Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ (original) (raw)
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O6-alkylguanine DNA lesions trigger apoptosis
Carcinogenesis, 1998
It is unclear whether alkylating agents induce apoptosis because they damage DNA, or because they damage other cellular targets. Isogenic Chinese hamster ovary (CHO) cell lines varying in the repair of O 6 -alkylguanine (O 6 AlkG) were examined for their propensity to undergo alkylationinduced apoptosis. Robust O 6 AlkG repair virtually eliminated the apoptogenic effects of N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) and 1,3-bis-(2-chloroethyl)-1nitrosourea (BCNU, carmustine), as did the expression of BCL-2. O 6 AlkG repair had no effect on apoptosis induced by tumor necrosis factor α or by γ-irradiation. We conclude that alkylating agents induce apoptosis by virtue of their ability to modify DNA bases and, more specifically, that O 6 AlkG lesions can trigger such programmed cell death.
Cancer research, 1977
determine the molecular basis for the cytotoxic action that produces the antitumor effect. Unfortunately, there is pres ently no direct way to identify with certainty the critical molecular lesions. It is, however, possible to obtain comrel ative evidence between molecular effects and cytotoxic effects in various cell types, which could lead to a probable identification of the critical lesions. In the current work, we studied DNA single-strand breaks and/or alkali-labile sites in BCNU-treated normal human embryo fibroblasts (Wl-38) and in an SV4O-transformed derivative (VA-13) that exhibited increased sensitivity to BCNU. Nitrosoureas react with biological macromolecules by 2 mechanisms: alkylation, which affects both proteins and nucleic acids; and carbamoylation, which affects proteins but not nucleic acids (3, 22). Since certain nitrosoureas, e.g., chlorozotocin, specifically lack the carbamoylation effect and yet retain antitumor potency (10), it is probably the alkylating activity that is primarily responsible for the antitumor action. Our working hypothesisis that the crucial activity stems from alkylation of DNA and that this activity may be reflected by effects on DNA macromolecular struc ture. Simple alkylnitrosoureas, such as methyl-or ethylnitro sourea, alkylate DNA at multiple sites (16, 20). Alkylations at guanmne-N-7 and adenine-N-3lead to depurination (15), and the resulting apuninic sites are susceptible to phospho diester cleavage by specific repair enzymes, as well as to cleavage by alkali (21). Alkali lability can also result from alkylation of DNA phosphate groups (19). Thus alkylation by simple nitrosoureas produces DNA lesions that can lead to enzymatically produced single-strand breaks and to al kali-labile lesions that can be converted to single-strand breaks by alkali. The sites of DNA alkylation by chloroe thylnitrosoureas, such as BCNU and CCNU, however, have not been fully determined.The production of single-strand breaks or alkali-labile sites by BCNU and CCNU have been reported previously in abstracts (6, 11). MATERIALS AND METHODS Cell Culture. Normal human embryonic lung fibroblasts (Wl-38, passages 16 to 32) and SV4O-transformed deriva tives (VA-13)were serially subcultured once a week at 1.5 to 2.0 x 10. cells in 75-sqcm plastic flasks (Falcon Plastics, Oxnard, Calif). The cells were grown in Eagle's basal medium (Grand Island Biological Co., Grand Island, N. V.), supplemented with 10% fetal calf serum (Flow Laboratories, 3744
DNA Repair, 2010
a b s t r a c t DNA-methylating agents of the S N 2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines. These adducts are repaired by base excision repair (BER). Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e.g. apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs). Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS). We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PK CS mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS. On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment. Since DSBs are expected to be formed specifically in the S-phase, we assessed the formation and kinetics of repair of DSBs by ␥H2AX quantification in a cell cycle specific manner. In the cytotoxic dose range of MMS a significant amount of ␥H2AX foci was induced in S, but not G1-and G2-phase cells. A major fraction of ␥H2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs. DSB formation following MMS treatment was also demonstrated by the neutral comet assay. Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair, while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype. Moreover, DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment. Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs. Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following S N 2-alkylating agents. (B. Kaina).
Cancer Research, 1985
The 9L-2, 9L-7, and 9L-8 cell lines, derived from the 9L in vivo rat brain tumor, were treated with nitrosoureas that can alkylate and cross-link DNA and carbamoylate intracellular molecules to various extents. Compared to 9L cells, 9L-2 cells were very resistant to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, and to 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-deoxyglucopyranose. The sensitivity of 9L-7 and 9L-8 cell lines to these drugs was intermediate between 9L and 9L-2. Treatment of 9L, 9L-2, 9L-7, and 9L-8 cell lines with 1,3-bis(frans-4-hydroxycyclohexyl)-1-nitrosourea produced approximately the same level of cell kill. Compared to 9L cells, 9L-2 cells are 10-fold more resistant to the cytotoxic effects, 34-fold more resistant to the induction of sister chromatid exchanges, and have 40% fewer DNA interstrand cross-links caused by treatment with 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea. In contrast, treatment of 9L and 9L-2 cells with 1-ethylnitrosourea produced approximately the same level of cell kill and induction of sister chromatid exchanges. Our results suggest that the resistance of 9L-2, 9L-7, and 9L-8 cells is related to DNA crosslinking and not to alkylation or carbamoylation.
