DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.     

Immobilized glycolipids from human diploid fibroblasts inhibit DNA synthesis of cultured human fibroblasts

Several previous studies have shown that glycolipids isolated from plasma membranes of cultured cells and added to cells in culture inhibit the growth rate in a concentration-dependent fashion. In order to investigate the possible involvement of glycolipids in the growth regulation of normal cells by cell-cell contacts, we tested the effect of immobilized glycolipids, isolated from human fibroblasts, on the DNA synthesis of freshly seeded fibroblasts. Gangliosides inhibited DNA synthesis to a great extent, whereas neutral glycolipids had only a minor effect. The degree of inhibition of DNA synthesis by immobilized gangliosides depended both on the cell density of the cultures from which the gangliosides were isolated and on the pretreatment of the immobilized gangliosides: Preincubation with DMEM without FCS of immobilized gangliosides, isolated from confluent cultures, resulted in a 75% inhibition of growth rate of embryonal human lung fibroblasts (FH109) cultured on immobilized gangliosides. Under the same conditions, gangliosides from sparse cultures reduced the growth rate by about 30%. On the other hand, the degree of inhibition exerted by immobilized gangliosides isolated from confluent cultures was found to be greatly reduced by preincubation with DMEM with FCS, whereas the slight inhibition of growth rate, exerted by gangliosides from sparse cultures, was found to be reversed into a slight stimulation of growth rate after preincubation with complete medium. Concomitantly with the reduction of the inhibition of DNA synthesis, it was found that the complete medium, used for preincubation of the gangliosides, was no longer able to support DNA synthesis to the same extent as untreated complete medium. The data suggest that gangliosides bind growth-supporting factors of the serum, gangliosides isolated from sparse cultures being more potent in the binding of these molecules than gangliosides isolated from dense cultures.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.     

Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in diploid human skin fibroblasts: an improved risk estimate in photochemotherapy

Cell killing and the induction of mutation were studied in dividing and non-dividing human skin fibroblasts as a result of treatment by 8-methoxypsoralen (8-MOP) and long-wave UV irradiation (UVA). The cytotoxic effect was highly dependent upon the duration of the UVA exposure. The frequency of mutations increased linearly with the UVA dose at concentrations of 10 and 0.25 microliter 8-MOP/ml, the latter representing the concentration in the skin during PUVA treatment. The number of mutations induced per unit dose (= per microgram 8-MOP/ml per joule UVA/m2) was calculated: for dividing cells this value was 3.3 X 10(-8) per cell and for non-dividing cells 0.6 X 10.8(-8) per cell. On the basis of these values the expected number of induced mutants in the human skin per session of photochemotherapy is 1.2 X 10(-5), and per 30 years of maintenance therapy 1.3 X 10(-2) per cell. A comparison was made between this frequency and the frequency to be expected from spontaneous mutation. In addition the significance of absence in patients of SCE induction by photochemotherapy is discussed.     

Reevaluation of DNA chain elongation rate in human diploid fibroblasts

The rate of DNA chain elongation in human diploid fibroblasts (IMR90) of different ages was examined by DNA fiber autoradiography and alkaline sucrose density gradient centrifugation. There was no difference in chain elongation rate in various population doubling level cells.     

DNA signals for G2 checkpoint response in diploid human fibroblasts

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included γ-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4–12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.     

Complement-dependent induction of DNA synthesis and proliferation of human diploid fibroblasts

The effects of fresh human serum (FHS) and heat-inactivated human serum (HHS) on the DNA synthesis and proliferation of human diploid fibroblasts were assessed. FHS activated significantly more quiescent fibroblasts to undergo DNA synthesis and proliferation than did HHS. The stimulatory effect occurred consistently over a serum concentration range of 0.1–10%. Using bromodeoxyuridine selective killing techniques, it was shown that this FHS stimulatory effect was on a specific subpopulation of fibroblasts unresponsive to HHS. The involvement of the complement system, and specifically of C1, was shown by the inability of Clq-depleted FHS to support enhanced DNA synthesis whereas Clq-depleted serum reconstituted with purified Clq was effective. Purified Clq did not restore activity when added to heated serum, nor was it mitogenic when tested in basal medium without serum. The addition of purified Clq to fresh serum inhibited the enhancement of DNA synthesis, and at Clq concentrations of 4γ/ml and greater, the fresh serum effects were abrogated. Thus, it appears that binding of the assembled C1 complex to the fibroblast surface was required for FHS-mediated enhancement of fibroblast proliferation, with Clq subcomponent serving as the recognition site. The results from several experiments indicated that antibody was not required for the complement-dependent fibroblast activation. FHS was not cytotoxic, and autologous serum was as effective as allogeneic sera. A 20-fold molar excess of Fab' from pooled human IgG did not alter the FHS effects. FHS from which IgG was more than 99% depleted was still effective. These results suggested an antibody-independent role for complement in the activation of a subpopulation of human diploid fibroblasts.     

Optimal parameters for the polybrene-induced DNA transfection of diploid human fibroblasts

Summary Recently it has been shown that Polybrene, in conjuction with dimethyl sulfoxide (DMSO) shock, can markedly increase frequency of DNA transfection of chicken embryo fibroblasts as compared with the frequency obtained with the standard calcium phosphate protocol. We have adapted this procedure for use with diploid human fibroblasts. Using plasmid DNA containing a dominant selectable marker gene (resistance to Geneticin), we have determined that treatment of the cells for 6 h in culture medium containing Polybrene at a concentration of 2 to 5 μg/ml, followed by a 4-min shock with 30% DMSO, resulted in the highest yield of transfectants, ca. 400/10⁶ cells treated with as little as 100 ng of plasmid DNA. The selective agent could be added immediately after the DMSO shock. This allows transfection and selection to be carried out in the same dishes and ensures that each clone represents a unique event. The pSV2neo plasmid was generously supplied by Dr. Paul Berg. This work was supported by U. S. Department of Energy contract EV 04659, National Institute of Environmental Health Sciences Postdoctoral Training Grant ES 07076, and a grant from the Michigan Osteopathic College Foundation.     

The spontaneous azaguanine-resistant mutants of diploid human fibroblasts

Summary The range of incidences of azaguanine-resistant colonies in cultures of fibroblasts from 16 unrelated humans was 0.4×10^-6 to 19×10^-6 and the mean value was 4.1×10^-6. A fluctuation test showed that most or all of the mutant colonies derived from mutations that occurred during in vitro proliferation of the fibroblasts and before exposure to azaguanine. The estimated rate of spontaneous mutation was 0.45×10^-6 to 1.8×10^-6 per cell generation. At least ten independent mutants, comprising two general classes, were studied. Class I mutants were a minority and resembled cells from boys having the Lesch-Nyhan syndrome: they had very little HG-PRT activity, showed maximum resistance to azaguanine and could not utilize hypoxanthine for growth. At least 90% of the mutants were in Class II: their apparent HG-PRT activities ranged between normal and Lesch-Nyhan amounts, they were partially sensitive to azaguanine and they could utilize hypoxanthine. Some Class II mutants resembled cells cultured from a family having an X-chromosomal mutant gene that does not cause the Lesch-Nyhan syndrome but does confer resistance to azaguanine, although the quantity of HG-PRT activity is apparently normal and hypoxanthine can be utilized. Electrophoretic differences between the HG-PRT activities of normal and mutant strains were not detected but other qualitative alterations were observed in some mutants.Paper No. 1558 from the Laboratory of Genetics.Supported by N.I.H. Grants GM-06983 and GM-15422 and by a grant from the Food Research Institute of The University of Wisconsin, Madison, Wisconsin.Supported by Grant He 753-1 from Die Deutsche Forschungsgemeinschaft.     

Cell population heterogeneity in the inducibility of DNA synthesis in human diploid fibroblasts

The initiation of nuclear DNA synthesis has been studied in cytochalasin B (CB)-induced binucleate human diploid fibroblasts (WI-38 cells). Mitotic cells from different passage levels were rendered binucleate by a brief pulse of CB. The cells were then washed free of the drug, and DNA synthesis was studied by [3H]thymidine labeling. The results showed that, in a small percentage of binucleate cells, one nucleus was labeled (S phase) and the other nucleus was unlabeled (G1 phase). There was no significant difference in the percentage of these cells with increasing passage levels. The results of this study suggest that some WI-38 cells retire from the cell cycle at different passage levels, and thereby become refractory to inducers of nuclear DNA synthesis generated by sister cells in S phase.     

Repair of radiation-induced DNA damage in nondividing populations of human diploid fibroblasts.

The occurrence of DNA repair in UV- (254 nm) and X-irradiated normal human diploid fibroblasts maintained in a quiescent, nondividing state using low serum (0.5%) medium was ascertained. Techniques that detect different steps of the excision repair process were used so that the extent of completion of repair at single sites could be determined. These included measuring the disappearance of pyrimidine dimers by chromatography, detecting repair synthesis by density-gradient and autoradiographic methods and detecting the rejoining of repaired regions and repair of x-ray-induced single-strand DNA breaks using alkaline sucrose gradients. Results show that dimer excision occurs and the subsequent steps of repair synthesis and ligation are completed. About 50% of the dimers formed by exposure to 20 J/m2 is excised in the initial 24-h post-UV period. DNA repair (unscheduled DNA synthesis) can be detected through a 5-d post-UV period. The fraction of damaged sites eventually repaired is not known. X-ray-induced single-strand DNA breaks are repaired rapidly.     

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.     

Isolation of ouabain-resistant human diploid fibroblasts

Seventeen clones resistant to the cytotoxic action of ouabain were isolated in culture by direct selection from 5 independent strains of diploid human fibroblasts. Resistant clones were recovered at frequencies on the order of 10^?7 per wild type cell selected from populations treated with the mutagen EMS, but no resistant cells were detected among 10⁸ unmutagenized cells. Most selected clones remained ouabain-resistant following further propagation in the absence of drug. The growth of wild type cells was inhibited by 50% at ouabain concentrations of 2–5 × 10^?8 M, while resistant clones required 15–180 fold higher drug concentrations to cause equivalent inhibition. Ouabain-resistant clones showed increased resistance of K+ transport function to ouabain inhibition that paralleled their increased resistance to growth inhibition. Initial experiments suggest that under selective conditions the resistant diploid fibroblasts differ significantly from wild type in binding of ³H-ouabain per unit surface area. The ouabain-resistant cells were similar to wild type in transport properties unrelated to ouabain inhibition. Resistant cells had normal karyotypes and senesced with a lifespan similar to control clones. The ouabain-resistant phenotypes of these diploid human fibroblast isolates apparently reflect point mutations that specifically affect the Na+/K+ transport ATPase with respect to ouabain-binding and/or response to bound ouabain.