Hypersensitivity to camptothecin in MSH2 deficient cells is correlated with a role for MSH2 protein in recombinational repair

Pietro Pichierri1,3, Annapaola Franchitto1,3, Rita Piergentili1, Claudia Colussi2 and Fabrizio Palitti1,4

1 Laboratorio di Citogenetica Molecolare e Mutagenesi, DABAC, Università degli Studi della Tuscia, Via S.Camillo de Lellis, 01100 Viterbo, Italy,
2 Laboratorio di Tossicologia Comparata ed Ecotossicologia, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy and
3 CNRS, UPR2169 `Instabilité Génétique et Cancer', Institut de Recherches sur le Cancer `André Lwoff', 7 Rue Guy-Moquet, BP8, 94801 Villejuif, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA mismatch repair (MMR) corrects DNA polymerase insertion errors that have escaped proofreading in order to avoid the accumulation of deleterious mutations. While the role of MMR in the correction of replication errors is well established, its involvement in the processing of DNA damage induced by chemical and physical agents is less clear. A role for some of the MMR proteins, such as MSH2, in the repair of double strand break (DSBs) through recombination has also been envisaged. Why MMR- deficient cells are sensitive to agents causing replication fork stalling and thus DSBs remains unclear. To verify a possible role of MSH2 in homologous recombinational repair, we have treated cells from knockout mice for the MSH2 gene and mouse colorectal carcinoma cells also defective for MSH2 with different doses of camptothecin, an agent known to interfere with DNA replication. In the absence of MSH2, we found a reduced survival rate accompanied by higher levels of chromosomal damage and SCE induction. Furthermore, MSH2–/– cells displayed an elevated spontaneous RAD51 focus-forming activity and a higher induction of RAD51 foci following camptothecin treatment. Thus, the absence of MSH2 could result in both spontaneous DNA damage and uncontrolled recombination events leading to the observed higher yield of chromosomal damage and the higher induction of RAD51 foci following CPT treatment. Therefore, our results suggest an involvement of MSH2 in the early events leading to correct RAD51 relocalization after the formation of DSBs specifically produced at the blocked replication fork.

Abbreviations: BrdUrd, 5-bromo-2'-deoxyuridine; ; CPT, camptothecin; ; DSBs, double strand breaks; ; HR, homologous recombination; ; MMC, mitomycin-C; ; MMR, DNA mismatch repair; ; SCEs, sister chromatid exchanges.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The primary role of post-replicative DNA mismatch repair (MMR) is to eliminate from the newly synthesized strand DNA polymerase errors, avoiding accumulation of deleterious mutations. MMR defects are associated with human cancer, in particular it is well established that MMR deficiency is linked to cancers of the colon, endometrium and ovary (for a review, see ref. 1).

While the role of MMR in the correction of replication errors is well known, its involvement in the processing of DNA damage, induced by chemical and physical agents, is less clear. MMR contributes to cell killing by DNA methylating agents (reviewed in ref. 2) and several investigations report a decreased sensitivity to the killing effects of DNA alkylating agents, accompanied by cellular hypermutability in MMR-deficient cells (3–5). On the contrary, contradictory data are available on the sensitivity to killing effects of DNA cross-linking agents. In fact, MMR deficiency is associated with cis-platinum resistance (6) but, more recently, sensitivity towards mitomycin-C (MMC) has been reported (7). The cytotoxicity of both cis-platinum and MMC is thought to be a consequence of the formation of DNA adducts which are poorly repaired and which block DNA replication and/or transcription (8–10).

