1 Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK and
2 Istituto Superiore di Sanitá, Laboratory of Comparative Toxicology and Ecotoxicology, Viale Regina Elena 299, 00161 Rome, Italy
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Abstract |
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Abbreviations: bzGua, O6-benzylguanine; DMSO, dimethyl sulphoxide; ES, embryonic stem; FISH, fluorescence in situ hybridization; HNPCC, hereditary non-polyposis colorectal cancer; MGMT, O6-methylguanineDNA methyltransferase; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; O6-meGua, O6-methylguanine; PBS, phosphatebuffered saline; SDS, sodium dodecyl sulphate; UV, ultraviolet; 3-meAde, 3-methyladenine.
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Introduction |
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In addition to its role as an editor of DNA replication, mismatch repair contributes to cell killing by some DNA damaging drugs (reviewed in ref. 8). The cytotoxicity of methylating agents such as N-methyl-N-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or their clinical counterparts, dacarbazine and temozolomide, is largely caused by their ability to introduce O6-methylguanine (O6-meGua) into DNA (reviewed in ref. 9). Two opposing DNA repair functions influence the lethality of this methylated base. A specific repair protein, O6-meGuaDNA methyltransferase (MGMT) counteracts the lethal effects of O6-meGua by catalysing its in situ demethylation (reviewed in ref. 10). In contrast, mismatch repair potentiates death by O6-meGua. Most cells are protected against DNA O6-meGua by MGMT but their resistance can be abolished by a specific MGMT inhibitor, O6-benzylguanine (O6-bzGua) (11,12). Mex cells, which do not express detectable MGMT, are very sensitive to methylating agents. Thus, persistent DNA O6-meGua is normally lethal. In contrast, mismatch repair-defective human cells are, without known exception, insensitive to the lethal effects of persistent O6-meGua. This resistance, known as methylation tolerance (13), is independent of MGMT and is retained even when the MGMT is inactivated by O6-bzGua (14). Tolerance can be conferred by defects in hMSH2 (14), hMSH6 (1517), hMLH1 (17,18) or hPMS2 (19). Current models for the involvement of mismatch repair in the lethality of methylating agents suggest that the hMutS and hMutL
complexes recognize incorrigible O6-meGua-containing base pairs. This recognition may lead to lethal processing (13) and/or to the direct activation of a cell cycle checkpoint (20,21). Because mismatch repair-defective Mex human cells are 50- to 100-fold more resistant to MNU or MNNG than their repair proficient counterparts, selection for drug resistance has been used to isolate human cell lines that are deficient in this repair pathway (17,22).
Mice in which one or both copies of a mismatch repair gene are inactive have been generated by targeted gene disruption (2328). Although many of their properties are consistent with HNPCC, they nevertheless display some anomalous phenotypes. Since much of our knowledge of the effects of mismatch repair deficiencies in human cells comes from the studies of repair defective human tumour cell lines, we investigated the properties of mismatch repair defective mouse tumour cell lines. Mismatch correction defective mouse cell variants were isolated by selection for tolerance to MNNG. A comparison of the properties of repair proficient and defective mouse cell lines with their human counterparts revealed a surprising intrinsic resistance to DNA methylation damage in mouse cells, which may be related to their lower mismatch repair capabilities.
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Materials and methods |
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Cell culture, survival determination and selection for methylation tolerance
The cell lines used in this study are listed in Table I. All of the starting cell lines were from the ICRF Cell Production (Clare Hall, UK) stocks except for A2780 and RH95021, which were kindly provided by Dr Robert Brown, University of Glasgow, UK, and Dr Hein te Riele, Amsterdam Cancer Centre, The Netherlands, respectively. All the tumour cell lines were cultured in Dulbecco's modified minimal essential medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) (complete medium). Cultures were incubated at 37°C in 5% CO2 and 95% relative humidity.
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Selection of MNNG-resistant variants of Colo26 was carried out as previously described (22). Briefly, separate cultures of exponentially growing cells were treated with 0.67 µM MNNG on day 1, 1 µM on day 3, 1.7 µM on day 9 and 2.3 µM on day 12. Surviving cells were isolated by single cell cloning.
MNU-resistant derivatives of A2780 were isolated by exposing cells to a single concentration of MNU (2 mM) in the presence of O6-bzGua (see above). Surviving cells were isolated by single cell cloning.
