Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells.

Removal of ultraviolet light induced cyclobutane pyrimidine dimers (CPD) from active and inactive genes was analyzed in cells derived from patients suffering from the hereditary disease Cockayne's syndrome (CS) using strand specific probes. The results indicate that the defect in CS cells affects two levels of repair of lesions in active genes. Firstly, CS cells are deficient in selective repair of the transcribed strand of active genes. In these cells the rate and efficiency of repair of CPD are equal for the transcribed and the nontranscribed strand of the active ADA and DHFR genes. In normal cells on the other hand, the transcribed strand of these genes is repaired faster than the nontranscribed strand. However, the nontranscribed strand is still repaired more efficiently than the inactive 754 gene and the gene coding for coagulation factor IX. Secondly, the repair level of active genes in CS cells exceeds that of inactive loci but is slower than the nontranscribed strand of active genes in normal cells. Our results support the model that CS cells lack a factor which is involved in targeting repair enzymes specifically towards DNA damage located in (potentially) active DNA.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Effect of B polA1- exrA- and recA-gene mutations on the reparation of single-strand DNA breaks induced by N-nitrosomethylurea]

The effect of different doses of N-methyl-N-nitrosourea (MNU) on a viability of bacterial cells with different defects in the systems of repair of UV-damages, and the MNU induction of single-strand DNA breaks (SS) were studied. The kinetics of both processes was investigated. There was a good correlation between the NMU sensitivity of bacterial cells and the number of SS in their DNAs. The most sensitive were the cells defective in DNA polymerase I. The optimal conditions for DNA repair in the strains under investigation were established. 90% of MNU-induced SS are repaired by DNA polymerase I and do not depend on protein synthesis. On the other hand, the exrA and recA dependent ways of SS repair depend on protein synthesis. The existence of an inducible recAexrA-dependent repair system of NMU-induced lesions in bacterial DNA is proposed.     

Methylation damage response in hematopoietic progenitor cells

The cellular response to methylation DNA damage was compared in multipotent CD34(+) hematopoietic stem cells and mature CD34(-) cells isolated from cord blood of the same donor. Cytofluorimetric analysis of freshly isolated cord blood cells indicated that both cell types were in the G0/G1 phase of the cell cycle. Quantitative RT-PCR identified a general trend towards high expression of several DNA repair genes in CD34(+) cells compared to their terminally differentiated CD34(-) counterparts. The overexpressed genes included members of the mismatch repair (MMR) (MSH2, MSH6, MLH1, PMS2), base excision repair (AAG, APEX), DNA damage reversal (O(6)-methylguanine DNA methyltransferase) (MGMT), and DNA double strand breaks repair pathways. These differences in gene expression were not apparent in CD34(+) and CD34(-) cells obtained following expansion of CD34(+) cells in a medium containing early acting cytokines. Early progenitor CD34(+) and early precursor CD34(-) cells form the two populations isolated under these experimental conditions, and both contain a significant proportion of cycling cells. The methylating agent N-methyl-N-nitrosourea (MNU) induced similar levels of apoptosis in these cycling CD34(+) and CD34(-) cells. Cytotoxicity required the presence of the MGMT inhibitor O(6)-benzylguanine and the timing of MNU cell death (48 and 72h) was similar in CD34(+) and CD34(-) cells. These data indicate that cycling CD34(+) and CD34(-) cells are equally sensitive to methylation damage. MGMT provides significant protection against MNU toxicity and MGMT and MMR play the expected roles in the MNU sensitivity of these cells.     

REV3 is required for spontaneous but not methylation damage-induced mutagenesis of Saccharomyces cerevisiae cells lacking O6-methylguanine DNA methyltransferase

O6-methylguanine (O6-MeG) DNA methyltransferase (MTase) removes the methyl group from a DNA lesion and directly restores DNA structure. It has been shown previously that bacterial and yeast cells lacking such MTase activity are not only sensitive to killing and mutagenesis by DNA methylating agents, but also exhibit an increased spontaneous mutation rate. In order to understand molecular mechanisms of endogenous DNA alkylation damage and its effects on mutagenesis, we determined the spontaneous mutational spectra of the SUP4-o gene in various Saccharomyces cerevisiae strains. To our surprise, the mgt1 mutant deficient in DNA repair MTase activity exhibited a significant increase in G:C-->C:G transversions instead of the expected G:C-->A:T transition. Its mutational distribution strongly resembles that of the rad52 mutant defective in DNA recombinational repair. The rad52 mutational spectrum has been shown to be dependent on a mutagenesis pathway mediated by REV3. We demonstrate here that the mgt1 mutational spectrum is also REV3-dependent and that the rev3 deletion offsets the increase of the spontaneous mutation rate seen in the mgt1 strains. These results indicate that the eukaryotic mutagenesis pathway is directly involved in cellular processing of endogenous DNA alkylation damage possibly by the translesion bypass of lesions at the cost of G:C-->C:G transversion mutations. However, the rev3 deletion does not affect methylation damage-induced killing and mutagenesis of the mgt1 mutant, suggesting that endogenous alkyl lesions may be different from O6-MeG.     

Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.     

Nuclear translocation of mismatch repair proteins MSH2 and MSH6 as a response of cells to alkylating agents

Mammalian mismatch repair has been implicated in mismatch correction, the prevention of mutagenesis and cancer, and the induction of genotoxicity and apoptosis. Here, we show that treatment of cells specifically with agents inducing O(6)-methylguanine in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, elevates the level of MSH2 and MSH6 and increases GT mismatch binding activity in the nucleus. This inducible response occurs immediately after alkylation, is long-lasting and dose-dependent, and results from translocation of the preformed MutSalpha complex (composed of MSH2 and MSH6) from the cytoplasm into the nucleus. It is not caused by an increase in MSH2 gene activity. Cells expressing the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O(6)-methylguanine, showed no translocation of MutSalpha, whereas inhibition of MGMT by O(6)-benzylguanine provoked the translocation. The results demonstrate that O(6)-methylguanine lesions are involved in triggering nuclear accumulation of MSH2 and MSH6. The finding that treatment of cells with O(6)-methylguanine-generating mutagens results in an increase of MutSalpha and GT binding activity in the nucleus indicates a novel type of genotoxic stress response.     

Degradation and repair of rat hepatoma cell DNA following exposure to gamma rays and methylnitrosourea]

Single-strand breaks induced in DNA of ascitic hepatoma cells by gamma-rays and N-methyl-N-nitrosourea (MNU), resp., may be effectively repaired. Double-strand breaks of DNA from MNU-treated hepatoma cells are also effectively repairable in vivo. Only a small part of double-strand breaks induced by gamma-rays in DNA of these cells is repaired in the postradiation period. The combined action of gamma-rays and MNU on the hepatoma cells causes a complete inhibition of repair of DNA and its further degradation. Under these conditions, inhibition of the repair of DNA synthesis and repression of DNA polymerase I activity is observed.     

Repair of oxidative DNA base lesions induced by fluorescent light is defective in xeroderma pigmentosum group A cells.

Fluorescent light (FL) has been shown to generate free radicals within cells, however, the specific chemical nature of DNA damage induced by FL has not previously been determined. Using gas chromatography/isotope dilution mass spectrometry, we have detected induction of the oxidative DNA lesions 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) in cultured cells irradiated with FL. We followed the repair of these lesions in normal and xeroderma pigmentosum group A (XP-A) cells. 5-OH-Cyt and FapyGua were repaired efficiently in normal cells within 6 h following FL exposure. XP-A cells were unable to repair these oxidative DNA base lesions. Additionally, to compare the repair of oxidative lesions induced by various sources, in vitro repair studies were performed using plasmid DNA damaged by FL, gamma-irradiation or OsO(4)treatment. Whole cell extracts from normal cells repaired damaged substrates efficiently, whereas there was little repair in XP-A extracts. Our data demon-strate defective repair of oxidative DNA base lesions in XP-A cells in vivo and in vitro.     

Quantitating the frequency of initiation and cH-ras mutation in in situ N-methyl-N-nitrosourea-exposed rat mammary gland

Mammary carcinogenesis is a multistep process consisting minimally of initiation and promotion/progression stages. The rate-limiting stage in the carcinogenesis process is undetermined but can in part be addressed by estimating the frequency of initiation, a heritable early event. Here, we use an in vivo limiting dilution transplantation assay to estimate initiation frequency in a rat mammary epithelial stem-like cell population that was exposed in situ to 50 mg/kg N-methyl-N-nitrosourea (NMU) administered i.v. We estimate that this dose resulted in the killing of 65% of exposed mammary cells. Known numbers of cells surviving NMU exposure were grafted into fat-pads of recipient rats in which the cells grew and differentiated into structurally and functionally normal mammary glands. Recipient rats were hormonally manipulated to provide maximal promotion of initiated cells. Mammary carcinomas developing at graft sites were quantitated over a 2-year period. Based on these results, we estimate that at least 1 surviving NMU-exposed mammary cell in 7,200 was initiated. Seventeen % of these graft site carcinomas had an activated H-ras oncogene with a G to A mutation in codon 12. This suggests that at least 1 mammary cell in 43,000 was mutated in this fashion by in situ exposure to NMU. These data suggest that cH-ras represents approximately 1 of 5 of the initiation events produced by NMU exposure of rat mammary glands.     

Characterization of the major DNA repair methyltransferase activity in unadapted Escherichia coli and identification of a similar activity in Salmonella typhimurium.

Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.     