Camptothecin (CPT) is a drug that shows anti-tumour activity in various animal tumour models (11,12). Camptothecin and its derivatives interfere with the DNA breakage–reunion reaction of topoisomerase I by stabilizing the intermediate enzyme-associated DNA single-strand breaks (12) and consequently inhibiting the rejoining activity of topoisomerase. It also interferes with DNA replication and transcription stabilizing the topoisomerase I–DNA cleavable complexes, thus leading to DSBs and cell death (13). Double strand breaks produced by CPT at stalled replication forks are repaired through recombination (14,15). Accordingly, CPT is also a strong inducer of sister chromatid exchanges (SCEs) (16), an S-dependent phenomenon associated with genetic recombination and dependent on the RAD51 homologous recombination protein (17). Also the interstrand cross-links generated by MMC are thought to be repaired mainly, even if not only, by homologous recombination (HR) (18,19). It has been reported that some of the MMR proteins could participate in double strand break (DSB) repair through recombination (20–24), as shown by the hyper-recombinational phenotype of the MMR-deficient cells (25,26). More recently a direct involvement of the yeast MSH2 repair protein in DSB repair has been reported (24,27).

In this study, we aimed to investigate whether MSH2 deficiency results in a higher sensitivity to CPT, an agent causing DSBs selectively at the stalled replication fork, on the basis of a possible role for the mammalian MSH2 protein in the recombinational repair.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell cultures
In this study we have used spontaneous transformed embryonic fibroblasts, from MSH2 gene knockout mice (MEF MSH2–/–) and their corresponding wild-type (MSH2+/+), as well as mouse colorectal carcinoma cells: colo26 (MSH2-proficient), colo5 (MSH2-deficient) and colo5#2 (MSH2-restored), the latter was recovered by the transfer of a single copy of human chromosome 2, which contains the wild-type MSH2 allele (28). We were not able to obtain stable primary cells from knockout mice, as after a few passages they underwent spontaneous crisis and then immortalization. However, we used embryonic cells within the fourth passage after immortalization in order to minimize any possible compromising of pathways influencing genome stability and cell survival (e.g. the p53 protein was not mutated in these cell lines; Pichierri,P., unpublished data). We were able to perform only a preliminary trial on primary embryonic fibroblasts observing consistent results with those obtained using immortalized cell lines.

All cell lines were cultured in Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Colo5#2 cell line was maintained in the presence of 200 µg/ml G418 (Gibco-BRL, Eggenstein, Germany) to select for cells containing the human chromosome, but all experiments were performed in the absence of this selective antibiotic. All the cell lines were incubated at 37°C in 5% CO2 (90% humidity nominal).

MSH2+/+ and MSH2–/– MEFs showed mean doubling times of 17 ± 2 h and 13 ± 1.5 h, respectively. No difference in the mean doubling time was observed among the colo cell lines.

Chemicals
Camptothecin (CPT) was obtained from Sigma (L'Isle d'Abeau, France). It was dissolved in dimethyl sulfoxide (DMSO) and a 2.5 mM stock solution was prepared and stored frozen at –20°C in the dark.

For treatment of cultures, CPT was added at the concentrations indicated such that the final DMSO concentration did not exceed 1%.

Determination of cell survival
To measure cell survival, MEFs and colo cell lines were seeded in complete medium (100 cells/6 cm dish) and pulse-treated with CPT 18 h later. CPT treatment lasted 2 h, after that cells were washed twice and 1 week later the surviving colonies were stained with Giemsa and counted. The results are presented as a fraction of the relevant control (surviving fraction).

G2-phase and S-phase treatments
For G2-phase treatment, exponentially growing cells were exposed to 10 or 20 µM CPT, together with 30 µg/ml 5-bromo-2'-deoxyuridine (BrdUrd) and 0.2 µg/ml colcemid, and collected after 2 h. BrdUrd was added to discriminate between cells treated in G2-phase (unlabelled) or S-phase (labelled), in order to score chromosomal aberrations only in the unlabelled population (29).

