DNA repair in cultured human fibroblasts does not decline with donor age

Human fibroblasts from young (3 days to 3 years) and old (84–94 years) donors were tested for their ability to repair DNA damage by measuring survival of colony formation following irradiation with ultraviolet (UV) light. Repair was also measured by the ability to reactivate herpes simplex virus following treatment of the virus with UV light, methyl methane sulfonate or 4,5′,8-trimethylpsoralen plus light. This virus was used as a probe of cellular repair capacity because survival of damaged virus is lower in repair-deficient cell lines [1]. Cell lines from both age groups exhibited comparable survivals following UV irradiation and failed to show increased sensitivity to irradiation in the presence of caffeine. Cells from both groups repaired damaged virus to equal extents. Proficient viral repair was observed under conditions in which cells were infected by either single or multiple viral genomes. These results suggest that DNA repair mechanisms which act on a variety of lesions (e.g. pyrimidine dimers, apurinic sites, alkylated bases, cross-links, etc.) do not decline with age. A model for biological aging resulting from the accumulation with age of unrepaired DNA damage is discussed.     

The repair of DNA damages induced in normal human skin fibroblasts exposed to simulated sunlight

The induction and repair of DNA damages produced by exposure of normal human skin fibroblasts to the simulated sunlight produced by a solar simulator were examined. The photoproducts measured were pyrimidine dimers, E. coli endonuclease III-sensitive sites, 6-4 photoproducts, Dewar isomers, DNA-protein crosslinks, and DNA single-strand breaks. The results of these experiments serve to form a basis for the quantitation of damages induced by exposure to sunlight.     

Semiautomated autoradiographic measurement of DNA repair in normal and xeroderma pigmentosum cultured human fibroblasts

Summary Assessment of DNA repair in cultured human fibroblasts by autoradiography may be facilitated by using semiautomated grain counting instruments. The instrument-determined number of autoradiographic grains per nucleus in cultured human skin fibroblasts was found to be linear in comparison to visual counts up to only 30 grains per nucleus. However, with two different instruments a greater range of linearity (to 100 to 120 grains per nucleus) was attained by measuring the grain surface area per nucleus. Semiautomated analysis of the grain surface area per nucleus yielded measurements of relative rates of unscheduled DNA synthesis after ultraviolet irradiation in xeroderma pigmentosum and normal human fibroblasts, which were reproducible and rapid.     

Induction and repair of DNA strand breaks in cultured human fibroblasts exposed to various phenols and dihydrodiols of benzo[a]pyrene

Cultured human fibroblasts from healthy donors were incubated for 30 min with nine different benzo[a]pyrene (BP) derivatives in the presence or absence of liver microsomes from 3-methylcholanthrene treated rats. The induction and repair of DNA strand breaks were analysed by alkaline unwinding and separation of double and single stranded DNA (SS-DNA) by hydroxylapatite chromatography immediately after the incubation or at various times after the treatment. In the absence of microsomes DNA stand breaks were detected in fibroblasts exposed to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP) and the three BP dihydrodiols (BP-4,5-, BP-7,8- or BP-9,10-dihydrodiol). After removal of the BP derivatives from the medium the DNA strand breaks disappeared within 24 h. alpha-Naphthoflavone (alpha-NF) caused a decrease in the induction of strand breaks by 1-, 3- and 9-OH-BP but did not affect the induction of strand breaks in cells exposed to BP-7,8-dihydrodiol. In the presence of microsomes DNA strand breaks were found after exposure to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP), as well as BP-7,8- and 9,10-dihydrodiol. In contrast BP-4,5-dihydrodiol did not induce strand breaks under these conditions. The induction of strand breaks by BP-7,8-dihydrodiol was enhanced in the presence of cytosine-1-beta-D-arabinofuranoside (AraC). In all cases the DNA strand breaks had disappeared 24 h after removal of the BP derivatives and microsomes except after treatment with BP-7,8-dihydrodiol.     

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.     

Dose-independent uniform distribution of UV-induced repair replicated DNA among DNA sequences of different reiteration frequencies in cultured human fibroblasts

The distribution of UV-induced repair replicated DNA sequences among highly reiterated, moderately reiterated, and single copy human DNA has been investigated by molecular reassociation at various UV doses. Results indicate that repair replicated DNA patches are uniformly distributed within highly repeated, moderately repeated, and single copy DNA sequences at every dose of UV radiation.     

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.     

Aphidicolin inhibits repair of DNA in UV-irradiated human fibroblasts

Aphidicolin, a specific inhibitor of DNA polymerase α, is shown to inhibit DNA repair in human diploid fibroblasts. Although aphidicolin has no apparent effect on the DNA of unirradiated cells, it causes a large number of strand breaks to accumulate in UV-irradiated cellular DNA. The number of breaks is the same as the number observed following a similar dose of ultraviolet light when cells are treated with arabinofuranosyl cytosine (araC) and hydroxyurea (HU), known inhibitors of repair. Moreover, two-dimensional paper chromatography shows that aphidicolin completely blocks removal of pyrimidine dimers. These observations are discussed in light of the proposed roles of DNA polymerases α β in DNA replication and repair and the action of aphidicolin on polymerase α.     