MGMT assay
MGMT activity in cell extracts was determined using [3H]MNU-treated DNA as previously described (29) or by measurement of transfer of 3H radioactivity to proteinase K-sensitive form. The limit of detection of the latter assay corresponded to ~0.03 units (pmole methyl groups per mg protein). Cell lines expressing <0.05 units of MGMT were considered to be Mex.
DNA alkylation
Approximately 2x108 exponentially growing Colo26, MNUcolo5, CMT167, HeLa and A2780 cells were each suspended in 10 ml serum-free growth medium. [3H]MNU (20 Ci/mmol, 5 mCi/ml in ethanol; Amersham International, Amersham, UK) was added to a final concentration of 0.1 mCi/ml. After incubation at 37°C for 60 min, cells were harvested by centrifugation, washed twice by pelleting and resuspension in 10 ml growth medium followed by a further wash in 10 ml PBS. Washed cells were suspended in 10 ml TE pH 8.0 and lysed by the addition of 0.2% sodium dodecyl sulphate (SDS)/1 mM EDTA. The viscosity of the lysate was reduced by shearing through a 19 g needle and 20 µg/ml RNase was added. After a 30 min incubation at 37°C, 50 µg/ml proteinase K was added and incubation continued for a further 60 min. After three phenol extractions, DNA was precipitated with ethanol, the concentration estimated from A260, hydrolysed in 0.1 N HCl (30 min, 70°C) and the radioactivity of the hydrolysates was determined by scintillation counting.
Mismatch repair assay
Substrates.
Mismatched heteroduplexes were constructed from molecules derived by subcloning a 211 bp PvuIPstI fragment of HK7 M13 (30) into the pBK-CMV phagemid (Stratagene, Cambridge, UK). The inserted region contains the `heteroduplex cassette' sequence that can be used to generate specific mismatches that inhibit restriction endonuclease cleavage. The standard substrate contained a T:C mispair in which the T was in the interrupted (c) strand and the C was located in the closed viral (v) strand. This mismatch was efficiently corrected in a long-patch, nick-dependent manner by extracts of human cells to regenerate an MluI restriction site (17).
To construct the substrate, purified RFI closed circular duplexes containing T at position 1030 were linearized by digestion with NdeI, denatured and annealed to an excess of viral DNA containing C at position 1030. The annealed products contain a single mispair, which is 580 bp 5' to a unique nick (Figure 1A). The nicked circular molecules were purified by electroelution from agarose gels. (A small amount of re-annealed duplex was present in most substrate preparations. The restriction endonuclease digestion products of this contaminant are well resolved from the diagnostic repair products and do not interfere with the assay.) After incubation with cell extract, DNA molecules are recovered and digested with MluI. Uncorrected substrate molecules contain a single MluI site (position 463). Cleavage of the restored MluI site at position 1030 of the corrected molecules produces a diagnostic fragment of 3.9 kb, which is well resolved from the uncorrected molecules that remain unit length (4.47 kb).
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Mismatch correction assay.
Extracts for mismatch correction assays were prepared from 15x109 exponentially growing cells and assays were carried out as previously described (17). Reactions (25 µl) contained 30 mM Hepes KOH, pH 8.0, 7 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM dNTP, 4 mM ATP, 40 mM phosphocreatine, 1 mg creatine phosphokinase (rabbit muscle type I), 80 ng mismatched or gapped DNA substrate and up to 200 µg cell extract. After a 60 min incubation at 37°C, reactions were terminated by the addition of 10 mM EDTA, 0.5% SDS. DNA was recovered from the reaction mixture and digested with the appropriate diagnostic restriction endonuclease. Digestion products were separated on 0.8% agarose gels in TAE buffer containing ethidium bromide and visualized under short wavelength UV light.
Gap-filling assays were carried out in an identical fashion except that the incubation time was reduced to 5 min. Products of the correction and gap-filling assays were quantified by scanning with a Gel-Doc system (BioRad, Hercules, CA) of the agarose gels. The data were processed by the computer program Molecular Analyst (Bio-Rad).
Western blotting
Cell extracts (150 µg) were denatured and separated on 8% SDS polyacrylamide gels. Proteins were transferred to nylon membranes, blocked by immersion in TBSTween (10 mM TrisHCl, pH 7.5, 150 mM NaCl, 0.05% Tween 80) containing 5% skimmed powdered milk. The blocked filter was incubated with primary antibody (1 µg/ml for hPMS2 and hMLH1, 0.1 µg/ml for hMSH2 or 1:1000 diluted antiserum for hMSH6) for 1 h at room temperature. After washing with TBSTween the appropriate secondary antibody was added. The blots were developed using the ECL detection reagents (Amersham International).