Alternatively, cells were exposed for 1 h to different doses of CPT (0.6 and 2.4 µM), washed twice and recovered in drug-free medium for 17 or 22 h, collecting metaphases with colcemid in the last 2 h. Immediately before CPT treatment, cells were pulse-labelled with 30 µg/ml BrdUrd for 30 min to monitor cell cycle progression. Based on our earlier preliminary experiments, CPT-treated cells undergo cell cycle arrest and re-enter into mitosis only at later recovery times. Sampling times were chosen on the basis of our experience in order to maximize collection of S-phase cells. Since S-phase cells are extremely sensitive to CPT, lower doses with respect to those used in G2-phase treatment were used.

Chromosomal damage was only scored in labelled cells (i.e. cells treated in S-phase), but only results from sampling times in which a similar percentage of labelled metaphases in the different cell lines was found (i.e. homogeneous populations) are presented. Comparison of the chromosomal damage in different sampling times between the wild-type and MSH2–/– MEFs was needed, since they presented a different cell cycle kinetic.

Sister chromatid exchanges analysis
Exponentially growing cells were exposed to 0.6 or 2.4 µM CPT for 1 h, washed twice and recovered in drug-free medium containing 3 µg/ml BrdUrd. Metaphase cells were collected 21 or 24 h after the addition of 0.2 µg/ml colcemid in the last 2 h. Slides prepared according to standard techniques were stained by the FPG technique (30) to visualize sister chromatid exchanges (SCEs). Fifty metaphase cells for each experimental point were scored. Sister chromatid exchanges were only scored in cells with 42 ± 2 chromosomes.

Cell cycle analysis
To evaluate the perturbation induced by CPT treatment on cell cycle progression, cells were treated for 2 h and then harvested at different time points. BrdUrd (45 µM) was added to the cultures together with CPT and remained until harvesting. BrdUrd labelling allows the analysis of cellular progression from S-phase into the other cell-cycle stages. Cells were processed for bivariate flow cytometry as follows: after overnight fixation with 70% ethanol at 4°C, cells were incubated in 4 N HCl and 0.5% Triton X-100 for 30 min at room temperature (RT) and then with FITC-conjugated anti-BrdUrd antibody (Dako) for 1 h at RT. Finally, cells were incubated in 1 µg/ml propidium iodide in PBS. Between each incubation, cells were washed with PBS containing 2% BSA and 0.02% Tween-20. After gating out cellular debris and aggregates, the percentage of cells in each cell stage was evaluated by analysing both DNA content and BrdUrd incorporation.

Chromosome preparations and analysis
Chromosome preparations were made according to standard procedures. Slides were prepared, for each experimental point, from colcemid arrested cells and used for the immunodetection of BrdUrd incorporation to discriminate between labelled (S-phase cells) and unlabelled (G2-phase cells) metaphases.

The frequency of chromosomal aberrations was scored in both labelled and unlabelled metaphases in cells with 42 ± 2 chromosomes. At least 100 unlabelled (G2-phase treatment) or labelled cells (S-phase treatment) were analysed in detail for chromatid-type aberrations according to the criteria already described (31). Using a fluorescence microscope to score chromosomal damage, we were unable to see gaps. However, chromatid breaks were easily identified; we defined a chromatid break as a discontinuity wider than the chromatid width.

Analysis of the induction of RAD51 foci
Cells cultured on cover slips were exposed to 0.6 or 2.4 µM CPT for 1 h and sampled after 2, 4 and 6 h of recovery. Cells were fixed in 4% paraformaldehyde-buffered solution and immediately processed for immunochemical detection of RAD51 as previously described (32), using goat polyclonal anti-RAD51 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and Alexa546-conjugated mouse anti-rabbit secondary antibody (Molecular Probes, Eugene, OR). For each experimental point at least 200 nuclei were examined and RAD51 foci scored by eye at a magnification of x100 using a Zeiss epifluorescence microscope. Only nuclei showing greater than five bright foci were considered as positive.

Statistical analysis
All the results are presented as mean ± SE and derived from at least three conclusive experiments based on several repeats showing a high reproducibility.

