Mismatch repair, G2/M cell cycle arrest and lethality after DNA damage

Gabriele Aquilina3, Marco Crescenzi3,1 and Margherita Bignami2

Laboratory of Comparative Toxicology and Ecotoxicology, Section of Chemical Carcinogenesis, Istituto Superiore di Sanita', Viale Regina Elena 299, 00161 Roma, Italy and
1 Laboratory of Molecular Oncogenesis, Regina Elena Cancer Institute, 00161 Roma, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The role of the mismatch repair pathway in DNA replication is well defined but its involvement in processing DNA damage induced by chemical or physical agents is less clear. DNA repair and cell cycle control are tightly linked and it has been suggested that mismatch repair is necessary to activate the G2/M checkpoint in the presence of certain types of DNA damage. We investigated the proposed role for mismatch repair (MMR) in activation of the G2/M checkpoint following exposure to DNA-damaging agents. We compared the response of MMR-proficient HeLa and Raji cells with isogenic variants defective in either the hMutL{alpha} or hMutS{alpha} complex. Different agents were used: the cross-linker N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), {gamma}-radiation and the monofunctional methylating agent N-methyl-N-nitrosourea (MNU). MMR-defective cells are relatively sensitive to CCNU, while no differences in survival between repair-proficient and -deficient cells were observed after exposure to {gamma}-radiation. Analysis of cell cycle distribution indicates that G2 arrest is induced at least as efficiently in MMR-defective cells after exposure to either CCNU or ionizing radiation. As expected, MNU does not induce G2 accumulation in MMR-defective cells, which are known to be highly tolerant to killing by methylating agents, indicating that MNU-induced cell cycle alterations are strictly dependent on the cytotoxic processing of methylation damage by MMR. Conversely, activation of the G2/M checkpoint after DNA damage induced by CCNU and {gamma}-radiation does not depend on functional MMR. In addition, the absence of a simple correlation between the extent of G2 arrest and cell killing by these agents suggests that G2 arrest reflects the processing by MMR of both lethal and non-lethal DNA damage.

Abbreviations: CCNU, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; FCS, fetal calf serum; ICL, interstrand DNA cross-links; MMR, mismatch repair; MMR, MMR-defective; MMR+, MMR-proficient; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; O6-meGua, O6-methylguanine; 6-TG, 6-thioguanine.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A primary function of the mismatch repair (MMR) pathway is to correct persistent DNA replication errors, to avoid the accumulation of deleterious mutations. Cultured human and rodent cell lines or mice with defects in MMR exhibit increased spontaneous mutation rates (16). Heterozygous germline mutations in MMR genes are associated with familial predisposition to colorectal carcinoma in hereditary non-polyposis colon cancer. Inactivation of growth control genes relevant to the neoplastic transformation process is thus accelerated by the mutator phenotype which arises following loss of the second MMR allele.

The genetic evidence for the role of MMR in ensuring genome stability is complemented by biochemical approaches. DNA containing mismatches is recognized and bound by either hMutS{alpha}, a heterodimer of hMSH2 and hMSH6 (7,8) or hMutSß, formed by hMSH2 and hMSH3 (9,10). Following the interaction with a second complex, hMutL{alpha}, a heterodimer of hMLH1 and hPMS2, the excision repair process is initiated (for a review see ref. 11).

While the role of MMR in the correction of replication errors is well defined, its involvement in the processing of DNA damage induced by chemical or physical agents and/or in checkpoint activation is less clear. This is an important point since the loss of MMR would then modulate mutagenic events which arise both during endogenous metabolic processes and as a consequence of the exposure to environmental carcinogens. MMR interacts with O6-methylguanine (O6-meGua) and 6-thioguanine (6-TG) and MMR-defective (MMR) cells are resistant to methylating carcinogens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) or the base analog 6-TG (1218). hMutS{alpha} can recognize and bind to synthetic oligonucleotides containing O6-meGua or 6-TG (or the methylated derivative of 6-TG, S6-methylthioguanine, which is the probable lethal lesion) (1922). Biological evidence indicates that MMR does not remove O6-meGua or 6-TG from DNA, but instead transforms these modified bases into lethal lesions (23). Resistance to cisplatin is also associated with loss of MMR (2428) and the ability of hMutS{alpha} to recognize one of the cisplatin DNA lesions, the intrastrand 1,2-diguanyl DNA cross-link (20,29) is in accordance with these observations.

