Combined mismatch and nucleotide excision repair defects in a human cell line: mismatch repair processes methylation but not UV- or ionizing radiation-induced DNA damage

M. O'Driscoll, S. Martinelli1, C. Ciotta1 and P. Karran2

Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK and
1 Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Interaction between long patch mismatch repair (MMR) and persistent DNA O6-methylguanine or 6-thioguanine (6-TG) is implicated in the cytotoxicity of methylating agents and 6-TG, respectively. Human cells with defective MMR tolerate DNA methylation damage and are cross-resistant to 6-TG. To determine whether MMR contributes to the lethal effects of persistent UV-induced DNA lesions, MMR deficiency was introduced into nucleotide excision repair (NER)-defective XP12RO cells. The doubly repair-defective cells, designated XP12ROB4, did not express detectable hMSH2 protein. They had the mutator phenotype, N-methyl-N-nitrosourea and 6-TG resistance typical of MMR-defective cells. Active MMR was not required for the cytotoxicity of UV light, and the hMSH2 defect did not detectably alter the survival of XP12ROB4. The level of spontaneous or UV-induced SCE was also similar in XP12RO and XP12ROB4, indicating that hMSH2 is not required for this recombination process. The combined deficiency in MMR and NER did not confer a significant degree of tolerance to ionizing radiation, and the survival of XP12RO and XP12ROB4 after {gamma}-radiation was similar. Although it recognizes and processes some persistent damaged or modified DNA base pairs, MMR is unlikely to serve as a general sensor of DNA damage.

Abbreviations: 6-meTG, 6-methylthioguanine; 6-TG, 6-thioguanine; 8-AzaG, 8-azaguanine; DTT, dithiothreitol; Mex, MGMT-deficient; MGMT, O6-methylguanine-DNA methyltransferase; MMR, mismatch repair; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; NER, nucleotide excision repair; O6-meGua, O6-methylguanine; SCE, sister chromatid exchange; TCR, transcription-coupled repair; XP, xeroderma pigmentosum.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The long-patch, nick-directed mismatch repair (MMR) pathway acts as an editor of DNA replication (reviewed in ref. 1) and potentiates the cytotoxicity of some DNA damaging drugs (reviewed in ref. 2). MMR involves the replacement of an extensive tract of the DNA strand which contains the mismatch. Biochemical studies indicate that the repair process is initiated by one of two heterodimers, hMutS{alpha} and hMutSß, which recognize and bind to mismatched DNA. hMutS{alpha} comprises hMSH2 and hMSH6. The components of the hMutSß complex are hMSH2 and hMSH3. A third heterodimer, hMutL{alpha}, which is composed of hMLH1 and hPMS2, participates in a later step and is also apparently essential for the correction process (1).

hMSH2, hMSH6, hMLH1 and hPMS2 are also implicated in the control of drug sensitivity. MMR potentiates the cytotoxicity of methylating agents such as N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The principal basis of the cytotoxicity of these agents and of their clinical counterparts, temozolomide and dacarbazine, is the ability to methylate DNA and, in particular, the O6 atom of guanine bases. The resulting O6-methylguanine (O6-meGua) can be demethylated by a specific DNA repair enzyme, O6-meGua-DNA methyltransferase (MGMT). MGMT expression is lost in cells of the MGMT-deficient (Mex) phenotype which, as a result, are hypersensitive to killing and to sister chromatid exchange (SCE) induction by methylating agents (reviewed in refs 3 and 4). This hypersensitivity is only apparent, however, in Mex cells which have a functional MMR pathway. MMR-defective Mex cells, derived either from repair defective tumors (57) or from cultured cell lines (811), are as resistant to methylating agents as their Mex+ counterparts. These methylation resistant, Mex cells are described as methylation tolerant because they are able to sustain unrepaired O6-meGua in their DNA. Selection of MNU or MNNG resistant clones of Mex cells has been used to generate MMR-deficient variants and cells with defects in hMSH6 (8,10) or the components of the hMutL{alpha} complex (911), have been reported among those selected for this form of methylation tolerance.