Further characterization of a cell-free system for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the involvement of DNA polymerase alpha in DNA repair.

DNA repair synthesis can be specifically measured in osmotically opened, confluent cultured human fibroblasts after exposure to DNA damaging agents such that both induction and mediation of DNA repair synthesis can take place in this cell-free system. Alternatively, by utilizing osmotically shocked, log phase cells and altering the DNA precursors, pH and ionic strength, replicative DNA synthesis can be specifically monitored. Autoradiographic studies show that virtually all of the nuclei from the lysates of the confluent, UV-iradiated cells are lightly labeled in the fashion characteristic of DNA repair. By contrast, only a fraction of nuclei is labeled in a population of unperturbed, opened log phase cells and the labeling is heavy and characteristic of replicative synthesis. Furthermore, equilibrium density gradient sedimentation shows that DNA synthesis in lysates of log-phase cells is semiconservative, whereas that with UV-irradiated cells is repair synthesis. This open cell system has been used to study the enzymology of DNA repair. Thus, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerases beta and gamma, does not inhibit either replicative or repair synthesis. By contrast, aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits DNA repair and replicative synthesis in both intact and permeabilized cells. Finally, phage T4 UV-exonuclease stimulates repair synthesis, but only when phage T4 UV-endonuclease is also added to the UV-irradiated nuclei.     

DNA repair in human fibroblasts treated with a combination of chemicals.

Excision repair of DNA damage was measured by the photolysis of bromodeoxy-uridine incorporated during repair in normal human and xeroderma pigmentosum group C fibroblasts (XP C) treated with a combination of the carcinogens N-acetoxy-2-acetylamino-fluorene (AAAF), and 4-nitroquinoline 1-oxide (4NQO). Repair was additive in normal and XP C cells treated with AAAF plus 4NQO, indicating that there are different rate limiting steps for removal of 4NQO and AAAF lesions.     

Maternal inheritance of mitochondrial DNA polymorphisms in cultured human fibroblasts

We have isolated the total cellular DNA from the cultured diploid fibroblasts of a six-member, three-generation human family. Using a specific radioactive probe for mitochondrial (mt) sequences we have identified new polymorphic variants in this family for the Hhal restriction endonuclease cleavage pattern of the mtDNA. The inheritance of these cleavage patterns verifies the maternal inheritance of mtDNA through all three generations.     

DNA damage and DNA repair in cultured human cells exposed to chromate

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.     

Stimulation by insulin of DNA synthesis in cultured mouse fibroblasts]

With the aid of autoradiography, the effect of insulin on entering S- from G1-period of the mitotic cycle and on the rate of DNA synthesis of the mouse fibroblasts (L), was studied,--in the cells incubated for 24 hr in serum-free medium. In these conditions the cells were temporarily blocked in G1-period. Insulin (100 mcU/ml) increased by 1.5-fold the amount of cells in S-period as well as caused a marked stimulation of DNA synthesis.     

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts.

Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblasts lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

Formation by bifunctional furocoumarins of DNA crosslinks and their repair in cultured mouse embryo fibroblasts

Formation of crosslinks in DNA by three bifunctional psoralen derivatives plus UVA light in mouse embryo fibroblasts was evaluated by a NaI density gradient centrifugation method. Psoralen was shown to be a more active cross-linking agent than 8-methoxypsoralen. As for 4,5',8-trimethylpsoralen, it needed much lower concentrations and much less 365 nm light fluence to yield high percentages of crosslinked DNA. Repair of adducts formed by these psoralen derivatives was studied by splitting the irradiation dose into two equal parts separated by variously long dark repair periods. It was shown that essentially only monoadducts formed during the first irradiation period were repaired. These mouse embryo fibroblasts seem unable to repair interstrand DNA crosslinks.     

Kinetics of ultraviolet induced DNA excision repair in rat and human fibroblasts

To obtain more information on the well-documented low excision-repair capacity of rodent cells in comparison with human cells, we have studied this form of DNA repair in UV-irradiated human and rat skin fibroblasts. For this purpose, we have determined (i) unscheduled DNA synthesis (UDS), using autoradiography, (ii) the number and size of repaired sites with the bromodeoxyuridine (BrdU) photolysis assay and (iii) the removal of Micrococcus luteus UV-endonuclease susceptible sites (ESS). We found rat cells to be quite capable of performing DNA-repair synthesis, as demonstrated by both UDS and BrdU photolysis, whereas they almost completely lacked the capacity to remove pyrimidine dimers, as indicated by the persistence of ESS. This discrepancy will be discussed in terms of the types of mechanisms by which mammalian cells may recognize and remove UV-induced photoproducts.