The concept of cell cycle checkpoints is now well established as a means of arresting the cell cycle in response to various threats, of which DNA damage is a prominent example. Since some confusion exists as to the exact definition of a checkpoint, in this paper we accept the definition of checkpoints as surveillance mechanisms that block cell cycle transitions (30). It has been suggested that MMR is part of the G2 checkpoint following some types of DNA damage (31,32). For example, G2 arrest induced by treatment with 6-TG or MNNG was not observed in hMLH1-defective HCT116 colon carcinoma cells unless active MMR was restored by chromosome transfer. In addition, G2 arrest (33) and apoptosis (34) were efficiently induced by O6-meGua-generating drugs only in MMR-proficient (MMR+) TK6 cells and not in their MMR derivative MT1.

While these observations might be consistent with an involvement of hMutS{alpha} and hMutL{alpha} in cell cycle checkpoint functions, the relative resistance of MMR cells to methylating agents and 6-TG complicates the analysis of cell cycle perturbations. We therefore used two alternative DNA damaging agents to determine whether MMR is required for checkpoint functions. We examined the cell cycle progression of cells treated with N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), which introduces DNA monoadducts as well as interstrand DNA cross-links (ICL), and {gamma}-radiation, which introduces several types of DNA damage including DNA strand breaks and modified DNA bases. MMR cells are generally hypersensitive to killing by CCNU, possibly because of an involvement of this pathway in the repair of ICLs (35). In contrast, MMR exerts little influence on killing by ionizing radiation and there are conflicting reports of minor protective or sensitizing effects conferred by loss of MMR (6,3638).

We compared the ability of CCNU and ionizing radiation to perturb the cell cycle of isogenic MMR+ and MMR clones derived from HeLa and Raji cells. The MMR defects were in either the hMutL{alpha} or the hMutS{alpha} complex (17,39). We did not observe a simple relationship between the extent of the G2 arrest and cell death but both MMR+ and MMR cells arrested in G2 following both types of damage. Our observations indicate that the G2/M checkpoint operates normally in MMR+ and MMR cells exposed to CCNU or to ionizing radiation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell cultures
HeLa cells and the MMR derivative clones were cultured in Dulbecco's modified minimal essential medium (Gibco BRL, Paisley, Scotland) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 µg/ml) (complete medium). Raji cells were cultured in RPMI 1640 supplemented with 15% FCS. Cultures were incubated at 37°C in 5% CO2 and 95% relative humidity.

Chemicals and {gamma}-ray irradiation
CCNU (Rhone-Poulenc, Neuilly sur Seine, France) was prepared fresh in 100% ethanol. MNU (Sigma, St Louis, MO) was dissolved in dimethylsulphoxide. CCNU and MNU solutions were diluted immediately before use in complete medium and in phosphate-buffered saline containing 20 mM HEPES (pH 7.4), respectively. Cells were irradiated with {gamma}-rays from a 60Co source at a dose rate of 6 Gy/min.

Determination of cell survival
To measure cell survival, HeLa and Clone 7 cells were seeded in complete medium (100 cells/6 cm dish) and treated 18 h later with CCNU for 1 h at 37°C or with MNU for 30 min at 37°C. Cells were then washed with phosphate-buffered saline, fed complete medium and 1–2 weeks later the surviving colonies were stained with Giemsa and counted. In the case of {gamma}-radiation, HeLa cultures were seeded in 25 cm2 flasks (2.5x105 cells), exposed to {gamma}-rays, trypsinized and diluted to clonal density (100 cells/6 cm dish). Raji cell lines were treated in suspension at 1x105/ml either with CCNU or {gamma}-radiation, diluted in multiwell plates (300 cells in 96 wells, cloning efficiency 30%) in complete medium and wells displaying cell growth were counted microscopically after 2 weeks.