MMR also mediates the cytotoxicity and clastogenicity of the base analog 6-thioguanine (6-TG) (1215) which is toxic via its incorporation into DNA. Methylation-tolerant, MMR-defective cells are frequently cross-resistant to this agent. It has been proposed that, once incorporated into DNA, 6-TG is converted to the ultimate cytotoxic base 6-methylthioguanine (6-meTG) by a non-enzymatic reaction with the intracellular methyl group donor S-adenosylmethionine (16). The methylated base miscodes during DNA replication to generate 6-meTG:T base pairs. One model to account for the resistance to methylating agents and 6-TG invokes futile attempts at correction of O6-meGua:T and 6-meTG:T base pairs by MMR (reviewed in ref. 17). These attempts are incomplete and generate cytotoxic DNA lesions. The structural similarities between O6-meGua:T, 6-meTG:T and mispairs between normal DNA bases suggest a plausible basis for the interaction of the modified purines with the MMR system. In agreement with this, hMutS{alpha} efficiently recognizes O6-meGua:T, and 6-meTG:T base pairs (18,19).

MMR defects have also been found in cells resistant to unrelated DNA modifying agents such as cisplatin and doxorubicin (20,21). Although certain cisplatin–DNA adducts may be recognized by hMutS{alpha} (19,22), there are no apparent common structural features among DNA lesions such as O6-meGua, doxorubicin–DNA adducts and the 1,2- and 1,3-diguanyl DNA intrastrand crosslinks, which are the major cisplatin DNA adducts. An alternative explanation for the association of defective MMR with high level drug resistance has therefore been advanced. In this model (8,14,23), MMR proteins act as general sensors for DNA damage, initiate cell-cycle checkpoints and thereby modulate the lethality of numerous structurally unrelated DNA lesions. This paper describes a study which tests the validity of the general sensor model.

Methylation-tolerant or MMR-defective human tumor cells are not generally resistant to UV irradiation (24,25). All the MMR-defective lines studied to date, however, are proficient in nucleotide excision repair (NER), which promotes the removal of lethal photoproducts. The human cell line XP12RO, derived from a xeroderma pigmentosum (XP) patient, has a genetic defect in NER. XP12RO cells do not produce detectable XPA protein (26), are unable to excise UV-induced photoproducts to a measurable extent and are highly sensitive to killing and SCE induction by UV radiation (25,27). Since UV photoproducts are common DNA lesions, they are likely targets for a putative general DNA damage sensor. We introduced an hMSH2 defect into XP12RO cells. The sensitivity of the doubly MMR- and NER-defective cells was then analyzed. The cells exhibited unchanged resistance to killing by UV and ionizing radiation, indicating that MMR does not contribute significantly to the cytotoxicity of some common DNA lesions. Induction of SCE by UV radiation was also not influenced by the MMR defect to a detectable extent. The data suggest that MMR is unlikely to be a general sensor of persistent DNA damage.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
All chemicals were obtained from Sigma (Poole, Dorset, UK) unless otherwise indicated. Recrystalized MNU, a kind gift of Dr Peter Swann (University College London, London, UK) was dissolved in 4 mM potassium acetate pH 4.0, and aliquots stored at –20°C. 6-TG, 8-azaguanine (8-AzaG) and hypoxanthine were each dissolved in 0.1 N potassium hydroxide. 14C-labeled hypoxanthine was used at a specific activity of 49.5 mCi/mMol.

Cells
The SV40-transformed fibroblast cell lines GM0637, XP12RO and its derivative XP12ROB4, and HeLaMR cells were routinely maintained in Dulbecco's modified Eagle's meduim supplemented with 10% fetal calf serum. Cell survival was determined by colony formation in 10 cm dishes. Cells were plated and allowed to attach for 3–6 h before drug treatment. For MNU, 6-TG and 8-AzaG, the drug was not removed after exposure. UV exposure was from a 254 nm germicidal lamp. The fluence of 1 J/m2/s was calibrated with a Latajet dosimeter. For SCE induction, a lower dose rate (0.1 J/m2/s) source was used.

Mutation frequency and rate
The mutation frequency at the HPRT locus was determined in 96-well dishes or in 10 cm plates. For each cell line, ~104 cells (103 cells for XP12ROB4) were seeded per well or 106 cells (105 cells for XP12ROB4) per plate in medium containing 30 µM 6-TG. A total of 10 dishes or plates were used per experiment. The cloning efficiency of each line was determined in parallel by seeding cells in normal medium.

The rate of mutation at the same locus was determined by fluctuation analysis. Twenty independent clones of each cell line were each expanded to ~107 cells which were then transferred to medium containing 30 µM 6-TG and distributed into 96-well dishes or 10 cm plates as above.

The mutation rate (µ) was calculated from the equation: µ = MC–1·ln2, where M = mutations per culture (–ln·Po), Po = fraction of cultures without mutants, C = total number of cells subject to selection.