Microcell transfer and fluorescence in situ hybridization (FISH)
The method for microcell chromosome transfer was slightly modified (31) from the one already described (32). Briefly, micronuclei of the monochromosomal hybrid A9 cells (8x105) were induced by treatment for 2 days with 0.06 µg/ml Colcemid (Sigma). The flasks were filled with serum-free DMEM containing 20 µg/ml cytochalasin B (Sigma) and placed into Sorval GSA rotor wells filled with warm water (30°C). The flasks were centrifuged at 7000 r.p.m. for 75 min at 34°C. The microcells were resuspended in DMEM and filtered through 8 and 5 µm polycarbonate filters (Nucleopore, Pleasanton, CA). The microcells were pelleted by centrifugation at 1400 r.p.m. for 20 min at 4°C and resuspended in medium containing 100 µg/ml phytohaemaglutinin (Sigma). The microcells were attached to recipient monolayers by incubation for 20 min at room temperature. The cultures were treated with 47% polyethylene glycol (Mr 1450; Sigma) for 1 min, extensively washed in serum-free medium and 24 h later seeded in selective medium (1 mg/ml G418). The G418 resistant microcell hybrids were isolated after 14 days of growth.
Slides for cytogenetic analysis were prepared according to standard procedures. Briefly, mitotic figures were accumulated by treatment with 4x107 M Colcemid (Sigma) and washed three times with methanol:acetic acid (3:1). FISH was performed using a biotinylated probe (Oncor, Gaithesburg, MD) for the alphoid sequences of human chromosome 2. Molecular hybridization and immuno-fluorescence detection of the probes were carried out according to the protocol provided by the manufacturer. Detection of the biotin-labelled probe was performed with fluoresceinatedavidin (FITCavidin; Vector, Burlingame, CA) and the intensity of fluorescence was amplified by a biotinylated anti-avidin antibody (Boehringer Mannheim, Mannheim, Germany) followed by an additional layer of FITCavidin. Slides were counterstained with propidium iodide. Two hundred nuclei per clone were analysed and the number of signals in each nucleus recorded.
TUNEL analysis
Cytospin preparations of 2x104 cells were air dried and fixed with formaldehyde (4% in PBS, pH 7.4) for 30 min at room temperature. Slides were incubated for 2 min on ice in a permeabilizing solution containing 0.25% Triton X-100 (Bio-Rad) and 0.1% sodium citrate. TUNEL assay was performed using the in situ Cell Death Detection kit, Fluorescein (Boehringer Mannheim) according to the manufacturer's instructions. Hoechst 33342 (1 µg/ml in PBS) was used to counterstain nuclei.
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Results |
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To investigate whether the level of resistance of Colo26 cells was typical of mouse cells, we examined two other cell lines, B16, a Mex mouse melanoma, and NIH3T3, a Mex+ non-transformed mouse embryonic fibroblast cell line (Table I). Survival after MNU treatment of the Mex B16 cells was similar to both Colo26 and to NIH3T3 cells depleted of MGMT by O6-bzGua treatment (Figure 2B
). Thus, in the absence of any repair of O6-meGua by MGMT, three mouse cell lines of different origins, Colo26, B16 and NIH3T3, showed similar levels of MNU sensitivity.
A fourth mouse cell line, CMT167, derived from a lung carcinoma, expressed MGMT levels that were close to the lower limit of detection (Table I). Despite this, CMT167 was significantly more resistant than Colo26 to MNU (Figure 2B
) and was comparable to MNUcolo5 and to MSH2/ RH95021 cells in this regard. The MNU resistance and extremely low levels of MGMT expressed by CMT167 suggested that CMT167 is a naturally methylation-tolerant tumour cell line.