Carcinogen-induced early molecular events and its implication in the initiation of chemical hepatocarcinogenesis in rats: chemopreventive role of vanadium on this process

Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molecular events in the initiation of chemical carcinogenesis and modulation of such events by chemopreventive agents could be an important step in limiting neoplastic transformation in vivo. Vanadium, a dietary micronutrient has been found to be effective in several types of cancers both in vivo and in vitro and also possesses profound anticarcinogenicity against rat models of mammary, colon and hepatocarcinogenesis. Presently, we report the chemopreventive potential of vanadium on diethylnitrosamine (DEN)-induced early DNA damages in rat liver. Hepatocarcinogenesis was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal (i.p.) injection of DEN (200 mg/kg body weight) at week 4. There was a significant induction of tissue-specific ethylguanines, steady elevation of modified DNA bases 8-hydroxy-2'-deoxyguanosines (8-OHdGs) (P<0.0001; 89.93%) along with substantial increment of the extent of single-strand breaks (SSBs) (P<0.0001) following DEN exposure. Supplementation of 0.5 ppm of vanadium throughout the experiment abated the formations of O(6)-ethylguanines and 7-ethylguanines (P<0.0001; 48.71% and 67.54% respectively), 8-OHdGs (P<0.0001; 81.37%), length:width (L:W) of DNA mass (P<0.01; 62.12%) and the mean frequency of tailed DNA (P<0.001; 53.58%), and hepatic nodulogenesis in preneoplastic rat liver. The study indicates that 0.5 ppm vanadium is potentially and optimally effective, as derived from dose-response studies, in limiting early molecular events and preneoplastic lesions, thereby modulating the initiation stage of hepatocarcinogenesis. Vanadium is chemopreventive against DEN-induced genotoxicity and resulting hepatocellular transformation in rats.     

G1 cell cycle regulatory proteins in chemically-induced rat mammary adenocarcinomas in vivo and tumor promotion-sensitive, -resistant and transformed mouse epidermal cells in vitro

Tumor promotion is characterized by selective proliferation of initiated cells resulting in their clonal expansion. Cyclin Dl is frequently upregulated in this process, but its expression does not necessarily correlate positively with cyclin A. In the present article, expression of G1 cell cycle regulatory proteins was systematically analyzed using two models of carcinogenesis: (a) N-methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas and normal rat mammary epithelial cells in vivo and (b) promotion-sensitive, -resistant, and transformed JB6 mouse epidermal cells in vitro. The results of this analysis revealed that p27Kipl negatively correlated with cyclin Dl. In addition, there were two types of correlations between p27Kipl and cyclin A. First, p27Kipl negatively correlated with cyclin A (type-l correlation). This scenario was observed in normal rat mammary epithelial cells in vivo and promotion-sensitive (P+) JB6 mouse epidermal cells, stimulated with phorbol ester (TPA) in vitro. Second, p27Kipl positively correlated with cyclin A (type-ll correlation). This correlation was observed in MNU-induced rat mammary adenocarcinomas in vivo and TPA-stimulated (P+) JB6 cells, treated with retinoic acid in vitro.     

Alkylation damage in DNA and RNA--repair mechanisms and medical significance

Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase (MGMT or AGT) that repairs the base in one step. However, the genotoxicity and cytotoxicity of O(6)-meG is mainly due to recognition of O(6)-meG/T (or C) mispairs by the mismatch repair system (MMR) and induction of futile repair cycles, eventually resulting in cytotoxic double-strand breaks. Therefore, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents are complex and require complex repair mechanisms. Thus, primary chloroethyl adducts at O(6)-G are repaired by AGT, while the secondary highly cytotoxic interstrand cross-links (ICLs) require nucleotide excision repair factors (e.g. XPF-ERCC1) for incision and homologous recombination to complete repair. Recently, Escherichia coli protein AlkB and human homologues were shown to be oxidative demethylases that repair cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues. Numerous AlkB homologues are found in viruses, bacteria and eukaryotes, including eight human homologues (hABH1-8). These have distinct locations in subcellular compartments and their functions are only starting to become understood. Surprisingly, AlkB and hABH3 also repair RNA. An evaluation of the biological effects of environmental mutagens, as well as understanding the mechanism of action and resistance to alkylating drugs require a detailed understanding of DNA repair processes.     

Demethylation of 3-methylthymine in DNA by bacterial and human DNA dioxygenases

Rare DNA lesions that are chemically stable and refractory to repair may add disproportionately to the accumulation of mutations in long lived cells. 3-Methylthymine is a minor lesion that is induced by DNA-methylating agents and for which no repair process has been described previously. Here we demonstrate that this lesion can be directly demethylated in vitro by bacterial and human DNA dioxygenases. The Escherichia coli AlkB and human ABH3 proteins repaired 3-methylthymine in both single-stranded and double-stranded polydeoxynucleotides, whereas the human ABH2 protein preferred a duplex substrate. Thus, the known substrates of these enzymes now include 3-methylthymine in DNA, as well as 1-methyladenine and 3-methylcytosine, which all have structurally similar sites of alkylation. Repair of 3-methylthymine by AlkB and ABH3 was optimal at pH 6, but inefficient. At physiological pH, 3-methylthymine, which is a minor methylated lesion, was more slowly repaired than the major lesion generated in single-stranded DNA, 3-methylcytosine. Our data suggest that 3-methylthymine residues in DNA will be repaired inefficiently in vivo and therefore may occur at a low steady-state level, but the residues should not gradually accumulate to high levels in long lived cells.