Cell cycle analysis
Cell cycle analysis was performed by flow cytometric measurements of the DNA content of the cells. For cell cycle analysis, cells were harvested at appropriate time points from treatment ({gamma}-irradiation or CCNU) and were stained at 4°C for at least 3 h in phosphate-buffered saline containing 0.1% Triton X-100, 5 µg/ml propidium iodide (Sigma) and 20 µg/ml RNase A (Boehringer-Mannheim, Indianapolis, IN). Cell cycle analyses were performed on an Epics XL analyzer (Coulter Corp., Miami, FL).

Western blotting
Cell extracts were prepared from 2x107 cells resuspended in a buffer containing 50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM DTT and 0.2% Triton X-100. Cell extracts (20–60 µg) were denatured and separated on 7.5% SDS–polyacrylamide gels together with a prestained low molecular weight marker (Bio-Rad, Hercules, CA). Proteins were transferred to nitrocellulose membranes (Bio-Rad) using a Trans-Blot cell apparatus (Bio-Rad) at 30 mA overnight at 4°C. The remaining protein binding sites of the nitrocellulose paper were blocked by immersion in TBS–Tween (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 30 min in the presence of 0.1% gelatin and an additional 30 min in the presence of 3–7% powdered skimmed milk. The blocked filter was incubated with primary antibody (1 µg/ml for the monoclonal antibodies anti-PMS2; Pharmingen, San Diego, CA) for 1h at room temperature on a rocker platform. After washing with TBS–Tween, the appropriate secondary antibody was added for an an additional 1 h. The blots were developed using the ECL detection reagents (Amersham Italia, Milano, Italy).


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Cell survival and cell cycle arrest after CCNU exposure of hMutS {alpha}- and hMutL {alpha}-defective Raji cells
Raji F12 and Raji 10 are MMR variants of Burkitt's lymphoma Raji cells. The Raji F12 cell line is defective in hMutS{alpha} (40), most likely in the hMSH6 gene product (17), while Raji 10 cells are defective in the hMutL{alpha} complex (17).

Raji F12 and Raji 10 cells are more sensitive to CCNU than their MMR+ parental cells and comparisons at the D37 value indicate an ~4- and 8-fold hypersensitivity, respectively (Figure 1AGo). We exposed Raji F12, Raji 10 and the parental cells to a range of CCNU concentrations (6–150 µM) and analysed the cell cycle distributions at daily intervals for 4 days. Figure 1BGo compares the cytofluorimetric profiles of Raji and their MMR derivatives treated at approximately equally cytotoxic CCNU concentrations (50 µM for Raji, 12.5 µM for Raji F12 and 6 µM for Raji 10). CCNU-treated parental cells accumulated in the S and G2 phases, most evidently at 2 days after treatment, and between days 3 and 4 the cell cycle profile of the surviving cells returned close to normal. The two MMR clones showed a more dramatic response to CCNU treatment, with >80% of the cells accumulated in the S or G2 phases at day 2 (Figure 1BGo). Neither population recovered a normal cycle distribution even 4 days after treatment and >30% of the population remained in G2 phase. Exposure to higher CCNU concentrations showed a dose-dependent increase in the fraction of cells arrested in G2 with no variations in the time at which growth arrest occurred (data not shown).




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Fig. 1. (A) Cytotoxicity of CCNU in Raji and methylation-tolerant, MMR derivatives. Raji ({circ}), Raji F12 (•) and Raji 10 ({blacksquare}) cells were treated for 1 h with increasing concentrations of CCNU. Three independent determinations of cell survival were performed and one representative experiment is shown. (B) Cytofluorimetric analysis of the cell cycle of MMR+ and MMR Raji cells. Raji cells and the MMR derivatives Raji F12 and Raji 10 were exposed to CCNU (50, 12.5 and 6 µM, respectively) and sampled for analysis at daily intervals. The percentages of cells in G1, S and G2 are shown.