Differences in the yield of aberrations and SCE induction were evaluated by chi-squared test comparing the observed results between wild-type and MSH2–/– cells. The value of abnormal cells was evaluated using the Fisher's test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of CPT treatment on cell survival
We compared the effects of different doses of CPT on cell survival in cell lines deficient in the MSH2 gene (Figure 1Go). Both MEF MSH2–/– and colo5 cell lines were more sensitive to CPT than their MSH2-proficient parental cell lines. This sensitivity was observed at all the doses used and increased in a dose-dependent manner. Sensitivity to CPT appeared higher in the colo5 cell line. Interestingly, the revertant colo5#2 showed cell survival closely similar to the parental colo26 cells. These data indicate that MSH2 loss confers hypersensitivity to CPT.



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Fig. 1. Cytotoxicity of camptothecin in wild-type (MEFs and colo26), MSH2-deficient (MEFs and colo5) or MSH2-deficient cells recovered with human MSH2 gene (colo5#2). Three independent determinations of cell survival were performed and the mean ± SE is presented.

 
Analysis of CPT-induced chromosomal damage in S- or G2-phase of the cell cycle
To verify whether the increased sensitivity of MSH2-defective cells to CPT is accompanied by higher levels of chromosomal damage, MEFs and colo cells were pulse-treated with CPT and analysed for the induction of chromosomal aberrations. It has been previously described that CPT induces chromosomal damage in the S-phase of the cell cycle but it also results in the formation of chromosomal aberrations when given in the G2-phase of the cell cycle, possibly due to interaction with transcription (33–36). Chromosomal damage was analysed in both BrdUrd-positive (labelled, S-phase cells) and -negative (unlabelled, G2-phase cells) metaphases (Tables I and IIGoGo, respectively). We found that CPT, in MSH2–/– cells, induced a higher yield of abnormal cells and chromosomal aberrations in S-phase cells at both doses used. Since MSH2–/– cells have shorter doubling times, chromosomal damage was compared in populations showing similar percentages of labelled mitotic cells (29). It is important to note that, even if the percentage of damaged cells was found to be elevated by only ~1.5-fold in MSH2-deficient cells, chromatid breaks doubled and chromatid exchanges were found ~4–6-fold higher with respect to MSH2+/+ cells. On the contrary, in the G2-phase of the cell cycle, CPT induced only slightly higher, but not statistically significant, chromosomal damage in MSH2- deficient cells (Table IIGo). It is worth noting that CPT treatments produced only chromatid breaks in both the MSH2-deficient and -proficient cells. The absence of chromatid exchanges after CPT exposure in the G2-phase of the cell cycle is consistent with other reports (37,38) and suggests that chromatid exchanges are mainly formed in S-phase cells after CPT treatment.


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Table I. Analysis of chromosomal damage induced by camptothecin in the S-phase of the cell cycle in MSH2 proficient and deficient cells
 

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Table II. Analysis of the chromosomal damage induced by CPT in the G2-phase of the cell cycle in MSH2-proficient and -deficient cells
 
Consistent with a sensitivity of S-phase cells to CPT, SCE induction by CPT was also significantly enhanced in MSH2-deficient cells with respect to wild-type and it was obtained at both dose levels used (Table IIIGo). Interestingly, we observed elevated levels of spontaneous SCEs in MSH2–/– cells (Table IIIGo).


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Table III. Sister chromatid exchange induction by camptothecin in MSH2-deficient and proficient cells
 
In conclusion, these data indicate that loss of MSH2 function is associated with increases in the level of chromatid exchanges induced by CPT treatment during S-phase.

Effects of CPT treatment on cellular progression
We then evaluated the possibility that CPT hypersensitivity of MSH2–/– cells was due to a different effect on the progression of cells through S-phase. After CPT treatment both cell lines showed an evident arrest in S-phase that appeared more prominent for early-S cells (Figure 2Go). A G2-phase arrest was also observed after 2 h in both cell lines. MSH2-proficient cells emerged from S-phase arrest after ~7 h of recovery, whereas MSH2-deficient cells consistently recovered from arrest within 5 h, with different cell cycle kinetics shown by the two cell lines (data not shown).



