Cytogenetic manifestations associated with the reversion, by gene amplification, at the HGPRT locus in V79 Chinese hamster cells

Some HGPRT spontaneous revertants were isolated from a mutant line (E2) of V79 Chinese hamster cells and phenotypically characterized. Dot-Blot hybridization with a 32P-labelled HGPRT probe revealed an increase in the number of HGPRT sequences in some of these revertants, suggesting the occurrence of gene amplification. Cytogenetic analysis performed in three of these revertants showed a characteristic abnormally banding region (ABR) on the elongated p arm of the X chromosome. In situ hybridization in one revertant (RHE2) showed that the amplified sequences reside on the p+ arm of the X chromosome in two different localizations. Because of the very probably clonal origin of the revertant, these features indicate that the amplified sequences might rearrange after their integration into the chromosome.     

Correlation between specific DNA-methylation products and mutation induction at the HGPRT locus in Chinese hamster ovary cells

Suspension cultures of Chinese hamster ovary (CHO) cells were exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) and assayed for mutation induction (6-thioguanine resistance) and for specific DNA adducts. DNA methylation at the 1-, 3- and 7-positions of adenine, the 3-, O6- and 7-positions of guanine, and phosphate was detected in cultures exposed to MMS, while MNU produced 3- and 7-methyladenine, 3-methylcytosine, 3-, O6- and 7-methylguanine, O4-methylthymidine and methylated phosphodiesters. When mutations induced by MMS and MNU were compared by linear correlation analysis with levels of each of these adducts, only O6-methylguanine displayed a strong correlation with mutations (r = 0.879, p less than 0.001). The relationship between O6-methylguanine and induced mutations in CHO cells is similar to that previously reported in CHO cells for O6-ethylguanine and mutations (Heflich et al., 1982) and indicates that alkylation-induced mutations at the HGPRT locus in CHO cells are primarily associated with O6-alkylguanine formation.     

Molecular analysis of mutation at the hprt locus of Chinese hamster V79 cells induced by ethyl methanesulphonate and mitomycin C

Mutation at the hprt locus of Chinese hamster V79 cells were induced by treatment with ethyl methanesulphonate (EMS), considered primarily a point mutagen and mitomycin C (MMC), a potent clastogen. EMS gave a dose-dependent induction of mutants while MMC induced a poor mutagenic response. Mutations were analysed using Southern and Northern blotting.Analysis of 9 EMS-induced and 4 spontaneous mutants yielded no detectable alterations in the hprt locus after digestion of DNA with 6 restriction enzymes. Mutants without detectable changes carried presumptive point mutations. In contrast, 4 out of 12 MMC-induced mutants had detectable alterations. 2 of these appeared to have lost the entire hprt gene while the other 2had prodable partial deletions. For these 4 deletion mutants no hprt mRNA was detected. 3 MMC-induced and 1 EMS-induced mutants had reduced levels of hprt mRNA. All the other mutants showed normal levels of hprt mRNA and the message detected was always of the correct size.It is suggested that the poor mutagenic response induced by MMC may be due to the lethal nature of large deletions involving both the hemizygous hprt locus and adjacent essential genes. This may lead to an underestimate of the mutagenicity of clastogenic agents such as MMC in the V79 HPRT mutation assay.     

Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.     

Mutagenicity evaluation of Schistosoma spp. extracts by the umu-test and V79/HGPRT gene mutation assay

Schistosomiasis has been suspected of being a risk factor for various types of cancers for sometime, e.g., bladder cancer, colorectal cancer and hepatic cancer. Among them, the etiological relationship between urinary schistosomiasis and bladder cancer is now widely accepted. However, mechanisms of the carcinogenesis are still unclear. Here, we tested the mutagenicity of the parasite extracts by the umu-test and hypoxanthine guanine phosphoribosyltransferase (HGPRT) gene mutation assay, which both overcome disadvantages of the Ames plate assay. Adult worm extracts and egg extracts of Schistosoma haematobium and Schistosoma mansoni were tested. Under our experimental conditions, neither worm nor egg extracts were shown to have any mutagenicity in both tests even in the presence of S9 mix. Our results suggest that there is very little possibility of immediate gene mutation due to the parasite-derived substances in schistosomiasis-related carcinogenesis.     