 
The above results show that functional MMR is not required for G2 arrest, since neither the hMutS{alpha}- nor the hMutL{alpha}-defective Raji cells displayed an impaired G2 arrest following CCNU treatment. On the contrary, at every CCNU concentration examined, their progress through the cell cycle was markedly more retarded than that of the parental population.

Cell survival and cell cycle arrest after CCNU exposure of hPMS2-deficient HeLa cells
We sought to generalize the above results by using a different cell line that bears another MMR-inactivating mutation. Thus we compared the effects of CCNU on cell survival and cell cycle progression in MMR+ HeLa cells and in the methylation tolerant, hMutL{alpha}-deficient Clone 7 (39). Western blotting analysis showed normal levels of the hMLH1, hMSH6 and hMSH2 proteins but no detectable expression of hPMS2, suggesting that the defect in Clone 7 resides in the hPMS2 gene (Figure 5BGo and data not shown). Clone 7 is 2-fold more sensitive to CCNU than the parental HeLa cells (Figure 2AGo; 35).



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Fig. 5. (A) Cytotoxicity of MNU in MMR+ and MMR HeLa cells. Exponentially growing cultures of HeLa ({blacklozenge}), Clone 7 ({square}) and Clone 7 no. 7 ({triangleup}) were treated with MNU and survival was measured as cloning efficiency. (B) Cell extracts were separated by SDS–PAGE and the levels of hPMS2 determined by immunoblotting.

 



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Fig. 2. (A) Cytotoxicity of CCNU in HeLa and a methylation tolerant, MMR derivative. HeLa ({blacklozenge}) and Clone 7 ({square}) cells were treated for 1 h with increasing concentrations of CCNU. Three independent determinations of cell survival were performed and one representative experiment is shown. (B) Cytofluorimetric analysis of the cell cycle of MMR+ and MMR HeLa cells. HeLa and MMR derivative Clone 7 cells were exposed to CCNU (25 and 12.5 µM, respectively) and analyzed at daily intervals. The percentages of cells in G1, S and G2 are shown.

 
Both MMR+ and MMR cells were exposed for 1 h to 12.5, 25 or 50 µM CCNU. A dose-related accumulation of cells in the S and G2 phases of the cycle was observed in both cell lines 1, 2, 3 and 4 days after treatment (data not shown). A comparison between cell cycle distributions of HeLa cells and Clone 7 at equitoxic CCNU concentrations (25 and 12.5 µM, respectively) is shown in Figure 2BGo. One day after treatment with 25 µM CCNU, there was a dramatic accumulation of HeLa cells in late S phase. One day later, a large fraction of the population (76%) was arrested in G2 phase. An even larger proportion of cells exposed to 50 µM CCNU accumulated in S phase, with a dose-dependent anticipation towards the early S phase (data not shown). Three to four days after treatment, the cell cycle arrest was only partially resolved in the parental cell population exposed to 25 µM CCNU.

Compared with the parental HeLa cells, Clone 7 exhibited a similar late S phase accumulation followed by G2 arrest 2 days after treatment with an equitoxic CCNU dose. In the following 2 days the G2 arrest was resolved and the surviving cells eventually adopted an apparently normal cell cycle distribution.

Thus, in two different cell types, loss of MMR does not prevent arrest and accumulation of cells in G2 after treatment with CCNU.

Effects of ionizing radiation on survival and cell cycle progression of MMR cell lines
Survival of CCNU is affected by the functional state of MMR. We sought to confirm that the MMR status does not affect the integrity of the surveillance mechanism using an agent whose lethality is minimally affected by MMR. Thus, progression through the cell cycle of Raji and HeLa cells exposed to ionizing radiation was examined. Cells were irradiated with 2, 4 or 6 Gy. No differences were detected in cell survival between the MMR clones and their parental Raji cells (Figure 3AGo). The cytofluorimetric profiles of Raji cells exposed to 2 Gy are shown in Figure 3BGo. The irradiated cells accumulated in G2 phase with no evidence of a G1/S arrest. The latter observation is compatible with the the G1/S checkpoint being abrogated due to a mutated p53 protein in Raji cells (41). The maximal accumulation in G2 occurred at 48 h; 1 day later the profiles more closely resembled those of unirradiated cells. At 4 and 6 Gy, the cells were still arrested in G2 or undergoing apoptosis 3 days after irradiation (data not shown).