Induction and scoring of SCE
Analysis of SCE induction was carried out essentially as described (28). Briefly, 1 day after seeding, cells were exposed to UV radiation at a dose rate of 0.1 J/m2/s and subsequently grown for 2 days in medium containing 5 µM 5-bromodeoxyuridine. Mitotic cells were accumulated, fixed and metaphase chromosomes stained and enumerated as described previously.

Isolation of methylation tolerant XP12RO
XP12RO cells (106 cells per 10 cm tissue culture dish) were treated with 100 µM MNU. Surviving cells which repopulated the dish were exposed to 200 µM MNU. Treatment with escalating MNU doses was continued over a period of 4 weeks at 100 µM increments up to a final concentration of 500 µM.

Enzyme assays
MGMT activity was determined as described previously (29). HPRT was assayed in cell extracts (106–107 cells) prepared in 10 mM Tris–HCl pH 7.4, 10 mM MgCl2, 30 mM KCl, 0.1 mM dithiothreitol (DTT) and 0.5% Triton X-100. Reactions (50 µl) contained 50 mM Tris–HCl pH 7.0, 9 mM MgCl2, 2 mM DTT, 2 mM phosphoribosyl pyrophosphate, 10 µCi/ml 14C-hypoxanthine and 0–25 µg protein. After 15 min at 37°C, reactions were terminated by heating at 70°C for 5 min and cooling on ice. Duplicate 10 µl aliquots were spotted onto DE81 paper (Whatman) which was then washed twice in 50 mM Tris–HCl pH 8.0, dried and the radioactivity determined by scintillation counting in 5 ml permablend (Packard, IL). One unit of enzyme activity catalyses the formation of 1 pmol IMP/min.

Western blotting
Nuclear extracts were denatured by boiling and separated on 8% SDS–polyacrylamide gels. Proteins were blotted onto PVDF membranes which were blocked for 1–3 h with a 5% solution of powdered milk. Proteins were analyzed by incubation with primary anti-hMSH2, -hMLH1 or -hPMS2 from Pharmingen. Immunoreactive proteins were visualized with the Amersham HRP detection kit using the secondary antibodies provided.


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 Introduction
 Materials and methods
 Results
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 References
 
Isolation of MMR-defective XP12RO
XP12RO fibroblasts are Mex and do not express detectable MGMT (<0.05 U/mg protein). They are sensitive to MNU, and have a D37 of ~30 µM, which is typical of Mex human cells (for comparison, the D37 of Mex+ HeLa cells, which express 1 U MGMT/mg protein, is 5 mM). XP12RO and the Mex NER-proficient GM0637 fibroblasts were equally sensitive to MNU, confirming that the NER pathway does not contribute significantly to the repair of potentially lethal methylation damage in these human cells (Figure 1aGo). MNU-resistant derivatives of XP12RO were isolated by exposure of XP12RO cells to escalating doses of MNU. One clone, XP12ROB4, exhibited the stable MNU resistance and cross resistance to 6-TG that is typical of methylation tolerant cells (Table IGo). In agreement with this, XP12ROB4 cells remained Mex and HPRT+ (<0.05 U/mg MGMT; 1.7 U/mg HPRT). By these criteria, XP12ROB4 cells exhibit a typical methylation-tolerant phenotype.



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Fig. 1. Sensitivity to MNU (a) and 6-TG (b). Cells, GM0637 ({square}), XP12RO ({blacksquare}) or XP12ROB4 ({bullet}), were treated at the drug concentrations shown. Survival was determined by colony formation after 14 days.

 

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Table I. Characteristics of XP12RO and XP12ROB4 cell lines
 
A mutator phenotype in XP12ROB4
Methylation tolerance in XP12ROB4 cells was associated with a substantial mutator phenotype. The frequency of HPRT mutants in XP12RO cultures was <10–7/cell (Table IIGo). The corresponding frequency in XP12ROB4 cells was 9x10–5/cell (Table IIGo), an increase of ~1000-fold. These data are compatible with a significant mutator effect in XP12ROB4 cells. The mutation rates in the two cell lines were compared by Luria-Delbrück fluctuation analysis. The rate of mutation to HPRT in XP12ROB4 cells was 7x10–6/cell/generation (Table IIGo). No HPRT mutants were recovered in two experiments with XP12RO, indicating a mutation rate of <10–8/cell/generation at this locus in the parental cells. Thus, the methylation-tolerant phenotype of XP12ROB4 confers a several-hundred-fold increase in spontaneous mutation rate at HPRT.