Isolation and characterization of spontaneously occurring mutations at the HPRT locus in V79 Chinese hamster cells

The aim of the present investigation was to screen for rare types of spontaneously occurring mutational events in order to provide information on the organization of the mammalian genome. For this purpose a hierarchical sequence of analyses is used with a first step utilizing a forward reverse mutation approach. The present paper deals with the characterization of 22 isolated mutants from 2 groups, 11 spontaneously appearing mutants and, in comparison, 11 ethyl methanesulfonate-induced mutants at the HPRT locus in V79 Chinese hamster cells, by means of reverse mutation analyses using selection with medium containing L-azaserine. Nine out of the 11 mutant clones of each group could be reverted either spontaneously or induced by treatments with ethyl nitrosourea (ENU), ICR191 or 5-azacytidine (5AC), which indicates that they were caused by point mutations. Two of the revertible mutant clones of spontaneous origin were found to be resistant to HAT but not HAsT medium. These 2 6TGrHATr mutants were the only mutants isolated which could be affected by 5AC with a significant increase in reversion frequency. Chromosome aberration analysis did not indicate any enhancement in aberration frequency in the X-chromosome by 5AC treatment. Studies on the mutagenicity at the OUA locus indicated that the 5AC- and ENU-induced mutation frequencies in these 2 mutants were comparable to the effects in the parent wild-type cell line. Their cellular incorporation of 3H-hypoxanthine was enhanced in the presence of aminopterin, but decreased with L-azaserine indicating that they were phosphoribosyl pyrophosphate (PRPP) mutants. On the basis of these results, it is hypothesized that reversion of these 2 6TGrHATr mutants may occur by a gene amplification mechanism and that this process may be facilitated by 5AC treatment.     

A comparison of induced mutation at homologous alleles of the tk locus in human cells.

Using a restriction fragment length polymorphism which can distinguish the two copies of the thymidine kinase (tk) gene in the TK6 human lymphoblastoid cell line, we have identified heterozygous subclones with alternate active alleles. Quantitative mutagenesis studies with X-rays revealed a markedly different response, depending on which homolog carried the active allele. The slopes of the dose-response curves differed by approximately 10-fold for mutation of the two alleles and this relationship held true for several independently isolated cell lines. Only one of the cell lines showed a different response to ethyl methanesulfonate. There were no differences among any of the cell lines at the X-linked hprt locus. Analyses of TK- mutants recovered from these cell lines indicated that the reduced yield of mutants from the one allele may be due, at least in part, to a lack of a specific class of TK- mutant, that is, the slow-growing mutants which have been associated with large-scale mutagenic events.     

Influence of hyperthermia on gamma-ray-induced mutation in V79 cells

Asynchronously growing V79 cells were assayed for mutation induction following exposure to hyperthermia either immediately before or after being irradiated with 60Co gamma rays. Hyperthermia exposures consisted of either 43.5 degrees C for 30 min or 45 degrees C for 10 min. Each of these heat treatments resulted in a survival level of 42%. For all sequences of combined treatment with hyperthermia and radiation, cell killing by gamma rays was enhanced. Mutation induction by gamma rays was enhanced when heat preceded gamma irradiation, but no increase was observed when heat was given after gamma exposures. Treatment at 45 degrees C for 10 min gave a higher yield in mutants at all gamma doses studied compared to treatment at 43.5 degrees C for 30 min. When heat-treated cells were incubated for different periods before being exposed to gamma rays, thermal enhancement of radiation killing was lost after 24 h. In contrast, only 5-6 h incubation was needed for loss of mutation induction enhancement.     

Effect of intercellular contact on DNA conformation, radiation-induced DNA damage, and mutation in Chinese hamster V79 cells

Chinese hamster V79 cells, when grown as small spheroids in suspension culture, are more resistant to killing by ionizing radiation than when grown as monolayers. We have attempted to determine whether this enhanced survival following irradiation is reflected in DNA damage and repair at the structural level (by measuring alkali-induced DNA unwinding rates from strand breaks) and at the functional level (by measuring resistance to forward mutation at the HGPRT locus). For a given dose of radiation, the unwinding of DNA in high salt/weak alkali was less complete for spheroid DNA than for monolayer DNA, and the rate of repair of radiation damage was faster in spheroid DNA. These differential responses were lost 8 hr after separation of spheroids into single cells, coinciding with loss of radioresistance measured by clonogenicity. In addition, spheroid cells showed fewer numbers of induced mutants per Gray, although, for a given level of survival, the mutation frequency for monolayers and spheroids was identical. These results suggest that conformational changes in DNA resulting from cell growth as spheroids might enhance repair of radiation-induced lesions.     

