Elsevier

DNA Repair

Volume 8, Issue 1, 1 January 2009, Pages 72-86
DNA Repair

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

https://doi.org/10.1016/j.dnarep.2008.09.003Get rights and content

Abstract

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-PKcs 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.

Introduction

Many environmental carcinogens and endogenously formed reactive metabolites damage DNA by methylation [1]. Methylating properties are also shared by anticancer drugs that are used in tumor therapy [2]. These agents, including temozolomide (TMZ), procarbazine, dacarbazine and streptozotocine, are highly cytotoxic; they are also mutagenic, recombinogenic and clastogenic. Although methylating agents induce 13 DNA adducts [3], only one of them, O6-methylguanine (O6MeG) that is induced in small amounts (maximally 8% of total methylation products), causes with high efficiency all these detrimental effects (for review see [4], [5]). O6MeG is repaired by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) [6], [7], [8], [9], and cells unable to repair O6MeG are highly sensitive to O6-methylating agents compared to MGMT competent cells [7], [10], [11]. Work with natural cell variants, MGMT transfectants and pharmacological MGMT inhibitors (for review see [5], [6], [8]) provided compelling evidence for a critical role of O6MeG in the genotoxic and cytotoxic response of cells upon methylation. If O6MeG is repaired, base N-methylations will also become important at high dose levels in inducing genotoxic and killing effects [12]. Cytotoxicity induced by methylating agents in cells deficient in MGMT is due to apoptosis, which indicates that O6MeG is a critical pro-apoptotic DNA adduct [13], [14], [15], [16].

O6MeG is also a highly potent recombinogenic lesion that induces sister chromatid exchanges (SCEs) at high frequency [7]. The conversion probability for SCEs was determined to be in the range of 30–42 O6MeG lesions per SCE [12], [17]. Contrary to SCEs, the conversion probability for chromosomal aberrations is much lower. Thus, about 10,000 O6MeG adducts are required for one chromatid break to be generated [12]. Both SCEs and aberrations induced by O6MeG are formed in the second cell cycle after treatment, i.e. DNA has to replicate twice in the presence of O6MeG in order for SCEs and aberrations to be formed [18]. Genotoxicity induced by O6MeG is absolutely dependent on cell proliferation, which is also true for apoptosis [19].

Cells defective in MutSα (MSH2 and MSH6) or MutLα (MLH1 and PMS2) are highly resistant to O6-methylating agents; they tolerate O6MeG at the expense of point mutations [20] and cancer [21], [22]. Thus, genotoxicity and cell death provoked by O6MeG lesions requires MutSα and MutLα dependent DNA mismatch repair (MMR) [21], [23], [24]. MutSα recognizes damaged DNA and binds to O6MeG/T mispairs [25]. A current model for O6MeG-induced genotoxicity taking into account these findings suggests that O6MeG mispairs with thymine in the first round of DNA replication. In the following cell cycle, the O6MeG/T mispair is subject to a futile MutSα-dependent MMR cycle, forming a secondary DNA lesion, presumably a gapped DNA structure [26], that blocks DNA replication. This gives rise to SCEs and, at lower frequency, to DNA double-strand breaks (DSBs), which results in chromosomal aberrations in the post-treatment cell cycle [13]. This model competes with another hypothesis stating that the activated MMR complex operating on O6MeG/T lesions activates apoptotic signaling on its own, which could occur via the activation of DNA damage kinase ATR/ATRIP [27].

In previous studies, it was shown that cells lacking ATM were more sensitive to MNNG, compared to the wild-type, displaying significantly higher levels of apoptosis and chromosomal aberrations [28]. ATM exhibits different roles in signaling and DNA repair, one of them is stimulation of HR [29]. Furthermore, TMZ requires MMR for inducing apoptosis [30] and activates ATR and ATM as well as the downstream Chk1, Chk2 and p53 response. With low dose TMZ in MGMT deficient cells ATR was specifically activated suggesting O6MeG lesions are able to trigger the ATR and Chk1 signaling pathway [31], [32].

DSBs have been shown to occur in wild-type cells, but not in MGMT proficient and MMR compromised cells in response to O6MeG [16]. DSBs were also shown to occur in other cell systems such as MGMT depleted human lymphocytes [19] and MGMT deficient glioma cells [33] upon treatment with methylating agents. In all these cell models they precede apoptosis, which is in line with the hypothesis that they are formed as a result of MMR activity at O6MeG/T lesions and blocked DNA replication forks. Whether they are the final trigger of cell death is likely, but still needs experimental verification.

