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DNA/RNA synthesis precursors are especially vulnerable to damage induced by reactive oxygen species occurring following oxidative stress. Guanosine triphosphates are the prevalent oxidized nucleotides, which can be misincorporated during replication, leading to mutations and cell death. Here, we present a novel method based on micro-Raman spectroscopy, combined with ab initiocalculations, for the identification, detection, and quantification of oxidized nucleotides at low concentration. We also show that the Raman signature in the terahertz spectral range (<100 cm–1) contains information on the intermolecular assembly of guanine in tetrads, which allows us to further boost the oxidative damage detection limit. Eventually, we provide evidence that similar analyses can be carried out on samples in very small volumes at very low concentrations by exploiting the high sensitivity of surface-enhanced Raman scattering combined with properly designed superhydrophobic substrates. These results pave the way for employing such advanced spectroscopic methods for quantitatively sensing the oxidative damage of nucleotides in the cell.

The comet assay is a versatile method for measuring DNA strand breaks in individual cells. It can also be applied to cells isolated from treated animals. In this review, we highlight advantages and limitations of this in vivocomet assay in a regulatory context. Modified versions of the standard protocol detect oxidized DNA bases and may be used to reveal sites of DNA base loss, DNA interstrand crosslinks, and the extent of DNA damage induced indirectly by reactive oxygen species elicited by chemical-induced oxidative stress. The assay is, however, at best semi-quantitative, and we discuss possible approaches to improving DNA damage quantitation and highlight the necessity of optimizing protocol standardization to enhance the comparability of results between laboratories. As a genotoxicity test in vivo, the in vivocomet assay has the advantage over the better established micronucleus erythrocyte test that it can be applied to any organ, including those that are specific targets of chemical carcinogens or those that are the first sites of contact of ingested or inhaled mutagens. We illustrate this by examples of its use in risk assessment for the food contaminants ochratoxin and furan. We suggest that improved quantitation is required to reveal the full potential of the comet assay and enhance its role in the battery of in vivoapproaches to characterize the mechanisms of toxicity and carcinogenicity of chemicals and to aid the determination of safe human exposure limits.

To determine prevalence, spectrum and genotype–phenotype correlations of MUTYHvariants in Italian patients with suspected MAP (MUTYH-associated polyposis), a retrospective analysis was conducted to identify patients who had undergone MUTYHgenetic testing from September 2002 to February 2014. Results of genetic testing and patient clinical characteristics were collected (gender, number of polyps, age at polyp diagnosis, presence of colorectal cancer (CRC) and/or other cancers, family data). The presence of large rearrangements of the MUTYHgene was evaluated by Multiplex Ligation-dependent Probe Amplification analysis. In all, 299 patients with colorectal neoplasia were evaluated: 61.2% were males, the median age at polyps or cancer diagnosis was 50 years (16–80 years), 65.2% had <100 polyps and 51.8% had CRC. A total of 36 different MUTYHvariants were identified: 13 (36.1%) were classified as pathogenetic, whereas 23 (63.9%) were variants of unknown significance (VUS). Two pathogenetic variants were observed in 78 patients (26.1%). A large homozygous deletion of exon 15 was found in one patient (<1.0%). MAP patients were younger than those with negative MUTYHtesting at polyps diagnosis (P<0.0001) and at first cancer diagnosis (P=0.007). MAP patients carrying the p.Glu480del variant presented with a younger age at polyp diagnosis as compared to patients carrying p.Gly396Asp and p.Tyr179Cys variants. A high heterogeneity of MUTYHvariants and a high rate of VUS were identified in a cohort of Italian patients with suspected MAP. Genotype–phenotype analysis suggests that the p.Glu480del variant is associated with a severe phenotype.

MUTYH, a human ortholog of MutY, is a post-replicative DNA glycosylase, highly conserved throughout evolution, involved in the correction of mismatches resulting from a faulty replication of the oxidized base 8-hydroxyguanine (8-oxodG). In particular removal of adenine from A:8-oxodG mispairs by MUTYH activity is followed by error-free base excision repair (BER) events, leading to the formation of C:8-oxodG base pairs. These are the substrate of another BER enzyme, the OGG1 DNA glycosylase, which then removes 8-oxodG from DNA. Thus the combined action of OGG1 and MUTYH prevents oxidative damage-induced mutations, i.e.GC-TA transversions. Germline mutations in MUTYHare associated with a recessively heritable colorectal polyposis, now referred to as MUTYH-associated polyposis (MAP). Here we will review the phenotype(s) associated with MUTYH inactivation from bacteria to mammals, the structure of the MUTYH protein, the molecular mechanisms of its enzymatic activity and the functional characterization of MUTYHvariants. The relevance of these results will be discussed to define the role of specific human mutations in colorectal cancer risk together with the possible role of MUTYH inactivation in sporadic cancer.