British Journal of Cancer, 1994
Inter-individual and cell-cell variability of repair of 06-alkylguanines (06-AlkGua) in nuclear DNA was studied at the single-cell level in peripheral lymphocytes from healthy donors and in leukaemic cells isolated from patients with chronic lymphatic leukaemia (CLL) or acute myeloid leukaemia (AML). Cells were pulse exposed to N-ethylor N-(n-)butyl-N-nitrosourea in vitro, and 06-AlkGua residues in DNA were quantified using an anti-(O6-AlkGua) monoclonal antibody and electronically intensified fluorescence. The kinetics of 06-AlkGua elimination revealed considerable inter-individual differences in 06-ethylguanine (O6_ EtGua) half-life (ti) values in DNA, ranging from 1.5 to 4.5 h (five AML patients), from 0.8 to 2.8 h (five CLL patients) and from 1.2 to 7.3 h (five healthy donors). The elimination from DNA of equimolar amounts of 06-butylguanine was generally 3-5 times slower in comparison with 06-EtGua. The t, values of individual samples varied in parallel for both DNA alkylation products. Upon preincubation with 06-benzylguanine, the activity of the DNA repair protein 06-alkylguanine-DNA alkyltransferase (AT) in both lymphocytes and leukaemic blasts was reduced to <1%. However, while the rate of 06-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT. Within individual samples, no major cell subpopulations were observed whose repair kinetics would differ significantly from the remaining cells.
Proceedings of the National Academy of Sciences, 1998
Alkylation of DNA at the O 6-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. The enzyme O 6-methylguanine-DNA methyltransferase repairs O 6-methylguanine as well as a minor methylated base, O 4-methylthymine, in DNA. Mouse lines deficient in the methyltransferase (MGMT) gene are hypersensitive to both the killing and to the tumorigenic effects of alkylating agents. We now show that these dual effects of an alkylating agent can be dissociated by introduction of an additional defect in mismatch repair. Mice with mutations in both alleles of the MGMT gene and one of the mismatch repair genes, MLH1, are as resistant to methylnitrosourea (MNU) as are wild-type mice, in terms of survival, but do have numerous tumors after receiving MNU. In contrast to MGMT ؊͞؊ MLH1 ؉͞؉ mice with decrease in size of the thymus and hypocellular bone marrow after MNU administration, no conspicuous change was found in MGMT ؊͞؊ MLH1 ؊͞؊ mice treated in the same manner. Thus, killing and tumorigenic effects of an alkylating agent can be dissociated by preventing mismatch repair pathways.
Molecular Pharmacology, 2009
The presence of DNA damage initiates signaling through the ataxia-telangiectasia mutated kinase (ATM) and the ATM-and the Rad3-related kinase (ATR), which phosphorylate, thus activating, the checkpoint kinases (Chk) 1 and 2, which leads to cell cycle arrest. The bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is cytotoxic primarily by inducing DNA monoadducts and ultimately, interstrand crosslinks, which block DNA replication. In this study, we investigated the activation of the ATR-Chk1 pathway in response to BCNU treatment and the dependency of this response on the DNA mismatch repair (MMR) capacity. Medulloblastoma cells were exposed to low and moderate doses of BCNU and the effects on this DNA damage signaling pathway examined. In response to BCNU, Chk1 was found to be phosphorylated at serine 345 and exhibited increased kinase activity. Caffeine and wortmannin, which are broad-spectrum inhibitors of ATM and ATR, reduced this phosphorylation. Cell cycle analysis further revealed an accumulation of cells in the S phase in response to BCNU, an effect that was attenuated by caffeine. Small interfering RNA knockdown of ATR also reduced Chk1 phosphorylation following exposure to BCNU. However, knockdown of ATM had no effect on the observed Chk1 phosphorylation, suggesting that ATR was primarily responsible for Chk1 activation. Analysis of Chk1 activation in cells deficient in MMR proteins MutLα or MutSα indicated that the DNA damage response induced by BCNU was independent of the MMR apparatus. This MMR-independent activation appears to be the result of DNA interstrand crosslink formation.
Balancing repair and tolerance of DNA damage caused by alkylating agents
Nature Reviews Cancer, 2012
Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for an organism's favorable response to alkylating agents. Furthermore, an individual's response to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity.
Proceedings of the National Academy of Sciences, 1980
Normal (IMR-90) and simian virus 40-transformed (VA-13) human embryo cells were treated with antitumor nitrosoureas, and the effects on cell viability and cell DNA were compared. All six nitrosoureas tested were more toxic to VA-13 cells than to IMR-90 cells as measured by decrease in cell proliferation or in colony formation. The nitrosoureas capable of generating alkylisocyanates produced a smaller difference between the cell types than did derivatives lacking this capacity. DNA damage was measured by alkaline elution in cells treated with four chloroethylnitrosoureas. Whereas VA-13 cells exhibited dose-dependent interstrand crosslinking, little or none was detected in IMR-90 cells. The IMR-90 cells, however, exhibited at least as much DNA-protein crosslinking as did VA-13 cells. The results can be interpreted in terms of a possible difference in DNA repair between the cell lines.