Comparative Analysis of DNA Repair in Stem and Nonstem Glioma Cell Cultures (original) (raw)
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Molecular Cancer Therapeutics, 2012
Glioblastoma multiforme (GBM) is the most common form of brain tumor with a poor prognosis and resistance to radiotherapy. Recent evidence suggests that glioma-initiating cells play a central role in radioresistance through DNA damage checkpoint activation and enhanced DNA repair. To investigate this in more detail, we compared the DNA damage response in nontumor forming neural progenitor cells (NPC) and glioma-initiating cells isolated from GBM patient specimens. As observed for GBM tumors, initial characterization showed that gliomainitiating cells have long-term self-renewal capacity. They express markers identical to NPCs and have the ability to form tumors in an animal model. In addition, these cells are radioresistant to varying degrees, which could not be explained by enhanced nonhomologous end joining (NHEJ). Indeed, NHEJ in glioma-initiating cells was equivalent, or in some cases reduced, as compared with NPCs. However, there was evidence for more efficient homologous recombination repair in glioma-initiating cells. We did not observe a prolonged cell cycle nor enhanced basal activation of checkpoint proteins as reported previously. Rather, cell-cycle defects in the G 1 -S and S-phase checkpoints were observed by determining entry into S-phase and radioresistant DNA synthesis following irradiation. These data suggest that homologous recombination and cell-cycle checkpoint abnormalities may contribute to the radioresistance of glioma-initiating cells and that both processes may be suitable targets for therapy.
The DNA damage/repair cascade in glioblastoma cell lines after chemotherapeutic agent treatment
International Journal of Oncology, 2015
Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose-and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas.
DNA damage response and repair: insights into strategies for radiation sensitization of gliomas
Future oncology (London, England), 2011
The incorporation of radiotherapy into multimodality treatment plans has led to significant improvements in glioma patient survival. However, local recurrence from glioma resistance to ionizing radiation remains a therapeutic challenge. The tumoricidal effect of radiation therapy is largely attributed to the induction of dsDNA breaks (DSBs). In the past decade, there have been tremendous strides in understanding the molecular mechanisms underlying DSB repair. The identification of gene products required for DSB repair has provided novel therapeutic targets. Recent studies revealed that many US FDA-approved cancer agents inhibit DSB repair by interacting with repair proteins. This article will aim to provide discussion of DSB repair mechanisms to provide molecular targets for radiation sensitization of gliomas and a discussion of FDA-approved cancer therapies that modulate DSB repair to highlight opportunities for combination therapy with radiotherapy for glioma therapy.
Glioblastoma Stem Cells: Radiobiological Response to Ionising Radiations of Different Qualities
Radiation Protection Dosimetry, 2015
Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor, with very poor prognosis. The high recurrence rate and failure of conventional treatments are expected to be related to the presence of radio-resistant cancer stem cells (CSCs) inside the tumor mass. CSCs can both self-renew and differentiate into the heterogeneous lineages of cancer cells. Recent evidence showed a higher effectiveness of C-ions and protons in inactivating CSCs, suggesting a potential advantage of Hadrontherapy compared to conventional radiotherapy for GBM treatment. In order to investigate the mechanisms involved in the molecular and cellular response of CSCs to ionising radiations, we irradiated two GBM stem cell (GSC) lines, named line # 1 and line # 83, derived from patients with different clinical outcome and having different metabolic profiles (as shown by NMR spectroscopy), with 137 Cs photons and with protons or C-ions of 62 MeV/u in the dose range 5-40 Gy. The biological effects investigated were: cell death, cell cycle progression, and DNA damage induction and repair. Preliminary results show a different response to ionising radiation between the two GSC lines for the different end points investigated. Further experiments are in progress to consolidate the data and to get more insights on the influence of radiation quality.
Cancer Letters, 2017
The role of histone deacetylase (HDAC) 4 and 6 in glioblastoma (GBM) radioresistance was investigated. We found that tumor samples from 31 GBM patients, who underwent temozolomide and radiotherapy combined treatment, showed HDAC4 and HDAC6 expression in 93.5% and 96.7% of cases, respectively. Retrospective clinical data analysis demonstrated that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome. In vitro experiments revealed that short hairpin RNA-mediated silencing of HDAC4 or HDAC6 radiosensitized U87MG and U251MG GBM cell lines by promoting DNA double-strand break (DSBs) accumulation and by affecting DSBs repair molecular machinery. We found that HDAC6 knock-down predisposes to radiation therapy-induced U251MG apoptosis-and U87MG autophagy-mediated cell death. HDAC4 silencing promoted radiation therapy-induced senescence, independently by the cellular context. Finally, we showed that p53 WT expression contributed to the radiotherapy lethal effects and that HDAC4 or HDAC6 sustained GBM stem-like radioresistant phenotype. Altogether, these observations suggest that HDAC4 and HDAC6 are guardians of irradiation-induced DNA damages and stemness, thus promoting radioresistance, and may represent potential prognostic markers and therapeutic targets in GBM.
Is inhibiting the DNA damage response the answer to treatment resistance in glioma stem cells?
Translational Cancer Research, 2016
Research efforts in the last decade have increased our understanding of the complexity and treatment resistance of aggressive stage IV gliomas (GBMs) that result in a dismal prognosis for GBM patients. GBMs have inherent plasticity; a single tumor consists of many phenotypes, including glioma stem-like cells (GSCs) that have the ability to recreate all tumor phenotypes. GSCs are particularly resistant to treatment, upregulating various growth factor receptor signaling pathways as well as the DNA damage response pathway and switching metabolic phenotypes when necessary. A whole raft of monoclonal antibodies and small molecule tyrosine kinase inhibitors targeting growth factor receptor pathways have shown promise in preclinical studies but have failed in clinical trials. More recently, inhibitors of the DNA damage response pathway, resulting in cell cycle checkpoint blockade, have demonstrated strong radiosensitizing effects, particularly in GSCs with mutated p53. This perspective covers the different mechanisms that underlie GSC treatment resistance and recaps our current understanding of the radiosensitizing effects of ATM inhibitors on GBMs and GSCs in particular.
BMC Cancer
Background Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells. Methods Two GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by γH2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations. Results We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-...
Cell Cycle Changes after Glioblastoma Stem Cell Irradiation: The Major Role of RAD51
International Journal of Molecular Sciences
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.
Cancer Stem Cells and Radioresistance: DNA Repair and Beyond
Cancers
The current preclinical and clinical findings demonstrate that, in addition to the conventional clinical and pathological indicators that have a prognostic value in radiation oncology, the number of cancer stem cells (CSCs) and their inherent radioresistance are important parameters for local control after radiotherapy. In this review, we discuss the molecular mechanisms of CSC radioresistance attributable to DNA repair mechanisms and the development of CSC-targeted therapies for tumor radiosensitization. We also discuss the current challenges in preclinical and translational CSC research including the high inter- and intratumoral heterogeneity, plasticity of CSCs, and microenvironment-stimulated tumor cell reprogramming.