Gabriele Aquilina

The Mammalian Mismatch Repair Pathway Removes DNA 8-oxodGMP Incorporated from the Oxidized dNTP Pool

Mismatch repair (MMR) corrects replication errors. It requires the MSH2, MSH6, MLH1, and PMS2 proteins which comprise the MutSalpha and MutLalpha heterodimers. Inactivation of MSH2 or MLH1 in human tumors greatly increases spontaneous mutation rates. Oxidation produces many detrimental DNA alterations against which cells deploy multiple protective strategies. The Ogg-1 DNA glycosylase initiates base excision repair (BER) of 8-oxoguanine (8-oxoG) from 8-oxoG:C pairs. The Myh DNA glycosylase removes mismatched adenines incorporated opposite 8-oxoG during replication. Subsequent BER generates 8-oxoG:C pairs, a substrate for excision by Ogg-1. MTH1-an 8-oxodGTPase which eliminates 8-oxodGTP from the dNTP pool-affords additional protection by minimizing 8-oxodGMP incorporation during replication. Here we show that the dNTP pool is, nevertheless, an important source of DNA 8-oxoG and that MMR provides supplementary protection by excising incorporated 8-oxodGMP. Incorporated 8-oxodGMP contributes significantly to the mutator phenotype of MMR-deficient cells. Thus, although BER of 8-oxoG is independent of Msh2, both steady-state and H(2)O(2)-induced DNA 8-oxoG levels are higher in Msh2-defective cells than in their repair-proficient counterparts. Increased expression of MTH1 in MMR-defective cells significantly reduces steady-state and H(2)O(2)-induced DNA 8-oxoG levels. This reduction dramatically diminishes the spontaneous mutation rate of Msh2(-/-) MEFs.

Unmasking a killer: DNA O(6)-methylguanine and the cytotoxicity of methylating agents.

Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells. Their effects can be ascribed to an ability to modify DNA covalently. Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O(6)-methylguanine (O(6)meG) as a potentially important DNA lesion. Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells - including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli - have defined the contributions of O(6)meG and other methylated DNA bases to the biological effects of these chemicals. More recently, the role of O(6)meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O(6)meG and the mismatch repair pathway. Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O(6)meG. We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent. Such exposure may increase the probability that the cells mismatch repair pathway becomes inactive. Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens.

The Oxidized Deoxynucleoside Triphosphate Pool Is a Significant Contributor to Genetic Instability in Mismatch Repair-Deficient Cells

ABSTRACT Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2−/− mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of −G and −A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.

Mismatch repair in correction of replication errors and processing of DNA damage.

The primary role of mismatch repair (MMR) is to maintain genomic stability by removing replication errors from DNA. This repair pathway was originally implicated in human cancer through an association between microsatellite instability in colorectal tumors in hereditary nonpolyposis colon cancer (HNPCC) kindreds. Microsatellites are short repetitive sequences which are often copied incorrectly by DNA polymerases because the template and daughter strands in these regions are particularly prone to misalignment. These replication‐dependent events create loops of extrahelical bases which would produce frameshift mutations unless reversed by MMR. One consequence of MMR loss is a widespread expansion and contraction of these repeated sequences that affects the whole genome. Defective MMR is therefore associated with a mutator phenotype. Since the same pathway is also responsible for repairing base:base mismatches, defective cells also experience large increases in the frequency of spontaneous transition and transversion mutations. Three different approaches have been used to investigate the function of individual components of the MMR pathway. The first is based on the biochemical characterization of the purified protein complexes using synthetic DNA substrates containing loops or single mismatches. In the second, the biological consequences of MMR loss are inferred from the phenotype of cell lines established from repair‐deficient human tumors, from tolerant cells or from mice defective in single MMR genes. In particular, molecular analysis of the mutations in endogenous or reporter genes helped to identify the DNA substrates for MMR. Finally, mice bearing single inactive MMR genes have helped to define the involvement of MMR in cancer prevention.

MISMATCH REPAIR DEFECTS AND O6-METHYLGUANINE-DNA METHYLTRANSFERASE EXPRESSION IN ACQUIRED RESISTANCE TO METHYLATING AGENTS IN HUMAN CELLS

Fifteen variants with ≥30-fold resistance toN-methyl-N-nitrosourea were isolated from the Burkitt’s lymphoma Raji cell line. Eight had received a single treatment with a highly cytotoxic dose. The remainder, including the previously described RajiF12 cell line, arose following multiple exposures to initially moderate but escalating doses. Surprisingly, methylation resistance arose in three clones by reactivation of a previously silent O 6-methylguanine-DNA methyltransferase gene. Five clones, including RajiF12, displayed the microsatellite instability and increased spontaneous mutation rates at the hypoxanthine-guanine phosphoribosyltransferase locus, consistent with deficiencies in mismatch repair. Defects in either the hMutSα or hMutLα mismatch repair complexes were identified in extracts of these resistant clones by in vitro complementation using extracts from colorectal carcinoma cell lines. Defects in hMutLα were confirmed by Western blot analysis. Remarkably, five methylation-resistant clones in which mismatch repair defects were demonstrated by biochemical assays did not exhibit significant microsatellite instability.