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Fig. 2. Analysis of cell cycle progression after CPT treatment. MSH2-deficient and -proficient MEFs were pulse-treated with 2.4 µM CPT, and 45 µM BrdUrd to label S-phase cells, and samples collected at the indicated recovery times (see Materials and methods). Vertical axis (logarithmic scale), BrdUrd incorporation; horizontal axis (linear scale), DNA content. The results shown are representative of three independent experiments, similar results were obtained after 10 µM CPT.

 
In conclusion, no difference in cellular progression was observed after CPT treatment in MSH2+/+ and MSH2–/– cells.

Induction of RAD51 foci
Given that hypersensitivity could be due to defects in the repair of replication-associated DSBs and that such DNA damage is repaired through the homologous recombinational pathway, we investigated whether RAD51 focus formation was altered in MSH2–/– cells following CPT treatment. A significantly higher spontaneous RAD51 focus-forming activity was observed in MSH2-deficient cells (Figure 3aGo). RAD51 foci were induced in both MSH2-proficient and -deficient cells after CPT exposure, but with a different kinetic. Induction of RAD51 foci increased with dose and in a time-dependent manner, reaching the highest value 6 h after treatment (Figure 3b and cGo). In MSH2–/– cells, RAD51 foci appearance was delayed and increased after 4 h of recovery. RAD51 foci induction was similar between MSH2-proficient and -deficient cells at 4 h of recovery, but it appeared higher in MSH2-deficient cells at 6h of recovery. Furthermore, at 6h of recovery MSH2-deficient cells not only showed a more elevated percentage of RAD51 positive nuclei, but also a significantly higher yield of foci per nucleus (Figure 3eGo).



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Fig. 3. Kinetics of RAD51 foci formation in wild-type and MSH2-deficient cells. Exponentially growing cells were either (a) untreated or pulse-treated with (b) 0.6 or (c) 2.4 µM CPT and fixed at the indicated recovery time. Cells were stained for RAD51 and scored for the presence of foci, as described in Materials and methods. The percentage of spontaneous RAD51 focus-forming activity was subtracted. The data are presented as mean ± SE of three independent experiments. (d) A representative pattern of RAD51 foci formation following 2.4 µM CPT treatment in both (A) MSH2-proficient and (B) MSH2-deficient cells; MSH2-deficient cells show a higher number of RAD51 foci/nucleus. (e) A table showing the number of RAD51 foci per nucleus after 1 h CPT treatment after different recovery times. **Statistically significant compared with the MSH2-proficient cell line (P < 0.01, chi-squared test).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A lot of evidence suggests that the collision of DNA replication forks with drug-stabilized topo I–DNA complexes induce DSBs at replication sites (39,40). From this point of view, CPT gives rise to DSBs quite exclusively in S-phase at the stalled replication fork. CPT-induced DSBs appear to be repaired by homologous recombination, as also demonstrated by the strong induction of SCEs that appear largely dependent upon the presence of an active RAD51-mediated recombinational repair pathway (17). It has been reported that recombinational repair requires the concerted action of several enzymes, including some of the MMR proteins (reviewed in ref. 41). Studies from the yeast model system suggest that MSH2 and MSH3 play an important role in recombinational repair of DSBs. In particular, the MSH2 protein participates in two specialized forms of recombinational repair: gene conversion and SSA (21,22,42). More recently, it has been suggested that, in yeast, MSH2/MSH3 interact with early intermediates at the DSB (24). In addition, it has been envisaged a role for the MMR proteins in the control of recombination between homeologous (divergent) sequences to prevent genetic exchange between divergent chromosome regions (43–45).