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Fig. 3. (A) Cytotoxicity of ionizing radiation in Raji and methylation-tolerant MMR derivatives. Exponentially growing cultures of Raji ({circ}), Raji F12 (•) and Raji 10 ({blacksquare}) cells were exposed to {gamma}-radiation and survival was measured as cloning efficiency in 96-well plates as described in Materials and methods. (B) Cytofluorimetric analysis of the cell cycle of {gamma}-irradiated MMR+ and MMR Raji cells. Cellular suspensions were exposed to 2 Gy of {gamma}-radiation and sampled for analysis at daily intervals. The percentages of cells in G2 are shown.

 
Irradiation also induced an efficient G2 arrest in MMR Raji F12 and Raji 10 clones (Figure 3BGo). Interestingly, at 48 and 72 h post-irradiation the percentages of cells accumulated in G2 were significantly increased in comparison with the parental cells. This extended G2 arrest was more pronounced in the hMutS{alpha}-defective Raji F12 clone than in the hMutL{alpha}-defective Raji 10 cells.

The MMR defect in Clone 7 did not detectably affect its survival of ionizing radiation in comparison with the parental HeLa cells (Figure 4AGo). Cell cycle progression analyses were performed after irradiation with 2, 4 and 6 Gy. For comparative purposes, since HeLa and Clone 7 cells are somewhat more resistant to {gamma}-rays than Raji cells, we present the profiles of the HeLa cells irradiated with 4 and 6 Gy (Figure 4BGo). In both HeLa and Clone 7, an efficient G2 arrest was observed at both doses. At all time points and at both doses examined, the proportion of MMR cells accumulated in the G2 phase was slightly greater.




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Fig. 4. (A) Cytotoxicity of ionizing radiation in HeLa and methylation tolerant, MMR derivatives. Exponentially growing cultures of HeLa ({blacklozenge}) and Clone 7 ({square}) cells were exposed to {gamma}-radiation and survival was measured as cloning efficiency. (B) Cytofluorimetric analysis of the cell cycle of {gamma}-irradiated MMR+ and MMR HeLa cells. Exponentially growing cultures were exposed to 4 and 6 Gy of {gamma}-radiation and subjected to analysis at daily intervals. The percentages of cells in G2 are shown.

 
In conclusion, these data demonstrate that active MMR is not required for efficient G2 arrest following DNA damage. In addition, we found that, under the conditions we tested, the survival of {gamma}-irradiated Raji and HeLa cells is not detectably affected by loss of MMR.

Analysis of cell cycle progression after MNU treatment in MMR+ and MMR cell lines
Tolerance of the cytotoxic effect of methylation damage is a general feature of MMR-defective cells (42). Raji F12 and Raji 10 cell population growth is completely arrested by a MNU concentration at least 30-fold higher than that required for Raji cells (17). Clone 7, by comparison of D37 values, is ~80-fold more resistant to MNU than the parental HeLa cell line (16).

Cell cycle progression of Raji, Raji F12 and Raji 10 cells was analysed after treatment with 2.5 µg/ml MNU, a concentration that completely inhibits cell growth in Raji cells, but does not affect proliferation of the MMR derivatives. No apparent alterations in cell cycle distribution are observed in the parental Raji cells after 24 h, while cells undergo a prominent accumulation in S phase 2 days after treatment and are almost all blocked in S/G2 the following day (Table IGo). As expected, the cell cycle progresses regularly at this MNU dosage in the two methylation-tolerant Raji derivatives.


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Table I. Cytofluorimetric analysis of the cell cycle of MMR+ and MMR human cell lines treated with MNU
 
Cell cycle alterations induced by methylation in HeLa cells were studied using a MNU dosage of 9 µg/ml. A slight increase in the S phase fraction is observed in HeLa cells 1 day after treatment. During the following 2 days the cells accumulate progressively in G2. At the same dosage, no cell cycle alteration was observed in the MMR derivative Clone 7 (Table IGo).