Given a role for DSBs in apoptosis induction and genotoxicity by O6MeG lesions, it would be expected that cells defective in DSB repair are more sensitive to O6-methylating agents and that this hypersensitivity is abrogated when O6MeG lesions are repaired by MGMT. DSBs are repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) [34]. The contribution of these pathways to DSB repair depends on the DNA damaging agent, cell type and cell cycle phase. Thus, in yeast HR is the major pathway of DSB repair whereas in mammalian cells NHEJ is believed to play the main role [35]. DSBs induced in G1 phase of the cell cycle, e.g. by ionizing radiation, are repaired by NHEJ which is thought to be error-prone giving rise to chromosomal aberrations. In contrast, HR repairs DSBs induced in lateS/G2 by an error-free recombination process [36]. For chemical agents that do not directly induce DSBs, it is less clear how DSBs are processed. It is believed that for replication blocking DNA lesions, such as inter- or intra-strand cross-links, abasic sites and bulky lesions, DSBs arise at blocked replication forks due to DNA cleavage by the heterodimeric Mus81-Eme1/Mms4 structure-specific endonuclease and that the formation of these DSBs initiates recombination [37].

In this work, we wished to provide an answer to the question of whether HR or NHEJ or both are involved in the protection against O6MeG lesions. Further, we wished to know whether defects in these pathways result in an altered genotoxic response. Comparing MGMT proficient and MGMT deficient DSB repair mutants, we show that HR defective cells, but not NHEJ mutants, are hypersensitive to O6MeG adducts. We further show that DSBs (as measured by γH2AX) are formed in MGMT deficient cells upon treatment with methylating agents [N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and TMZ] and that HR, but not NHEJ, is the major pathway for repairing them. We also provide evidence that this results in a high frequency of SCEs without cell killing effects, indicating that HR leading to SCEs causes significant protection against cell death. Therefore, HR can be considered a mechanism for tolerating O6MeG adducts. A minor fraction of DSBs appears to be repaired by NHEJ and, as a result, the level of protection mediated by NHEJ is much lower to that mediated by HR. If HR cannot occur, DSBs accumulate, which results in chromatid-type aberrations and finally cell death. The data obtained clearly suggest that HR is an important component in the cellular defense against the cytotoxicity and genotoxicity of simple methylating agents such as MNNG and TMZ. The data suggest that modulation of HR mediated DSB repair has a significant impact on the effectiveness of temozolomide and other methylating anticancer drugs, which has important therapeutic implications.

Section snippets

Cell lines and culture conditions

The well characterized Chinese hamster cell lines V79B and XR-V15B-defective in Ku80 [38], CHO-K1 and xrs1-, xrs5-defective in Ku80 [39], CHO-9 and XR-C1-defective in DNA-PKcs [40], V79 and irs1-defective in Xrcc2 [41], V79 and V-C8 defective in Brca2, and V-C8+Brca2 (V-C8 containing a BAC with the murine Brca2 gene) [42] were used in this study. All cell lines were cultured in RPMI-1640 containing 10% fetal bovine serum and kept at 37 °C in a humidified atmosphere of 93% air and 7% CO2.

Transfection of cells with MGMT

MGMT

HR, but not NHEJ, strongly protects against the cytotoxic effects of temozolomide

To determine whether HR or NHEJ protects cells against the cytotoxic effects of temozolomide, we compared a panel of DSB repair-defective cells as to their ability to form colonies following treatment with increasing doses of TMZ (Fig. 1). All nine cell lines (wild-type and the corresponding mutants) included in the study were MGMT deficient. The xrs1 and xrs5 cell lines derived from CHO-K1 are defective in Ku80 while the XR-C1 cell line derived from CHO-9 is defective in DNA-PKcs; these cells

Discussion

This study was aimed at elucidating the role of HR and NHEJ in the repair of DSBs induced during the processing of O6MeG lesions. We utilized mutants defective in well-characterized pathways of DSB repair, namely Brca2 and Xrcc2 that are involved in HR [51], [52], and Ku80 and DNA-PKcs that are involved in NHEJ [53], [54]. All these mutant cell lines lack MGMT activity. Therefore, we stably transfected all of them with MGMT in order to substantiate that the effects observed are due to O6MeG.

Conflict of interest

We, the authors of the paper “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”, would like to state that there is no conflict of interest.

Acknowledgements

We gratefully acknowledge Tina Brachetti, Andrea Piee-Staffa and Georg Nagel for technical assistance. Work was supported by Deutsche Forschungsgemeinschaft (DFG grants to BK, KA724,13-3,4) and Stiftung Rheinland-Pfalz.

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