Chemotherapy treatment is known to generate oxidative stress, hence the effect of several chemotherapeutic agents were tested. We discovered interesting differences in the sensitivity to chemotherapeutic drugs (Temozolomide, Withaferin A and Sulforaphane) between the PAX6 expressing and non-expressing cells. CONCLUSIONS:The U251 N PAX6 knock out cell lines generated can be used as a tool to study the molecular functions and mechanisms of PAX6 as a tumor suppressor with regard to tumor progression and treatment of glioblastoma.

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Cell Cycle Changes after Glioblastoma Stem Cell Irradiation: The Major Role of RAD51.

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Cell Cycle Changes after Glioblastoma Stem Cell Irradiation: The Major Role of RAD51.

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