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Table II. Mutation frequency and rate of XP12RO and XP12ROB4 cell lines
 
The high spontaneous mutation rate is consistent with methylation tolerance in XP12ROB4 cells arising by loss of mismatch repair.

XP12ROB4 cells are defective in hMSH2
Expression of MMR proteins by XP12RO and XP12ROB4 was analyzed by western blotting. hMSH2 was not detectable in extracts of XP12ROB4 cells (Figure 2aGo). They resembled extracts of Jurkat cells, which are known to be deficient in this protein (30). hMSH2 was present to approximately similar extents in extracts of the parental XP12RO cells, the normal human fibroblast cell line GM0637 and the hMLH1-defective tumor cell-line HCT116. A second essential MMR protein, hMLH1, was present at similar levels in XP12RO and XP12ROB4. As expected, this protein was absent from extracts of the colorectal carcinoma cell line HCT116 (31) (Figure 2bGo). The levels of the hPMS2 MMR protein were comparable in XP12RO and XP12ROB4 (data not shown). We conclude that the methylation-tolerant phenotype of XP12ROB4 cells is a consequence of a defect in MMR which arises from greatly reduced or absent expression of the essential hMSH2 protein.



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Fig. 2. Western blot analysis of MMR proteins. Cell extracts were separated by PAGE, blotted and probed with anti-hMSH2 (a) or anti-hMLH1 (b) antibody. Blots were also probed with anti-PCNA as a loading control.

 
Cell survival following UV and ionizing radiation
XP12RO cells exhibited the expected sensitivity to UV radiation when compared with the NER-proficient GM0637 (Figure 3aGo). They were also more sensitive to mitomycin C (Figure 3bGo) and nitrogen mustard (data not shown) both of which introduce bulky DNA monoadducts as well as DNA interstrand crosslinks. These properties are consistent with the XPA defect in XP12RO. The UV sensitivity of XP12ROB4 cells did not differ significantly from that of their XP12RO parents (Figure 3aGo). Thus, cell killing by persistent UV-induced DNA damage, in contrast to that induced by MNU and 6-TG, is not altered to a detectable extent by the introduction of the hMSH2 defect.



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Fig. 3. Sensitivity to various DNA damaging agents. Cells, GM0637 ({square}), XP12RO ({blacksquare}), XP12ROB4 ({bullet}) or HeLa ({circ}), were UV irradiated at a dose rate of 1 J/m2/s (a), treated with mitomycin C for 24 h (b) or {gamma}-irradiated at a dose rate of 1 Gy/s (c). Survival was determined by colony formation.

 
XP12RO were slightly more sensitive to ionizing radiation than Mex, NER- and MMR-proficient HeLaMR cells. The introduction of the hMSH2 defect into XP12RO did not significantly alter its sensitivity to ionizing radiation (Figure 3cGo).

SCE induction by UV radiation
SCE induction was measured in XP12RO and XP12ROB4 exposed to 0–0.8 J/m2 UV light. The total SCE scored and the frequencies of SCE per chromosome are presented in Table IIIGo. UV induced a dose-dependent increase in SCE in each cell line. XP12RO cells were found to be very sensitive to UV-induced SCE. This is in agreement with the general sensitivity of XP cells, including XP12RO, previously reported by others (25,32). There was no significant difference in the sensitivities of XP12ROB4 and XP12RO to SCE induction by UV radiation. At each of the four UV doses used, which covered a range of survival down to 0.1% for both cell lines, the frequency of SCE in XP12RO was indistinguishable from that in XP12ROB4 (Figure 4Go). In addition, no difference was apparent in the frequencies of spontaneous SCE in the two cell lines, which were in good agreement with published values for XP12RO cells (25). The hMSH2 defect of XP12ROB4 cells does not measurably increase their susceptibility to spontaneous or to UV-induced SCEs.


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Table III. UV-induced SCE in XP12RO and XP12ROB4 cells
 


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Fig. 4. SCE induction in XP12RO ({blacksquare}) and XP12ROB4 ({bullet}). Cells were irradiated at a dose rate of 0.1 J/m2/s. and SCE were scored as described in Materials and methods. The inset shows the cell survival determined in parallel by clonal assay.