MNU-tolerant human cell lines
In order to compare these results with methylation-tolerant human cells, we used tolerant variants of the HeLaMR and A2780 cell lines. HeLaMR cells are Mex and are very sensitive to MNU. The previously described methylation-tolerant derivative, HeLaClone 6 (22), is ~100-fold more resistant to this agent (Figure 2C, Table I
). The ovarian carcinoma cell line A2780 is Mex+ and is resistant to MNU (Figure 2C
, Table I
). When A2780 cells were treated with MNU in the presence of O6-bzGua, they exhibited the expected hypersensitivity of MGMT-depleted human cells and were similar to HeLaMR cells (Figure 2C
, Table I
). Methylation-tolerant variants of A2780 were selected by treatment with MNU. These variants had become highly resistant to MNU in the presence of O6-bzGua, as expected for a typical methylation-tolerant human cell line. One example, MNUA2780, is shown in Figure 2C
. The MNU resistance of both MNUA2780 and HeLaClone 6 fell within the expected range for methylation-tolerant human cells (Figure 2C
, light grey shaded sector). There are two notable features of the data presented in Figure 2
. Firstly, the MNU resistance of the methylation-tolerant mouse and human cell lines is similar. Secondly, the three mouse cell lines are significantly (
20-fold) more MNU resistant than both the human cell lines when the contribution of MGMT is abolished by O6-bzGua treatment (Figure 2C
, dark grey shaded sector).
In summary, we observed major differences between mouse and human cells in their response to a methylating agent. First, human cells were much more sensitive to killing than mouse cells. Despite this large (~20-fold) difference, methylation-tolerant mouse and human cells were similar in their sensitivity to MNU.
Mismatch repair activities in cell extracts
Extracts of Colo26 cells corrected several different mispairs (including T:G, A:C, A:G and A:A) in a nicked circular duplex substrate. Repair of a T:C mismatch was typical and an example is shown in Figure 3. Correction was directed to the nicked strand of the duplex, and little or no correction of the intact strand was observed (data not shown). This activity was absent in extracts of RH95021 mouse fibroblasts, which are MSH2/. Repair of the T:C mismatch in this substrate is therefore by the MSH2-dependent long-patch, nick-directed repair pathway. Consistent with their methylation-tolerant phenotype, extracts of MNUcolo5 and CMT167 did not detectably correct the mispair and resembled MSH2-defective RH95021 cells in this regard. Extracts of MNUcolo2, a variant that had received an identical MNNG treatment but retained normal MNU sensitivity (Table I
), were fully repair-proficient and were comparable to Colo26. Methylation-tolerant HeLaClone 6 (33) and MNUA2780 extracts were also deficient in mismatch correction whereas extracts of their parental HeLaMR (data not shown) and A2780 cells efficiently repaired the mismatch (Figure 3
).
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Identification of the mismatch repair defects
Mismatch correction was not restored by mixing extracts of MNUcolo5 and CMT167 (Figure 4A), suggesting that their repair defects lie in the same protein or protein complex. Addition of extracts of human HCT116 or DLD-1 cells, defective in hMLH1 and hMSH6, respectively, partially complemented both MNUcolo5 and CMT167. Neither extract was complemented by Jurkat or LoVo cell extracts in which neither the hMSH2 (34) nor hMSH6 protein is detectable (Figure 4A
and data not shown). HCT116, but not LoVo, Jurkat or DLD-1 extracts, also complemented the repair defect in RH95021 (Figure 4A
). This is in agreement with the absence of detectable MSH2 or MSH6 protein in RH95021 extracts as determined by western blotting (data not shown).
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Western blotting indicated that both hMLH1 and hPMS2 proteins, which were present in A2780 extracts, were undetectable or significantly reduced in extracts of MNUA2780 cells. hMSH2 and hMSH6 levels were comparable in the two cell lines (Figure 4B). The methylation-tolerant phenotype of MNUA2780 therefore results from a defect in the hMutL
complex.
Thus, complementation of the mismatch repair defects of CMT167 and MNUcolo5 indicates that their methylation tolerance is the result of a defect in the MutS complex, most likely a mutation in MSH2. Analysis by western blotting indicates that MNUA2780 cells are methylation-tolerant because they are deficient in the hMutL
complex.
Reversion of methylation tolerance in mouse cells by transfer of human chromosome 2
Mismatch repair defects in human tumour cell lines can be corrected by the introduction of an appropriate single chromosome (18,35). The in vitro mismatch correction assay suggested a defective MutS complex in MNUcolo5 cells. Human chromosome 2, which contains both the hMSH2 and hMSH6 genes, was introduced into MNUcolo5 by microcell fusion in order to investigate whether methylation tolerance in mouse cells could be reversed by expression of the human repair complex. The presence of the human chromosome was confirmed in G418-resistant clones of MNUcolo5 by hybridization with a human-specific centromeric probe for chromosome 2 (data not shown). One G418-resistant clone, in which 50 and 36% of the cell population showed 1 and 2 hybridization signals for the human chromosome 2, respectively, was chosen to analyse survival after MNU treatment. Figure 5
(a representative experiment of three) shows that human chromosome 2 reversed the MNU resistance in this clone to close to that of the parental Colo26 cells. These observations confirm that the human mismatch repair proteins can participate in processing of DNA methylation damage by the mouse mismatch repair pathway. Significantly, restoration of mismatch correction did not confer the level of MNU sensitivity that is characteristic of the human cells we examined.