Methylglyoxal-induced mutation to 6-thioguanine resistance in V79 cells

Methylglyoxal, at concentrations ranging from 0.16 to 1.5 mM, caused a dose-dependent increase in the frequency of HGPRT-deficient mutants in V79 cells. Its mutagenic activity was reduced when V79 cells were co-cultured with freshly isolated rat hepatocytes.     

Lack of activity of the bacterial mutagen emodin in HGPRT and SCE assay with V79 Chinese hamster cells

Genotoxicity of emodin was studied in the Salmonella/microsome assay, the sister-chromatid exchange (SCE) assay and the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) forward mutation assay with V79 Chinese hamster cells. In the Salmonella/microsome assay, emodin was found to be positive in TA97, TA100 and TA1537 in the presence of liver homogenate. In TA1537 a weak direct mutagenicity was also observed. In both mammalian test systems, no genotoxicity was found either with or without metabolic activation.     

Gene amplification as a mechanism of reversion at the HPRT locus in V79 Chinese hamster cells

Spontaneous phenotypic revertants of hypoxanthine phosphoribosyl-transferase (HPRT) temperature-sensitive V79 Chinese hamster cells were selected by plating a temperature-sensitive mutant in HAT medium at 39 degrees C. The incidence of such revertants was approximately 2 X 10(-4) per cell. The majority of the revertants examined had increases of between three- and tenfold in their specific activity of the enzyme, and they were able to grow continuously in the presence of HAT medium at 39 degrees C. When the revertants were cultivated in the absence of HAT, they recovered their HAT-sensitive phenotype and their lowered level of HPRT. Three of the revertants were examined for their temperature inactivation profiles, and all were found to have profiles identical to the ts parent, and quite different from the V79 wild type. The kinetic properties of the cell lines were studied: the Km for both PRPP and hypoxanthine was significantly different in the temperature-sensitive cells but was not significantly altered in the revertants with respect to the ts mutants. A specific antibody to Chinese hamster brain HPRT was employed in immunoprecipitation experiments. By measuring the point at which the immunoprecipitation of the antibody to HPRT was overcome by increasing concentrations of cell supernatant, it was possible to estimate the relative amount of enzyme molecules in the cell lines. From these data, it could be concluded that the revertants overproduced an enzyme with the same immunological properties as the ts line. Southern blots of the Hind III restricted DNA from the ts mutant and two revertant cell lines were examined with an HPRT cDNA probe. This established that the HPRT gene was amplified twofold in one of the revertants, and threefold in the other. However, if the revertants were reintroduced into nonselective medium, the gene copy number declined to one. Finally, northern blots of RNA extracted from the various cell lines demonstrated that the HPRT mRNA was augmented 1.5-fold in one revertant and 1.4-fold in the other. Reintroduction into non-selective medium resulted in a decline in mRNA level for the second mutant, whereas the first mutant appeared to be stabilized. We conclude that gene amplification and concomitant amplification of messenger RNA and enzyme levels are mechanisms of phenotypic reversion at the HPRT locus in Chinese hamster cells.     

Influence of caffeine on X-ray-induced killing and mutation in V79 cells

Effects produced by caffeine on X-irradiated Chinese hamster V79 cells depended on the growth conditions of the cells. For exponentially growing cells, nontoxic concentrations of caffeine decreased the shoulder width from the survival curve, but the slope remained unchanged. The yield of mutants under the same conditions also remained unaffected. In case of density-inhibited cells, delaying trypsinization for 24 h after X irradiation increased the survival and decreased the yield of mutants. The presence of caffeine during this incubation period inhibited such recovery and significantly increased the yield of X-ray-induced mutants.     

Further studies on tetracycline-induced mutation in V79 Chinese hamster cells

The interaction between ultraviolet light and tetracycline in producing cell killing and mutation has been studied in V79 Chinese hamster cells. It has been established that these agents act independently of each other. Cycloheximide altered the response to tetracycline in the fractionation experiment: when cycloheximide was not present, fractionation of TC treatment resulted in a higher mutation yield but no change in survival level; in the presence of cycloheximide, however, mutation was greatly reduced but survival increased. The results were taken to indicate that for tetracycline action to take place, de novo protein synthesis during tetracycline treatment was necessary. Caffeine had no influence on tetracycline-induced lethality or mutagenicity. This observation was considered to suggest that tetracycline did not affect cellular repair processes.