Brca2/Xrcc2 dependent HR, but not NHEJ, is required for protection against O⁶-methylguanine triggered apoptosis, DSBs and chromosomal aberrations by a process leading to SCEs

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• 作者：Wyn ; P. Roos ; Teodora Nikolova ; Steve Quiros ; Steffen C. Naumann ; Olivia Kiedron ; Mał ; gorzata Z. Zdzienicka ; Bernd Kaina
• 关键词：Apoptosis ; DNA double-strand breaks ; Aberrations ; SCEs ; Brca2 ; XRCC2 ; NHEJ ; MGMT ; Homologous recombination ; Temozolomide
• 刊名：DNA Repair
• 出版年：2009
• 出版时间：1 January 2009
• 年：2009
• 卷：8
• 期：1
• 页码：72-86
• 全文大小：1655 K

文摘

O⁶-methylguanine (O⁶MeG) is a highly critical DNA adduct induced by methylating carcinogens and anticancer drugs such as temozolomide, streptozotocine, procarbazine and dacarbazine. Induction of cell death by O⁶MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O⁶MeG. Here, we compared the response of cells mutated in HR and NHEJ proteins to temozolomide and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (DNA-PK_cs mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of O⁶-methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific hypersensitivity was due to O⁶MeG lesions. MNNG provoked H2AX phosphorylation 24–48 h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The γH2AX foci formed in response to O⁶MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O⁶MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O⁶MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving cancer therapy with methylating agents.