Processing of O<sup>6</sup>-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles

Quirós, Steve; Roos, Wynand P.; Kaina, Bernd

doi:10.4161/cc.9.1.10363

Cited by 135 publications

(148 citation statements)

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“…Although MGMT is a key protector for both O 6 -methylating and O 6 -chloroethylating agents, the mode of action of these anticancer drugs is quite different. Using the same synchronized system, we showed previously that O 6 MeG gives rise to DSB formation and G 2 arrest in the second cell cycle following treatment, 27 which is explained on the basis of the finding study, which made use of a reporter assay, showed that homology-directed cross-link repair is dependent on DNA replication. 33 Further analyzing the fate of cells that were arrested in G 2 , we became aware that a certain fraction finally underwent apoptosis, which occurred with a significant delay of > 16 h following the onset of G 2 arrest (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…25 Previously, it has been shown that methylating agents, such as MNNG and temozolomide, induce cell death triggered by the DNA damage O 6 -methylguanine following the passage through two DNA replication cycles, and that DSB formation and their repair by homologous recombination (HR) and, to a much lesser extent, by nonhomologous end-joining (NHEJ) in the second post-treatment cell cycle is involved. [26][27][28] In this study, we analyzed the cell cycle dependence of ACNU, Cell Cycle volume 11 issue 14 junction resolution (XRCC3). 24 The BRCA2, Rad51 and XRCC3 mutants proved to be extremely sensitive to ACNU; toxic effects were already observed with the lowest tested concentrations of 1 and 2.5 μM ACNU (Fig.…”

Section: Experiments With Synchronized Cellsmentioning

confidence: 99%

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Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

et al. 2012

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C hloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto-and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of γH2AX foci , which co-stained with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that γH2AX foci were not induced in G 1 but increased significantly in S phase and remained at a high level in G 2 , where a fraction of cells became arrested and underwent, with a delay of > 12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O 6 -chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O 6 -chloroethylguanine-triggered

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Section: Discussionmentioning

confidence: 99%

Section: Experiments With Synchronized Cellsmentioning

confidence: 99%

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

et al. 2012

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“…This was surprising because N-alkylation lesions also contribute to cytotoxicity (Roos et al, 2009b) and, theoretically, NBN could be involved in this pathway. O 6 MeG has previously been identified as the critical killing lesion for S N 1 methylating agents that causes the formation of DSBs and evokes the DNA damage response in the second cell cycle after treatment (Roos et al, 2009a;Quiros et al, 2010). Because NBN represents an essential element in the trimeric MRN complex together with MRE11 and Rad50, which recognizes DSBs and helps ATM/ATR to become activated, it is reasonable to conclude that the increased sensitivity of NBN mutant cells to S N 1 agents is due to impaired recognition and/or repair of DSBs induced by O 6 MeG adducts.…”

Section: Discussionmentioning

confidence: 99%

“…Although accounting for less than 8% of total base modifications, methylation of guanine at the O 6 -position is most critical because it is mutagenic and cytotoxic. The toxicity of O 6 -methylguanine (O 6 MeG) is dependent on DNA mismatch repair (MMR) (Karran and Bignami, 1994) and DSB formation (Ochs and Kaina, 2000;Roos et al, 2009a), which occurs in the second S-phase after its induction (Quiros et al, 2010). The model of toxicity states that in the first DNA replication cycle O 6 MeG mispairs with thymine.…”

Section: Introductionmentioning

confidence: 99%

Nijmegen Breakage Syndrome Protein (NBN) Causes Resistance to Methylating Anticancer Drugs Such as Temozolomide

Eich

Roos²,

Dianov³

et al. 2010

Mol Pharmacol

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Methylating agents are first-line therapeutics for gliomas and malignant melanomas. They attack DNA at various sites, and both O 6 -methylguanine and N-methylated base adducts contribute to the killing response. The mechanism of cellular defense against these agents primarily involves O 6 -methylguanine-DNA methyltransferase (MGMT) and base excision repair (BER). Here, we determined whether a key protein involved in DNA double-strand break (DSB) recognition and signaling, nibrin (NBN alias NBS-1), plays a role in the cellular defense against methylating agents. Comparing NBN mutated fibroblasts and lymphoblastoid cells from patients suffering from Nijmegen breakage syndrome, we show that NBN mutants are clearly more sensitive to N-methyl-NЈ-nitro-N-nitrosoguanidine and temozolomide than the corresponding wild-type cells. Hypersensitivity was due to the induction of both apoptosis and necrosis. The mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 were expressed at a similar level in the cell lines and BER was not affected by NBN mutation. Because MGMT expression abrogated the hypersensitivity of NBN mutated cells, we conclude that O 6 -methylguanine-derived lesions are responsible for triggering the response. Down-regulation of NBN in melanoma cells by small interfering RNA rendered them more sensitive to temozolomide, suggesting that NBN is a novel modulator of temozolomide sensitivity. Because NBN is part of the MRN complex, which recognizes DSBs, the data strongly indicate that MRN is critically involved in DSB processing after O 6 -methylguanine induction. The data provide first evidence that NBN is involved in the cellular defense against O 6 -methylguanine-inducing agents such as temozolomide and identify NBN as a critical target of methylating anticancer drug resistance.

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“…DNA polymerases attempt to repair this gap by filling it with newly synthesized DNA, reintroducing the mismatched thymidine opposite the O 6 -methylguanine adduct and subsequently requires MMR again. The continuous failing at removing the erroneous pairing will result in a permanent single strand gap that will cause a DSB during the DNA replication in the following S-phase, which induces apoptosis of the cell [55]. As can be concluded from this event, the MMR pathway can be a crucial factor in resistance to methylating agents besides MGMT activity.…”

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Methylation of DNA repair genes and the efficacy of DNA targeted anticancer treatment

Julsing¹,

Peters²

2014

Oncol Discov

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Cancer is considered both a genetic and epigenetic disease. The best studied epigenetic mechanism in carcinogenesis is DNA methylation, a mechanism which inactivates tumor suppressor genes by methylating their promoters. A subclass of tumor suppressor genes that are often methylated in the process of carcinogenesis are the DNA repair genes. Accumulation of DNA damage is associated with cancer and thus inactivation of DNA repair genes promotes cancer formation. Methylation of DNA repair genes seems to be unique compared to the methylation of other tumor suppressor genes in that it not only promotes tumorigenesis, but at the same time influences the sensitivity of tumors to chemotherapies that are based on agents that damage DNA to kill cancer cells. This literature study summarizes the current knowledge regarding epigenetic inactivation of DNA repair genes and the subsequent progress that has been made coping with the acquired chemotherapy resistances.

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Processing of O⁶-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles

Cited by 135 publications

References 33 publications

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

Nijmegen Breakage Syndrome Protein (NBN) Causes Resistance to Methylating Anticancer Drugs Such as Temozolomide

Methylation of DNA repair genes and the efficacy of DNA targeted anticancer treatment

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Processing of O6-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles

Cited by 135 publications

References 33 publications

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

Nijmegen Breakage Syndrome Protein (NBN) Causes Resistance to Methylating Anticancer Drugs Such as Temozolomide

Methylation of DNA repair genes and the efficacy of DNA targeted anticancer treatment

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Processing of O⁶-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles