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PubMed1982

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts.

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on cell growth status. Exponentially growing cells treated with 10(-3)-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 x 10(-4). However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10(-3)-0.6 LF/ml), the survival decreased to 2 x 10(-6) and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10(-3) mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.

Cell Division↗

PubMed1982

Genetic control of budding-cell resistance in the diploid yeast Saccharomyces cerevisiae exposed to gamma-radiation.

The gamma-radiation response of stationary and budding cells of wild-type diploid strains (RAD) and radiation-sensitive strains rad2, 6, 9, 18, 50-55, 57 and rec4 was studied. As compared with the wild-type strains, mutants generally showed enhanced sensitivity in both stages of the cell cycle. Budding-cell resistance was totally absent from rad50-55 strains. Mutants rad6, 9 and 18 showed some degree of budding-cell resistance. The response of rad2 and rec4 strains was identical with that of the corresponding wild-type strains. These results suggest that the pathway dependent upon the expression of RAD50-55 loci functions more efficiently in budding cells compared with the pathway dependent on RAD2 and RAD6, 9 and 18 loci. Recombination between sister chromatids appears to play an important role in budding-cell resistance, and this process is under the control of the RAD52 repair pathway. The relationship between the repair pathways associated with budding-cell resistance and post-irradiation cellular recovery (LHR) is discussed.

Cell Cycle↗

PubMed1982

Mutation assay at the thymidine kinase locus in diploid human lymphoblasts.

A thymidine kinase heterozygote was isolated from a diploid human lymphoblast line which forms colonies with high efficiency in microtiter dishes. We show that this cell line, called TK6, can be mutated from a TK+/- to TK-/- state by diverse mutagens, including ethyl methanesulfonate, butyl methanesulfonate, nitrosomethylurea, UV light, ICR-191, 4-nitroquinoline oxide, fluorodeoxyuridine, benzo[a]pyrene and aflatoxin B1. We report here the experiments required to demonstrate the applicability of this new line in quantitative assays of mutation in human cells. Mitotic recombination between the centromere and the tk locus could not be induced by either dimethylsulfoxide or phorbol-12-myristate-13-acetate.

Cell Line↗

PubMedDate not supplied

Effect of growth rate on phenotypic expression of 6-thioguanine resistance in human diploid fibroblasts.

The influence of growth rate on the kinetics of phenotypic expression of 6-thioguanine resistance has been investigated by establishing 3 protocols for the maintenance of mutagenized stock cultures of human diploid fibroblasts. These protocols differed in the frequency with which subculture was performed during the phenotypic expression period. The resultant growth kinetics varied widely from continual exponential proliferation to density-inhibited growth for much of the expression period. With all of the subculture protocols, a maximum mutation frequency was reached by 9 days after exposure of confluent cultures to 6 J/m2 of 254 nm UV light. The maximum frequency was similar in all experimental groups and remained stable up to 17 days after irradiation, the last time point tested. These results indicate that continual proliferation is not required for optimal expression of 6-thioguanine resistance.

Cell Division↗

PubMed1983

Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts.

We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts. Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation. In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35 000 cells per 100-mm petri dish. The induced mutation frequency increased linearly with dose over the range of 3-9 J/m2; the D0 of the survival curve was 4.2 J/m2. Delaying subculture to low density for 1.5-24 h after irradiation produced unexpected alterations in induced mutation frequencies. An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3 h. This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6 h as compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels. These data suggest that the repair of potentially mutagenic damage is a complex phenomenon which can lead to an increase or decrease in mutation frequency as a function of holding time. Although the decline in mutation frequency observed following longer holding intervals is consistent with the notion of an error-free process, we hypothesize that the increased mutation frequency produced by a short holding period reflects the existence of a cell-mediated process which enhances the mutagenic potential of at least some UV-induced DNA photoproducts.

DNA Repair↗

PubMed1983

Ultraviolet light-induced mutation of diploid human lymphoblasts.

Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited 3H-TdR uptake into DNA. A shoulder of about 6 J/m2 and a D0 of 6 J/m2 was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m2; above 6 J/m2, complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m2 caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation. Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 J/m2), moderately toxic fluences (8.8 J/m2) and toxic fluences (17.6 J/m2). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show an enhanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation.

Cell Line↗

PubMed1984

Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts.

The mutagenic specificities of ethylnitrosourea (ENU), X-rays (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene (BPDE), ICR-191, and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) were analyzed and compared in diploid human fibroblasts and Salmonella typhimurium. In the human fibroblasts, we compared the frequency of diphtheria toxin (DT)-resistant mutants, presumably induced in the gene coding for elongation factor-2, with the frequency of 6-thioguanine (TG) resistance induced by mutations in the gene coding for hypoxanthine(guanine)phosphoribosyltransferase (HPRT). Recovery of DT-resistant (DTr) cells requires that the mutant EF-2 retain the ability to carry on protein synthesis since the normal EF-2 will be inactivated by DT selection. Therefore, the DTr mutation cannot involve major changes in the gene. In contrast, cells can acquire TG resistance by any mechanism which eliminates HPRT activity, e.g., base substitution, frameshift, deletion, loss of chromosomes. Each agent was assessed by calculating the ratio of the slopes of the dose-response plots (induced variant frequency as a function of dose of the agent used) for the two markers (DTr/TGr variants.). In S. typhimurium we examined the reversion frequency in four histidine-requiring strains bearing forward mutations of the frameshift (TA1538, TA98) or missense (TA1535, TA100) type. ENU, which was predominantly a base substitution mutagen in the bacteria, gave a ratio of DTr to TGr variants of 1.5. As expected of an agent inducing gross chromosomal changes, X-rays induced no revertants in bacteria and in human cells gave a ratio of 0.1. ICR-191 which was predominantly a frameshift mutagen in bacteria gave a ratio of 0.15. In the set of bacterial strains containing the plasmid pKM101, BPDE reverted both frameshift and base substitution mutations. It did not cause reversions in the other set of strains. In human cells BPDE gave a response similar to ENU, i.e., a ratio of DTr/TGr variants of 1.5. As reported by others, N-AcO-AAF was predominantly a frameshift mutagen in bacteria. However, in the human cells it gave a ratio of DTr/TGr variants of 1.5, similar to ENU and BPDE. These results suggest that in human cells, BPDE and N-AcO-AAF, like ENU, yield predominantly base substitutions, while ICR-191 and X-rays largely produce mutations by mechanisms which result in more extensive alterations in the gene.

Carcinogens↗

PubMed1985

Confluent holding leads to a transient enhancement in mutagenesis in UV-light-irradiated xeroderma pigmentosum, Gardner's syndrome and normal human diploid fibroblasts.

The influence of confluent holding periods of 0-24 h of UV-light-induced mutagenesis has been investigated in several human cell strains including xeroderma pigmentosum complementation group A (XPA), Gardner's syndrome (GS) and normal human diploid fibroblasts (NHDF). These cells strains were chosen for the variety of their responses in cytotoxicity experiments. Confluent cultures of NHDF exposed to UV light exhibited a time-dependent increase in survival when subculture was delayed up to 24 h after irradiation. GS and XPA fibroblasts showed no such increase. In dose-response experiments, GS cells from 3 kindreds were moderately hypersensitive to cell killing by UV light whereas XPA cells were strongly hypersensitive. Confluent cultures of GS cells were slightly and XPA markedly hypermutable to 6-thioguanine resistance (6-TGR) when the cells were subcultured immediately after UV exposure. When allowed confluent holding periods of 1.5-24 h, GS, XPA and NHDF all exhibited a transient enhancement of mutagenesis such that a 5-10-fold increase in mutation frequency was observed in cells subcultured at 6-9 h after irradiation as compared to cells subcultured at 3-6 h. A decline in mutation frequency prior to the mutagenesis peak was observed in GS and normal cells but not in XPA. After 24 h of confluent holding, the mutation frequency in irradiated GS and NHDF had returned to near background levels although XPA mutation frequencies remain similar to those observed in immediately subcultured cells. A model to explain these overall results is discussed.

Cell Survival↗

PubMed1985

Relationship between lesions photoinduced by mono- and bi-functional furocoumarins in DNA and genotoxic effects in diploid yeast.

The induction of genetic effects was studied in a diploid strain of Saccharomyces cerevisiae (D7) after treatments with the monofunctional furocoumarins 7-methylpyrido[3,4-c]psoralen (MePyPs), pyrido[3,4-c]psoralen (PyPs) and 3-carbethoxypsoralen (3-CPs) and the bifunctional furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in the presence of 365-nm radiation. The DNA photobinding of radioactively labelled MePyPs, 3-CPs, 5-MOP and 8-MOP was determined in parallel. The DNA-photobinding capacity was highest for MePyPs followed in decreasing order by 5-MOP, 3-CPs and 8-MOP. At a concentration of 5 microM and 4.2 kJ/m2 of 365-nm radiation approximately 160, 66, 60 and 16 adducts per 10(6) base pairs were formed by MePyPs, 5-MOP, 3-CPs and 8-MOP, respectively. The activity of MePyPs and PyPs for the induction of lethal effects lay in the same range as that of 5-MOP whereas 8-MOP was 3 times less active and 3-CPs showed very little activity. For the induction of mitotic gene conversion and genetically altered colonies including mitotic crossing-over the order of activity was about the same as that observed for the induction of lethal effects: MePyPs greater than 5-MOP greater than PyPs greater than 8-MOP much greater than 3-CPs. Nuclear reversions were induced most effectively by 5-MOP, 8-MOP being about 3 times less effective. Up to 4 and 6 kJ/m2 of 365-nm radiation, MePyPs and PyPs, respectively, were less mutagenic than 8-MOP but became more mutagenic at higher doses. At equal survival, the pyridopsoralens were, however, clearly less mutagenic than the bifunctional furocoumarins 8-MOP and 5-MOP. By plotting the genetic data versus the number of lesions induced in DNA, it was shown that the monoadducts induced by the monofunctional furocoumarins MePyPs and 3-CPs exert a relatively low potential for the induction of lethal and nuclear genetic events as compared to photoadditions induced by the bifunctional furocoumarins 8-MOP and 5-MOP. However, at a very high density, the monoadducts induced by MePyPs became as lethal and as mutagenic as the mixture of mono- and biadducts induced by 8-MOP and 5-MOP probably due to overloading of cellular repair capacities.(ABSTRACT TRUNCATED AT 400 WORDS)

Cross-Linking Reagents↗

PubMedDate not supplied

Cytogenetic changes induced in human diploid fibroblasts by tsA58 SV40 at permissive and restrictive temperatures.

Human diploid fibroblasts have been transformed by ts A58 SV40. At the permissive temperature, apparent chromosome and chromatic rearrangements were observed in a high percentage of cells and the frequency of SCE increased. If the transformed phenotype returned to normal at the restrictive temperature these alterations also returned to normal levels. Chromosome banding demonstrated many apparent chromosomal rearrangements in which diffuse staining material was joining intact chromosomes end-to-end and forming pseudostructural abnormalities. Homogeneously staining regions associated with gene amplification or virus-induced alterations in the coiling and stickiness of telomeric regions are possible mechanisms.

Cell Transformation, Viral↗

PubMed1990

Frequency of spontaneous and induced recessive mutations in a diploid strain of Aspergillus nidulans.

The spontaneous and UV-induced frequencies of recessive mutations have been studied in a diploid strain of Aspergillus nidulans, by the p-fluoro-phenylalanine (FPA) and 8-azaguanine (8-AZA) resistance tests, on either resting or germinating conidia. Observed frequencies are in the order of magnitude of those expected, which have been calculated considering the observed mutation frequencies in the haploid strain as well as the mitotic recombination frequencies. We also review some papers which claim to have found higher rates of recessive mutations in mammalian cell lines; in some cases no really higher rates are evident and the authors' conclusions often rest on misinterpretation of their own data.

Aspergillus nidulans↗

PubMedDate not supplied

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O6-methylguanine (O6-MeG) and O4-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O6-MeG is repaired by O6-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O6 position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 microM, O6-benzylguanine (O6-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O6-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 microM in the presence or absence of O6-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O6-BzG (25 microM) was also present in the appropriate half of the cells during the 24 h immediately following exposure to MNNG. This 27-h exposure to O6-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenicity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 microM and 4 microM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 x 10(-6) and 240 x 10(-6), respectively; in the cells with abundant AGT activity, these values were 10 x 10(-6) and 20 x 10(-6), respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G.C with 92% being G.C----A.T. In both sets, approximately 73% of the premutagenic lesions were located in the nontranscribed strand.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1993

Molecular analysis of ethylene oxide-induced mutations at the HPRT locus in human diploid fibroblasts.

Ethylene oxide (EtO)-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were characterized in 28 independently derived 6-thioguanine-resistant human diploid fibroblast clones using polymerase chain reaction-based techniques and Southern blot analysis. Sequence analysis revealed one single base pair deletion and 13 base substitutions, nine of which were transversions: five AT-->TA, three GC-->TA and one GC-->CG. Four mutants were found to have GC-->AT transitions. Seven of the point mutations caused splicing errors. Six occurred in splice site sequences and one created a new splice acceptor site 16 bp upstream of exon 9. Three splice mutations were localized at the same site in the splice donor sequence of intron 8. Fourteen mutants had large HPRT gene deletions. In seven mutants the entire HPRT gene was deleted. The remaining deletion mutants had a truncated HPRT gene, where one or several exons were lost. These results show that EtO induces many different kinds of HPRT mutations, among which as many as 50% are large deletions.

Base Sequence↗

PubMed1983

The suppression of myogenic functions in heterokaryons formed by fusing chick myocytes to diploid rat fibroblasts.

Heterokaryons were formed by fusing differentiated chick skeletal myocytes to fibroblasts derived from skin, lung or heart cultures. The heterokaryons were analyzed for the synthesis of skeletal myosin light chains, acetylcholine receptor, total CPK activity and the ability to spontaneously fuse to form myotubes. Whereas all of the above myogenic functions were expressed in control heterokaryons formed between myocytes and myoblasts, all were extinguished in the crosses between myocytes and fibroblasts. These results confirm that the suppression of myogenic functions previously observed in cell hybrids involving fibroblastoid tumor cells also occurs in heterokaryons isolated using biochemical inhibitors between diploid fibroblasts and chick skeletal myocytes.

Animals↗

PubMed1984

Interactions between diploid embryonal carcinoma cells and early embryonic cells.

Two diploid embryonal carcinoma (EC) cell lines, P10 and P19, differ in their response to the embryonic environment. P10 produces mostly normal chimeras following injection into blastocysts, whereas P19 produces mostly abnormal chimeras. In this study, P10 cells were aggregated with morulae, and all resulting fetuses were chimeric with very large contributions from the EC cells. However, all embryos were abnormal. Following aggregation of P19 cells with morulae, very few embryos were recovered and they were all non-chimeric. Both P10 and P19 were capable of forming functional gap junctions with morula cells and with the ICM of the blastocyst but not with trophoblast, showing that differences in the ability to make junctional contact with the embryo cannot explain the differences between the two cell lines.

Animals↗

PubMed1976

Altered protein metabolism in arrested populations of aging human diploid fibroblasts.

Protein synthesis and turnover were measured in human diploid fibroblasts which were arrested in an essentially nonmitotic state by reducing the serum concentration in the incubation medium to 0.5%. Through the first 4 days of the arrested period both early and late passage cells lost about 20% of their cellular protein. There was a reduction in the rate of protein synthesis at both passage levels during this period, but there was no significant age-related difference in the synthetic rate or the rate of protein turnover. After day 4 both early and late passage cells maintained a constant protein content, but late passage cells did this while processing more protein through faster rates of both synthesis and turnover than did early passage cells. These results support those theories of cellular senescence which predict altered protein metabolism as a major consequence of the aging process.

Cell Survival↗

PubMed1978

Unscheduled dna synthesis in confluent and mitotically arrested populations of aging human diploid fibroblasts.

Unscheduled DNA synthesis, used as a measure of excision repair following exposure to ultra-violet irradiation, was determined in confluent and arrested human diploid fibroblasts and correlated with in vitro age. Confluent cultures exhibited identical levels of unscheduled DNA synthesis at all in vitro ages. Cells arrested by lowering the serum concentration of the incubation medium exhibited similar levels of unscheduled DNA synthesis as did confluent cells during the first one-third of the cells' characteristic in vitro lifespan. During the last two-thirds of the lifespan, however, arrested populations exhibited a 30 to 50% increase in the amount of detectable DNA repair. This apparent increase in ability to perform unscheduled DNA synthesis was not time or dose dependent and could not be attributed to alterations in precursor pools. It was postulated that the increase may be correlated with changes in DNA structure.

DNA↗

PubMed1978

Age related alterations in the chromosomal proteins from human diploid fibroblasts.

Histone and non-histone chromosomal proteins from human diploid fibroblasts of different in vitro ages were extracted and subjected to SDS--polycrylamide gel electrophoresis. The proteins were taken from cells maintained in three distinct culture states: preconfluent (log phase of growth), confluent (stationary phase of growth), and arrested (presumptive G0 phase). Age associated alterations in the incorporation of radioactive amino acids were detected in the fractionated proteins in every culture state but were less pronounced during the arrested state. Age related difference detected under growth conditions may reflect variations in the proliferative nature of the populations. Differences seen during the arrested state may be indicative of basic changes in the chromosomal protein complement of different age populations.

Amino Acids↗