 

    Discussion
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 Materials and methods
 Results
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XP12ROB4 combines defects in three DNA repair functions: MGMT, MMR and NER. XP12ROB4 cells exhibited the increased MNU and 6-TG resistance expected of a methylation-tolerant, MMR-defective cell line and did not express detectable hMSH2. Consistent with their MMR deficiency, XP12ROB4 cells exhibited a mutator phenotype. The mutator effect was more extensive than is normally associated with methylation tolerance and the mutation rate was comparable with that found in some repair-defective tumor cell lines. It is not clear at present whether this unusually high mutation rate simply reflects the absence of hMSH2 expression in XP12ROB4 or is related in some way to the simultaneous absence of three important DNA repair pathways.

The hMSH2 defect in XP12ROB4 did not detectably alter its sensitivity to UV radiation as measured by clonal survival or by susceptibility to SCE induction by UV radiation. It has been noted previously that NER-proficient methylation-tolerant cells are not cross-resistant to UV radiation (24). Similarly, the UV survival of NER-proficient, MMR-defective human tumor cell lines is generally comparable with that of NER- and MMR-proficient cells (25). There is a suggestion that MMR may participate in transcription-coupled repair (TCR) of UV-induced DNA damage to the transcribed DNA strand (33). Since TCR is dependent on the XPA protein, we are unable to address this problem in XP12ROB4. Thus, although we cannot exclude the possibility of MMR/UV photoproduct interactions which do not result in significant alteration in cell survival, hMutS{alpha} is clearly not required to transduce signals from cytotoxic DNA photoproducts and to activate cell death.

In addition to its lethal effects, DNA O6-meGua is also a potent inducer of SCEs in Mex cells which are proficient in MMR (28,34). UV radiation is also an efficient inducer of SCEs and its effect is particularly marked in NER-deficient XP cells (25,32). Thus, persistent UV photoproducts are among the DNA lesions which initiate exchanges, and SCEs provide a sensitive alternative marker for the effects of persistent DNA damage. The production of SCEs by methylating agents requires that MMR processes DNA O6-meGua. XP12RO and XP12ROB4 were equally sensitive to SCE induction by UV radiation. This rules out a significant role for MMR in processing persistent UV photoproducts into SCE intermediates. Taken together with the absence of significant changes in susceptibility to UV-induced killing, it seems most likely that UV-induced DNA damage is not recognized or processed by MMR. This is consistent with the reported inability of the purified hMutS{alpha} mismatch recognition factor to recognize UV DNA photoproducts (35). It is in direct contrast to the requirement for MMR participation in both methylating agent induced death and SCE induction, and the recognition of O6-meGua-containing base pairs by hMutS{alpha} (19).

Results from biological and biochemical experiments concur that hMSH2 is not required for NER in human cells (35,36). Recent studies in yeast also exclude a major role for the Saccharomyces cerevisiae MSH2 protein in processing UV-induced DNA damage but suggest that it might participate in a minor, possibly recombinational, repair pathway (37). This is consistent with the likely role of MSH2 in double-strand break rejoining by the single-strand annealing or gene conversion pathways (38,39). The properties of XP12ROB4 suggest that if similar minor hMSH2-dependent recombinational repair pathways operate in human cells, they differ from SCE and any effects on UV sensitivity fall below the level of detection of our assays.

MMR can influence the survival of cells exposed to ionizing radiation although not in a consistent fashion. MLH1-defective mouse cells are slightly more resistant to {gamma}-irradiation than MMR competent controls (40). In contrast, hMLH1 deficiency in the human tumor cell line HCT116 confers a slight {gamma}-ray sensitivity (41). The reasons for the contradiction between these findings are unclear. They may simply indicate a different balance of repair and death promoting pathways in the two types of cell. Both studies suggest, however, that MMR is likely to be a relatively minor determinant of cellular sensitivity to ionizing radiation. This is consistent with the unaltered sensitivity of XP12ROB4 to {gamma}-irradiation.

In conclusion, O6-meGua and 6-meTG are cytotoxic only if two criteria are fulfilled: (i) the methylated bases must persist in DNA and (ii) the MMR pathway must be active. Our observations with XP12RO and XP12ROB4 cells confirm these requirements and indicate that NER does not influence the interaction of MMR with these types of DNA damage. The properties of XP12ROB4 cells indicate that any interaction of MMR with persistent UV-induced DNA damage does not detectably influence the cell's sensitivity to killing or to SCE induction. The data exclude a significant role for MMR as a general sensor of DNA damage. They are more consistent with a model in which certain damaged base pairs exhibit structural features which mimic mispairs between undamaged DNA bases and initiate inappropriate, and ultimately lethal, processing.


    Notes
 
2 To whom correspondence should be addressed Email: karran{at}icrf.icnet.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 19, 1999; revised February 10, 1999; accepted February 11, 1999.