Retinitis pigmentosa (RP) is a genetically heterogenous group of inherited retinal degenerative diseases resulting from photoreceptor cell death and affecting 1 million persons globally. Although oxidative stress has been implicated in the pathogenesis of RP, the mechanisms by which oxidative stress mediates photoreceptor cell death are largely unknown. Here, we show that oxidation of nucleic acids is a key component in the initiation of death-signaling pathways in rd10mice, a model of RP. Accumulation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) increased in photoreceptor cells, and especially within their nuclei, in rd10mice as well as in Royal College of Surgeons rats, another model of RP caused by different genetic mutations. Vitreous samples from humans with RP contained higher levels of 8-oxo-dG excreted than samples from nondegenerative controls. Transgenic overexpression of human MutT homolog-1, which hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, significantly attenuated 8-oxo-dG accumulation in nuclear DNA and photoreceptor cell death in rd10mice, in addition to suppressing DNA single-strand break formation, poly(ADP-ribose) polymerase activation, and nuclear translocation of apoptosis-inducing factor. These findings indicate that oxidative DNA damage is an important process for the triggering of photoreceptor cell death in rd10mice and suggest that stimulation of DNA repair enzymes may be a novel therapeutic approach to attenuate photoreceptor cell loss in RP.

Tumor resistance to cytotoxic chemotherapy drugs and their toxicity to normal cells are major clinical obstacles to anticancer therapy effectiveness. Alterations in various DNA repair pathways play a key role in the development of both mechanisms of drug resistance and toxicity. Since deregulation of the DNA damage response and alterations in DNA repair pathways are relatively common in human cancer, the knowledge of these alterations in cancer cells would be an important predictive factor for the clinical response to chemotherapy and a useful guide in designing an appropriate therapeutic strategy. This review is focused on the mismatch repair (MMR) pathway and the O6-methylguanine-DNA-methyltransferase (MGMT) repair protein. In particular, we examine how inactivation of these DNA repair mechanisms might affect the response of tumor cells to chemotherapy, with a special emphasis on agents inducing methylation and oxidative DNA damage and interstrand DNA cross-links (ICLs). In addition, we provide novel experimental evidence indicating that MMR is required for efficient repair of ICLs via stabilization of RAD51 containing repair intermediates. Finally, we discuss possible emerging therapeutical strategies for treating MMR-defective tumors.

Due to major developments in genetics over the past decade, molecular biology tests are serving promising tools in early diagnosis and follow-up of cancer patients. Recent epidemiological studies revealed that the risk for each individual to develop cancer is closely linked to his/her own genetic potentialities. Some populations that are defective in DNA repair processes, for example in Xeroderma pigmentosum or in the Lynch syndrome, are particularly prone to cancer due to the accumulation of mutations within the genome. Such populations would benefit from the development of tests aimed at identifying people who are particularly at risk. Here, we review some data suggesting that the inactivation of mismatch repair is often found in endometrial cancer and we discuss molecular-based strategies that would help to identify the affected individuals in families with cases of glandular malignancies.

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. © 2001 Wiley-Liss, Inc.

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. © 2000 Wiley-Liss, Inc.

Mutagenesis and neoplastic transformation assays on mammalian cells in culture have been extensively used for quantitative estimates of the activity of carcinogens, in spite of the limitations that such in vitro systems have when compared with in vivosystems for tumor induction. In order to assess the validity of these correlations, a series of studies was undertaken in our laboratory with the BALB/3T3 Cl A31-1-1 mouse embryo cell line. Different carcinogens were found to induce dose-dependent frequencies of transformation, including the direct-acting alkylating agent N-methyI-N'-nitro-N-nitrosoguanidine (MNNG) and carcinogens that were metabolically activated by these cells through different pathways (benzo[a]pyrene, 3-methylcholanthrene, aflatoxin B1, and benzidine). Their respective level of activity on a molar basis was different from that obtained in standard Salmonella+ S9 mutagenesis tests. Studies currently underway indicate the possibility of lowering the serum content in the medium considerably, thereby reducing a major variable in the assay. Methods were established for the induction of ouabain-resistant (ouar) mutants in these cells. Studies were conducted by applying 30-min MNNG exposures to cells that were synchronized by serum deprivation followed by serum-induced release from growth block. While maximal induction of mutants occurred in the S phase, the transformation frequency remained constant for treatments in G1and early or late S. In subsequent studies, cytotoxicity, alkali-labile DNA lesions, ouarmutations, and neoplastic transformation were analyzed concurrently in this cell line after cells were exposed to two concentrations of MNNG and the exposures were protracted for different time periods (30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). A marked temporal dissociation was found in the exposure times required to induce maximal frequencies of mutations and of transformation. Cytotoxicity increased for periods up to 100-200 min; mutations reached a maximal induction level after a much shorter exposure time (30-60 min); DNA damage detected by alkaline elution was already maximal by 30 min. Transformation frequencies, however, reached maximal levels only after exposure periods 1-3 hr longer than those required for maximal mutation. The ratio of transformation to ouarmutation frequencies was 3.7 for short treatment times (30-60 min), but it increased to more than 20 for exposure times of 240 min or longer. These studies support the hypothesis that a single gene mutational event is not sufficient to account for the expression of neoplastic transformation.