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Cell killing by other DNA damaging agents
Since the higher MNU resistance of mouse cells is not due to less methylation of DNA, we investigated whether it reflected a general resistance to DNA damaging agents, perhaps as a result of less efficient engagement of the pathways of cell death. Loss of mismatch repair in our methylation-tolerant mouse or human cell lines was not accompanied by significant changes in sensitivity to UV or -radiation (Table I
). In addition, the sensitivities of Colo26 and HeLa cells to ionizing radiation were essentially identical and within the range of published values for cultured mouse and human cells. A2780 cells were somewhat radiosensitive. This may be related to their functional p53/p21 pathway (36). The survival of Colo26 mouse cells after UV irradiation was indistinguishable from that of human HeLa or A2780 cells (Table I
). Notwithstanding the
-radiation sensitivity of A2780, which was not accompanied by hypersensitivity to UV radiation, we conclude that the increased resistance to MNU of mouse cells does not reflect a general resistance to DNA damaging agents.
In an alternative approach, we determined the ability of the mouse and human cells to undergo apoptosis in response to DNA damaging agents. After exposure to 25 J/m2 UV light, Colo26, MNUcolo5 and A2780 cells underwent apoptosis to similar extents at 1 and 2 days after irradiation (Figure 6A). It appears that the pathway(s) of apoptosis that is engaged following UV irradiation is intact, and similar levels are triggered in the mouse and human cells.
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Mismatch repair levels
Because the differential sensitivity of the human and mouse cell lines to MNU was not readily explained by different levels of DNA damage or ability to engage death pathways, we considered the possibility that mouse cells might have a lower capacity for long-patch mismatch repair with which to process DNA O6-meGua. Extracts of human cells were consistently 2- to 3-fold more efficient at mismatch correction than mouse cell extracts. Typical examples of the correction assay are presented in Figure 7A. As a control, we examined the ability of the same extracts to fill a small single-stranded gap in a DNA duplex that was identical to the mismatch repair substrate except that the mismatch was replaced by a short single-stranded region. The extents of repair and gap-filling were quantitated following separation of MluI digestion products on agarose gels. The mismatch repair efficiency was normalized to the latter in order to minimize differences due to extract quality. In general, the levels of gap filling were closely similar in human and mouse cell extracts. Data derived from extracts of two mouse cell lines, Colo26 and NIH3T3, and two human lines, Raji and HeLa, are presented in Figure 7A
. Figure 7B
presents a comparison of Colo26 and HeLa extracts in which T:C mismatch repair is normalized to gap-filling activity. Thus, mouse cell extracts are consistently 2- to 3-fold less proficient at mismatch repair than human cell extracts. These differences are not accounted for by extract quality.
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Since active mismatch repair is essential for the cytotoxicity of DNA methylation damage, we conclude that a reduced capacity for long-patch, nick-directed mismatch repair may contribute to the relatively high MNU resistance of mouse cells.
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Discussion |
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An important difference between mouse and human cells, however, is that loss of mismatch correction in mouse cells confers only a relatively modest increase in methylation resistance. The increase is minor because mouse cells are intrinsically more refractory to killing by MNU than human cells. We observed consistently higher MNU resistance in several mouse cell lines of different types and from different genetic backgrounds. There are additional observations that suggest that discrepant methylation sensitivity is a general feature of mouse and human cells. Examples of MGMT-independent methylating agent resistance in mouse and hamster cells can be found in published reports (37,38), although the differences between rodent and human cells have gone unremarked. Mex human and hamster cells can be protected against methylating agent cytotoxicity by expression of a transfected Escherichia coli Ada gene (the homologue of MGMT). The degree of protection afforded is, however, very different (20- to 40-fold for human cells compared with ~5-fold for hamster cells) (3842). These differences cannot be ascribed to different levels of Ada gene expression. In addition, the extent of methylation tolerance in a mismatch recognition-defective hamster cell line is modest (12-fold) in comparison with the 50- to 100-fold observed in mismatch repair-defective derivatives of human HeLa (22), Raji (17), TK6 (15) or A2780 cells (this study).