A single human chromosome 7, which contains the hPMS2 gene, was introduced by microcell fusion into Clone 7. The resulting hybrid Clone 7 no. 7 displayed an almost complete reversion of sensitivity to cell killing by MNU (Figure 5AGo). Restoration of hPMS2 protein expression in Clone 7 no. 7 was confirmed by western blot (Figure 5BGo). The timing and extent of cell cycle alterations induced by MNU in Clone 7 no. 7 are similar to those observed in the parental HeLa cells (Table IGo).

Thus, cell cycle alterations induced by MNU are strictly dependent on a functional MMR system in both the cellular systems analysed. These alterations, abolished in MMR-defective lines, are restored in a HeLa-derived clone by the introduction of an extra chromosome 7, which complements the MMR defect. The earliest effect of methylation damage in MMR+ cells is accumulation in S phase, more prominent in Raji than in HeLa cells, followed by G2 arrest. Little or no effect is observed before day 2, consistent with the previous observation that replication inhibition induced by MNU is delayed until the second S phase after treatment (43).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have used isogenic cell lines to investigate the interactions between a cell cycle checkpoint and MMR. Previous studies have suggested that activation of the G2/M checkpoint following treatment with MNNG, 6-TG or cisplatin is dependent on hMLH1 or hMSH6 (31,32,44). There are at least two general ways in which MMR might be involved in checkpoint functions. The mismatch repair proteins, following DNA damage recognition, might interact directly with checkpoint proteins to activate cell cycle arrest. Alternatively, mismatch repair is required to process certain types of DNA damage into a form which is capable of triggering the checkpoint response.

According to the current model of methylation toxicity (42) O6-meGua is not able to form a good complementary match either with cytosine or with thymine and the resulting imperfect base pairing is recognized and processed by MMR proteins. Futile attempts to repair the DNA distortion create breaks in the newly synthesized strand, i.e. the strand opposite to O6-meGua. These breaks are converted into double-strand breaks at the following S phase, the second one after treatment (35,45,46). Consistent with this model and with several previous reports (34,43,47), we observed little or no cell cycle perturbation before 48 h after MNU treatment. In apparent contrast to this model, a clear delay of the first S/G2 after treatment was described in some cell lines, both MMR+ and MMR, using a very high dosage of methylating agent (32). We observed a similar cell cycle alteration treating MMR cells with MNU doses corresponding to ~1% survival (data not shown). The fact that these early perturbations of the cell cycle at a high level of methylation damage are also displayed by MMR cells, which lack cytotoxic processing of O6-meGua, suggests that this effect might be due to other methylation products.

To minimize the complications arising from the introduction of lethal DNA damage by active MMR, we investigated the effects of CCNU and ionizing radiation. MMR does process CCNU damage in a poorly defined way, but the participation of MMR slightly improves cell survival, possibly through an involvement in the removal of potentially lethal ICLs. Therefore, in contrast to its role in the processing of methylation damage, MMR does not transform CCNU-induced DNA modifications into lethal lesions. Equitoxic concentrations of CCNU provoked efficient G2 arrest in both MMR+ and MMR Raji and HeLa cells. Comparison of cell cycle distributions following exposure to several CCNU concentrations confirmed that the ability to undergo G2 arrest in response to CCNU damage is independent of active MMR in these two different cell types.