We have studied whether spontaneous intrachromosomal recombination is altered in methylation tolerant human cells with a defect in mismatch repair. Somatic recombination was analysed in HeLaMR cells containing the vector pTPSN, which carries two copies of the gene for hygromycin resistance. The hygromycin genes are both inactivated by an inserted HindIII linker but hygromycin-resistant clones can arise by recombination. The spontaneous rate of recombination in a clone of HeLaMR cells containing a single integrated copy of pTPSN (HeLaG1) was 3.1 × 10−6/cell per generation. Two methylation tolerant variants from HeLaG1 cells (clone 12 and clone 15) were isolated by exposure to MNNG. Clone 12 cells exhibited a 16-fold increase in spontaneous mutation rate at the HPRT gene and extensive microsatellite instability at both mono- and dinucleotide repeats. Microsatellite instability limited to mononucleotide repeats was found in clone 15, whereas the mutation rate at HPRT was not significantly affected. A mismatch binding defect in extracts of clone 15 could be complemented by exogenous GTBP but not by purified hMSH2 protein. These data suggest that clone 15 is defective in GTBP. Extracts of clone 12 were unable to correct a single C:T mispair and complementation by extracts of human colorectal carcinoma cells with known deficiencies in mismatch repair indicated a defect in hMutLα. Western blotting with antibodies against different human mismatch repair proteins showed that clone 12 cells did not express hPMS2 protein, but expression of hMLH1, hMSH2 and GTBP appeared normal. The spontaneous recombination rate of clone 12 was 19-fold higher than the parental HeLaG1 cells, whereas no increase was observed in clone 15. Analysis of individual recombinants showed that hygromycin resistance arose exclusively by gene conversion. Our data indicate that mismatch correction regulates somatic recombination in human cells.

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.

IEC-18 cells, a cell line derived from the ileum of rat intestine, have the characteristics of normal cells since they have a contact inhibited cell growth, do not form colonies in soft agar and are not tumorigenic when injected in nude mice. IEC-18 cells were transfected with nuclear oncogenes, c-myc, v-myc and SV40 T antigen in order to obtain immortal cell lines. Independent clones were isolated and characterized for the growth properties. Expression of v-myc altered the morphology of the cells and shortened the doubling time. A slow growth together with a low cloning efficiency was associated with the expression of SV40 T antigen. No changes either in growth or in morphology were observed in c-myc-expressing IEC-18 cells. Expression of these nuclear oncogenes did not result in the neoplastic transformation of the IEC-18 cells, since none of the clones lost the anchorage dependence or were able to form tumors in vivo. The c-myc-containing IEC-18 cells were unable to secrete in the growth medium TGF a and exposure to TGF ß inhibited the growth rate by 30%. All these observations are consistent with the conclusion that the expression of nuclear oncogenes does not lead to the neoplastic transformation of these cells.

Human cell lines resistant to N-methyl-N-nitrosourea (MNU) were previously assigned to two complementation groups. Members of group I are defective in mismatch correction [S. Ceccotti, G. Aquilina, P. Macpherson, M. Yamada, P. Karran, M. Bignami, Processing of O6-methylguanine by mismatch correction in human cell extracts. Current Biol. 6 (1996) 1528–1531]. To identify the mechanism responsible for the less pronounced phenotype of the second complementation group, we characterized the persistence of MNU-induced O6-methylguanine (O6-meGua) and mutation induction at the hypoxanthine guanine phosphoribosyl-transferase (HPRT) locus. Group II clones are unable to repair the premutagenic base O6-meGua and are as mutable by MNU as group I clones and the parental HeLaMR cells. MNU-induced SCE were undetectable in group I clones and drastically reduced in group II in comparison with the parental cells. These observations are consistent with a defective processing of DNA methylation damage by members of both groups. Group II clones exhibit a moderate spontaneous mutator phenotype at the HPRTgene but significant instability at mononucleotide repeat microsatellites. Introduction of a single human chromosome 2 (but not of chromosome 3 or 7) into group II cells partially reverts both MNU resistance and the increased spontaneous mutation rate. The properties of group II variants are consistent with methylation tolerance and a partially defective mismatch repair. We propose that members of group II are defective in the chromosome 2-based mismatch correction gene GTBP/hMSH6.

On the assumption that a cluster of five his genes (eight cistrons) in S. coelicolor corresponds to an operon, a genetic analysis of a constitutive mutant was carried out. This strain has a multi-site mutation localized at the (conventional) right end of the his cluster and is derepressed for at least two enzymes coded by genes of the cluster. The study of suitable heterozygous clones (heteroclones), showed the mutation to be cis-dominant, suggesting that the operator region is affected. Most likely the strain has a deletion connecting the his operon to an adjacent amm (ammonium requirement) operon as demonstrated by its inability to utilize nitrate as nitrogen source and to complement or recombine with two his cistrons and a near-by amm mutation.

Up to 6% of N-methyl-N′-nitro-N-nitrosoguanidine-induced back mutations in the hisAlocus of Streptomyces coelicolorwere forward mutations (comutations) in another closely linked hislocus.