Sensitivity to DNA cross-linking chemotherapeutic agents in mismatch repair-defective cells in vitro and in xenografts

Together with tolerance to killing induced by methylating agents, loss of mismatch repair (MMR) has previously been found to be associated with hypersensitivity to the DNAcross‐linking agent 1‐(2‐chloroethyl)‐3‐cyclohexyl‐nitrosourea(CCNU) in several human tumor cell lines (Aquilina et al., 1998 ). Here, we have investigated whether MMR might act as an efficient repair pathway and provide protection against the clastogenicity induced by CCNU and whether the hypersensitivity of MMR‐defective cells is extended to other cross‐linking agents. An increase in cell killing and in the frequency of micronuclei was observed after CCNU exposure in 2 hPMS2‐defective clones (clones 6 and 7) compared with the parental HeLa cells. Introduction of a wild‐type copy of chromosome 7 in clone 7 led to re‐expression of the hPMS2 protein and brought survival and chromosomal damage upon CCNU exposure to parental levels. Our data indicate that MMR protects against the clastogenic damage induced by this drug. The hPMS2‐defective HeLa cells were also hypersensitive to killing by mitomycin C. Mitomycin C sensitivity was confirmed in an hMLH1‐defective clone derived from Raji cells and in msh2‐defective mouse embryo fibroblasts derived from knock‐out mice. hPMS2‐defective and parental HeLa cells were transplanted into nude mice, and the animals were treated with mitomycin C. While parental cell growth rate was unaffected, the growth of MMR‐defective tumor was significantly reduced. Our results indicate that the in vitro hypersensitivity to mitomycin C conferred by loss of MMR is paralleled in vivo and may have implications for the chemotherapy of MMR‐defective tumors. Int. J. Cancer 85:590–596, 2000.

A Human Cell-Based Assay to Evaluate the Effects of Alterations in the MLH1 Mismatch Repair Gene

We describe a new approach to investigate alterations in the human MLH1 mismatch repair (MMR) gene. This is based on complementation of the phenotype of a MLH1-defective subclone of the ovarian carcinoma A2780 cells by transfection of vectors encoding altered MLH1 proteins. Measurements of resistance (tolerance) to methylating agents, mutation rate at HPRT, microsatellite instability (MSI), and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. The approach was validated by transfecting cDNA of wild-type (WT) MLH1, cDNAs bearing two previously identified polymorphisms (I219V and I219L) and two with confirmed hereditary nonpolyposis colorectal cancer (HNPCC) syndrome mutations (G224D and G67R). A low-level expression of two MLH1 polymorphisms partially reversed methylation tolerance and the mutator phenotype, including MSI. Higher levels of I219V resulted in full restoration of these properties to WT. Increased expression of I129L did not fully complement the MLH1 defect, because there was a simultaneous escalation in the level of oxidative DNA damage. The findings confirmed the important relationship between deficient MMR and increased levels of oxidative DNA damage. Mutations from Italian HNPCC families (G224D, G67R, N635S, and K618A) were all ineffective at reversing the phenotype of the MLH1-defective A2780 cells. One (K618A) was identified as a low penetrance mutation based on clinical and genetic observations.

Processing of O6-methylguanine by mismatch correction in human cell extracts

Human cell extracts perform an aberrant form of DNA synthesis on methylated plasmids [1], which represents processing of O6-methylguanine (O6-meG). Here, we show that extracts of colorectal carcinoma cells with defects in the mismatch repair proteins that normally correct replication errors do not carry out this synthesis. hMSH2-defective LoVo cell extracts (hMSH for human MutS homologue) performed O6-meG-dependent DNA synthesis only after the addition of the purified hMutS alpha mismatch recognition complex. Processing of O6-meG by mismatch correction requires PCNA and therefore probably DNA polymerase delta and/or epsilon. Mismatch repair-defective cells withstand O6-meG in their DNA [2], making them tolerant to methylating agents. Methylation-tolerant HeLaMR clones, with a mutator phenotype and a defect in either mismatch recognition or correction in vitro, also performed little O6-meG-dependent DNA synthesis. Assays of pairwise combinations of tolerant and colorectal carcinoma cell extracts identified hMLH1 as the missing mismatch repair function in a group of tolerant clones. The absence of processing by extracts of methylation-tolerant cells provides the first biochemical evidence that lethality of DNA O6-meG derives from its interaction with mismatch repair.

Urinary and faecal mutagenicity in Sprague-Dawley rats dosed with the food mutagens quercetin and rutin

The natural flavonoid quercetin was administered to Sprague-Dawley rats by ip injection or gastric intubation of a single dose of 500, 1000 or 2000 mg/kg body weight. Mutagenicity assays with Salmonella typhimurium strain TA98 showed moderate mutagenic activity in the urines and faecal extracts but not in plasma samples from the treated animals. The mutagenic activity detected in the urines accounted for about 0.5% of the administered dose, irrespective of the route of administration and the dose level. Higher mutagenicity was demonstrated in faecal extracts. Rutin (quercetin-3-O-rutinoside) was administered by gavage and ip injection at 2000 mg/kg. Although the chemical was inactive as a mutagen in vitro, significant mutagenicity was detected in the urines and faecal extracts of the treated rats. Such activity was similar to that detected after administration of free quercetin in a dose some four times lower (by weight).

Spontaneous mutations ataprt locus in a mammalian cell line defective in mismatch recognition

Clone B is a CHO cell line that showns a moderate mutator phenotype as a consequence of a defect in mismatch recognition. To identify the classes of mutation that accumulate spontaneously in a functional gene, we isolated and sequenced 54 clone B spontaneous mutants at the adenine phosphoribosyltransferase gene. This spectrum was compared to 42 mutants collected in the parental cells. Rates of AT→TA transversions and frameshifts were strikingly increased in clone B (almost eight- and sixfold, respectively). Minor increases were also observed for GC→TA transversions and GC→AT transition rates. Frameshifts occurred in repeated sequences, and a large proportion were losses of 2 bases occurring in dinucleotide runs of a type similar to microsatellite sequences. AT→TA transversions clustered in regions of secondary structure and their formation might be explained by slippage-mediated mechanisms. These data indicate that an important function of mismatch recognition is in repair of extrahelical bases generated by misalignment during DNA replication.