We considered several possible factors that might influence the relative methylation resistance of mouse cells. A trivial one, that the cells sustain less potentially lethal DNA damage, is excluded by our dosimetry data, which indicate that the levels of methylation in mouse and human cells are closely comparable. These are consistent with the simple nature of the methylation reaction and the absence of any requirement for metabolic activation of MNU. In addition to O6-meGua, other methylated bases, which are considered potentially cytotoxic, principally 3-methyladenine (3-meAde), will also be present at similar levels in MNU-treated mouse and human cells. Although it is possible that enhanced repair of 3-meAde by mouse cells might contribute to their relative MNU resistance, we consider it unlikely. Repair of 3-meAde is mediated by the product of the AAG gene, a DNA glycosylase that catalyses its removal from DNA (43). The levels of 3-meAde-DNA glycosylase are similar in human and rodent cells. They are, if anything, somewhat lower in mouse and hamster than in human cell extracts (4446). In addition, the complete absence of detectable 3-meAde-DNA glycosylase from AAG/ embryonic stem (ES) cells confers only a 2- to 4-fold increase in sensitivity to methylating agents (47). This change is small compared with the >20-fold difference in MNU resistance between mouse and human cells, and the observation supports the conclusion that different efficiencies of repair of 3-meAde are unlikely to be responsible for the relative MNU resistance of mouse cells.
Our data suggest that the MNU resistance of mouse cells is not due to defects in apoptotic pathways. Firstly, UV radiation induced similar levels of apoptosis in mouse and human cells. In addition, human and mouse cells were both susceptible to MNU-induced apoptosis, indicating that this pathway was intact and able to transduce death signals. Furthermore, we found no evidence for a systematically higher resistance to all types of DNA damage in mouse cells. With a single exception, the mouse and human cells were equally sensitive to two unrelated DNA damaging agents, UV and ionizing radiation. Finally, both MNU and -radiation provoke cell cycle arrest in the G2 phase. Since mouse and human cells exhibit similar sensitivity to
-radiation, it is unlikely that the differences observed for methylation damage are due to a relaxed cell-cycle checkpoint control in mouse cells.
We note that ES cells do not share the generally high resistance of differentiated mouse cells to DNA methylation damage. Although mouse ES cells without MGMT are extremely sensitive to MNNG (23,47), this hypersensitivity most likely reflects a general sensitivity to diverse DNA damaging agents. It has been suggested that ES cells are unusually efficient at activating apoptosis following exposure to many different types of cytotoxic agent (48).
Since active long-patch mismatch repair is a requirement for the cytotoxicity of O6-meGua in human cells, the higher intrinsic resistance of rodent cells may reflect less processing of the methylated DNA base. This possibility is supported by our observation that mouse cell extracts reproducibly carried out less long-patch, nick-directed mismatch repair than their human counterparts. In addition, repair by mouse cell extracts is apparently limited by a factor that could be provided by human cell extracts but is not one of the components of the hMutS and hMutL
complexes. The differences in repair capacity were small, however, and may not in themselves be sufficient to explain fully the relative MNU resistance of the mouse cells. An alternative possibility is that the mouse MutS
recognition factor might initiate processing of O6-meGua less efficiently than its human counterpart, perhaps because of a lower affinity for the incorrigible base pairs. The hMutS
complex recognizes G:T and O6-meGua:T base pairs (49,50). The affinity of mouse mismatch recognition factor(s) for O6-meGua-containing base pairs is unknown. We are currently addressing this question using mismatch recognition complexes purified from human and mouse cells.
Our findings have some implications for the use of mice as models of human cancer. Mice are used as hosts for human tumour xenografts to evaluate the potential of therapeutic agents. Clearly, since mouse tissues have an intrinsically higher resistance to DNA methylation damage than human tumour cells, methylating agents will be less toxic to the mouse host than to a human host. The use of such agents in xenograft experiments is therefore likely to indicate an erroneously high therapeutic index, not because of a high sensitivity of the tumour but because of low toxicity to the host. In addition, knockout mice are used as models for HNPCC. If the long-patch mismatch repair pathway is less efficient in mouse cells, the phenotypes of knockout mice are likely to be an incomplete reflection of HNPCC. Such differences may contribute to the apparent absence of a significant predisposition to tumour development in mice that are heterozygous for mismatch repair defects. They might also influence the types of tumours that develop in homozygously repair-defective mice, which do not, in general, reflect the tumours in HNPCC patients.
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Acknowledgments |
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Notes |
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4 These authors contributed equally to this work
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References |
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