We found that MSH2-deficient cells are more sensitive to CPT treatments, as shown by the lower cell survival. This effect appears to be the result of the MSH2 deficiency since transfection with the human chromosome 2, which bears the MSH2 gene, returns CPT-sensitivity to wild-type levels. Sensitivity to CPT is also observed at the chromosomal level. MSH2-deficient cells show a very much higher sensitivity to CPT-induced chromosomal damage, particularly when treated in S-phase. Several authors (16,37,46,47) have reported the effect of CPT at the chromosomal level. CPT does not induce chromosomal damage in G1-phase, whereas it induces chromatid-type aberrations during the S- and G2-phase of the cell cycle. However, the difference observed in the induction of chromosomal damage in the G2-phase of the cell cycle between MSH2-proficient and -deficient cell lines appears very much lower than that observed in S-phase, and is not statistically significant. Since hypersensitivity to CPT has been correlated either to defects in checkpoint response and/or DNA damage signalling (38,48–50), we sought to determine whether MSH2-deficient cells properly activate checkpoint response after CPT exposure. We found that MSH2-deficient cells correctly arrest and resume S-phase progression following CPT treatment, excluding the fact that the sensitivity of MSH2–/– cells could be accounted for by a defect in the checkpoint machinery, thus suggesting that it most likely derived from a deficiency in the repair process. CPT-induced DNA damage causes re-localization of the RAD51 recombination protein at the sites of DSBs (15). Our data show a different behaviour in the induction of RAD51 foci in the absence of MSH2. Appearance of RAD51 foci is delayed in MSH2–/– cells by ~2 h, and after 6 h dramatically overtakes that of wild-type. Not only the percentage of RAD51 positive nuclei, but also the number of foci per nucleus, is elevated in MSH2-deficient cells. Such a late response in RAD51 re-localization may be consistent with a possible early action of MSH2 during the HR of DSBs (24). In the absence of MSH2, it is possible that RAD51 could be more difficulty recruited at the sites of DNA damage, resulting in the later appearance of foci. However, once RAD51 foci are formed, MSH2 seems to be no longer crucial for the process of repair at the stalled replication fork. The larger induction of RAD51 foci observed in MSH2-deficient cells after CPT treatment is also consistent with the observed higher induction of chromatid exchanges and SCEs.

Taken together, these findings could be explained by the proposed role of MMR proteins in controlling the recombination between divergent DNA sequences (hoemologous recombination) (44,45), or with a possible elevated spontaneous yield of DNA damage. In fact, in MSH2–/– cells the absence of an active MMR could give rise to a persistence of unrepaired modified bases, which could be a site of replication fork stall. Replication fork stall and collapse trigger HR to reinitiate DNA synthesis (reviewed in ref. 51). Consistent with this latter hypothesis, we found higher levels of spontaneous induction of RAD51 foci and SCEs in MSH2–/– cells. On the other hand, the observed higher induction of chromatid exchanges could be accounted for by an elevated rate of recombination events between divergent sequences, consistent with a higher number of RAD51 foci per nucleus. The excess in recombination events after CPT treatment might actually result in the observed lower survival rate shown by MSH2-deficient cells, possibly through the activation of an apoptotic programme. In fact, it has been reported that uncontrolled recombination might cause cell death either in the yeast model system or in mammalian cells (15,52).

We propose that the higher sensitivity towards CPT observed in MSH2-deficient cells might be a consequence of the combined effect of elevated spontaneous DNA damage, which may result from the unrepaired base damage, and the absence of efficient control of homologous recombination. Furthermore, our data suggest a role for MSH2 in the early events leading to correct RAD51 relocalization after DSBs selectively produced at the blocked replication fork.


    Notes
 
4 To whom correspondence should be addressed Email: palitti{at}unitus.it Back

P.Pichierri and A.Franchitto contributed equally to this work


    Acknowledgments
 
We thank Dr M.Bignami for providing the MSH2+/+ and MSH2–/– cell lines. This work was partially supported by grants from MURST and from the EC.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 29, 2001; revised July 26, 2001; accepted July 28, 2001.