Early Chk1 Phosphorylation Is Driven by Temozolomide-Induced, DNA Double Strand Break- and Mismatch Repair-Independent DNA Damage

Faculdade de Medicina, Universidade de São Paulo, Brazil
PLoS ONE (Impact Factor: 3.23). 05/2013; 8(5):e62351. DOI: 10.1371/journal.pone.0062351
Source: PubMed


Temozolomide (TMZ) is a DNA methylating agent used to treat brain cancer. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair during DNA replication and trigger cycles of futile mismatch repair (MMR). Futile MMR in turn leads to the formation of DNA single and double strand breaks, Chk1 and Chk2 phosphorylation/activation, cell cycle arrest, and ultimately cell death. Although both pChk1 and pChk2 are considered to be biomarkers of TMZ-induced DNA damage, cell-cycle arrest, and TMZ induced cytotoxicity, we found that levels of pChk1 (ser345), its downstream target pCdc25C (ser216), and the activity of its upstream activator ATR, were elevated within 3 hours of TMZ exposure, long before the onset of TMZ-induced DNA double strand breaks, Chk2 phosphorylation/activation, and cell cycle arrest. Furthermore, TMZ-induced early phosphorylation of Chk1 was noted in glioma cells regardless of whether they were MGMT-proficient or MGMT-deficient, and regardless of their MMR status. Early Chk1 phosphorylation was not associated with TMZ-induced reactive oxygen species, but was temporally associated with TMZ-induced alkalai-labile DNA damage produced by the non-O6-methylguanine DNA adducts and which, like Chk1 phosphorylation, was transient in MGMT-proficient cells but persistent in MGMT-deficient cells. These results re-define the TMZ-induced DNA damage response, and show that Chk1 phosphorylation is driven by TMZ-induced mismatch repair-independent DNA damage independently of DNA double strand breaks, Chk2 activation, and cell cycle arrest, and as such is a suboptimal biomarker of TMZ-induced drug action.

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    • "Expression of MGMT protein, a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ [3], [5], [6], [7]. On the other hand, deficiency of DNA MMR proteins also contributes to TMZ resistance in GBM [7], [8]. "
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    ABSTRACT: Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O6-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.
    PLoS ONE 08/2014; 9(8):e104538. DOI:10.1371/journal.pone.0104538 · 3.23 Impact Factor
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    ABSTRACT: Alkylating agents are a common frontline therapy for the treatment of several cancers including pediatric glioblastoma (pGBM), a devastating lethal tumor in children. Unfortunately, many tumors are resistant to this therapy and traditional mechanisms of resistance including MGMT promoter methylation fail to fully explain treatment resistance in pGBM. We sought to identify ways of sensitizing tumor cells toalkylating agents while leaving normal glia and neural stem cells unharmed; increasing therapeutic response while minimizing toxicity. An siRNA screen targeting over 240 DNA damage response genes identified novel sensitizers to alkylating agents, namely temozolomide. In particular, the base excision repair (BER) pathway, including DNA-3-methyladenine glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM) were identified in our screen. ATM, MPG and BER were required for allowing tumour cells to repair damaged DNA and survive exposure to temozolomide. Patients with high expression of MPG had poorer overall survival compared to MPG low expressing patients and that MPG was one the most commonly amplified genes of the BER pathway in pGBM. Combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo using several orthotopic mouse models of pGBM. Further, we identified, several small molecule inhibitors of BER that effectively sensitized pGBM cells to clinically relevant doses of TMZ and prolonged survival in vivo. Inhibition of ATM and BER cooperate to sensitize tumour cells to alkylating agents, impairing tumour growth in vitro and in vivo with no toxicity to normal cells providing an ideal therapeutic window.
    Canadian Journal of Neurological Sciences/Journal Canadien des Sciences Neurologiques, Halifax, Nova Scotia, Canada; 06/2014
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    ABSTRACT: Isocitrate dehydrogenase 1 (IDH1) mutations occur in most lower-grade glioma, and not only drive gliomagenesis but are associated with longer patient survival and improved response to temozolomide (TMZ). To investigate the possible causative relationship between these events, we introduced wild-type (WT) or mutant IDH1 into immortalized, untransformed human astrocytes, then monitored transformation status and TMZ response. TMZ-sensitive parental cells exhibited DNA damage (gamma-H2AX foci) and a prolonged G2 cell cycle arrest beginning 3 days after TMZ (100μM, 3hr) exposure and persisting for greater than 4 days. The same cells transformed by expression of mutant IDH1 exhibited a comparable degree of DNA damage and cell cycle arrest, but both events resolved significantly faster in association with increased, rather than decreased, clonogenic survival. The increases in DNA damage processing, cell cycle progression, and clonogenicity were unique to cells transformed by mutant IDH1, and were not noted in cells transformed by WT IDH1 or an oncogenic form (V12H) of Ras. Similarly these effects were not noted following introduction of mutant IDH1 into Ras-transformed cells or established GBM cells. They were, however, associated with increased homologous recombination and could be reversed by the genetic or pharmacologic suppression of the homologous recombination DNA repair protein RAD51. These results show that mutant IDH1 drives a unique set of transformative events that indirectly enhance homologous recombination and facilitate repair of TMZ-induced DNA damage and TMZ resistance. The results also suggest that inhibitors of HR may be a viable means to enhance TMZ response in IDH1 mutant glioma.
    Neuro-Oncology 07/2014; 74(17). DOI:10.1158/0008-5472.CAN-14-0924 · 5.56 Impact Factor
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