A similar conclusion can be drawn from the analysis of irradiated cells. We did not observe any effects of MMR deficiency on {gamma}-ray sensitivity in Raji or HeLa cells. Contradictory effects of MMR deficiency on killing by ionizing radiation have been reported in human and mouse cells, but these effects are anyhow minor (3638). One report suggested that expression of the hMLH1 protein is required for competence of the G2/M cell cycle checkpoint (38). None of the defective cell lines show impairment of the G2/M checkpoint. Although Raji 10 cells are defective in hMutL{alpha} function, we do not know whether their defect resides in hMLH1. Thus, though it seems unlikely, we cannot formally exclude the possibility that the observations of Davis and co-workers are due to a specific role of MLH1 in the G2/M checkpoint. Alternatively, the apparent discrepancy with our conclusions might be due to methodological reasons. All systems employed to investigate the cell cycle consequences of loss of MMR have their shortcomings. In the absence of appropriate parental populations, chromosome-mediated transfer of the relevant MMR gene is often used to correct defective human tumour cells. However, transfer of a whole chromosome (38; the present work) might produce unbalanced expression of genes important in regulation of the cell cycle, for example chromosome 3-based topoisomerase IIß (48), which could have unpredictable effects on the phenomena under investigation. On the other hand, embryo fibroblasts derived from knockout mice (38) might not be directly comparable with human cells, due to subtle differences in the cell cycle controls (49,50) and DNA repair (51,52). Finally, selection of methylation-tolerant variants by exposure to a mutagen might induce, together with inactivation of MMR, other changes in the genome. Our results show that two agents, CCNU and {gamma}-radiation, which introduce widely different types of DNA damage, are able to trigger the G2/M checkpoint in a MMR-independent fashion. We reach this conclusion from experimental observations in two different isogenic cell systems (HeLa and Raji) and in three independent cell lines with defects in two MMR complexes. Our data support a model in which neither interaction with nor activation by MMR complexes are required for the G2/M checkpoint to exert its functions.

Although MMR is not required to trigger the G2/M checkpoint, differences in cell cycle progression are apparent between MMR+ and MMR cells exposed to CCNU. Equimolar CCNU concentrations induce a more prominent G2 arrest in MMR cells than in their parental counterparts (Figures 1B and 2BGoGo and data not shown). This result is consistent with the hypothesis that MMR is involved in the repair of DNA lesions induced by CCNU. Chloroethylnitrosoureas such as CCNU induce a variety of DNA lesions, which are the substrates of several repair pathways, including O6-meGua-DNA methyltransferase, 3-methyladenine DNA glycosylase and nucleotide excision repair (53). The best characterized cytotoxic lesions induced by CCNU are ICLs and monoadducts at the 7 position of guanine (53,54). The delay in progression through S phase which is evident after CCNU treatment is reasonably explained by the presence of ICLs, which are probably severe impediments to DNA replication (55). The enhanced G2 arrest in MMR cells does not allow, however, discrimination between the persistence of unrepaired ICLs or monoadducts.

One presumptive function of the G2 checkpoint is to delay progression into mitosis of damaged cells, thus providing more time to repair lethal DNA damage and enhancing survival. At similar levels of toxicity, however, MMR cells generally show enhanced G2 arrest following CCNU treatment and {gamma}-irradiation. This suggests that G2 arrest might reflect the cumulative effects of lethal and non-lethal DNA lesions.

Ionizing radiation introduces numerous modifications into DNA, such as single- and double-strand breaks, abasic sites and several modified DNA bases. A role for MMR in transcription-coupled repair of ionizing radiation-induced damage has been proposed following the observation that hMSH2-defective cells were unable to remove oxidative damage, including thymine glycols, from the transcribed strand of an active gene (36). There is also some recent evidence suggesting that MMR might process relatively non-lethal DNA damage. For example, 8-oxoguanine is a well-tolerated although mutagenic DNA lesion (56). The accumulation of 8-oxoguanine in the DNA of Msh2-defective mouse embryonic stem cells exposed to low level radiation is suggestive of a role of MMR in modulating a chronic oxidative stress (57). Our observations would then be consistent with the recognition and processing by MMR proteins of this DNA modification. In conclusion, we favour a model in which the role of MMR is to modulate the amount of lethal as well as non-lethal damage induced by CCNU and ionizing radiation without a direct control on the cell cycle surveillance mechanism.


    Acknowledgments
 
We would like to thank particularly Dr P.Karran for helpful discussions and Dr E.Dogliotti for critically reviewing the manuscript. This work was partially supported by the Associazione Italiana Ricerca sul Cancro.


    Notes
 
2 To whom correspondence should be addressed Email: bignami{at}iss.it Back

3 The first two authors contributed equally to this work. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 14, 1999; revised August 4, 1999; accepted August 16, 1999.