High sensitivity of the ultraviolet-induced p53 response in ultraviolet-sensitive syndrome, a photosensitive disorder with a putative defect in deoxyribonucleic acid repair of actively transcribed genes

Previously, we reported a new category of photosensitive disorder named ultraviolet-sensitive syndrome (UVs S) [T. Itoh, T. Fujiwara, T. Ono, M. Yamaizumi, UVs syndrome, a new general category of photosensitive disorder with defective DNA repair, is distinct from xeroderma pigmentosum variant and rodent complementation group 1, Am. J. Hum. Genet. 56 (1995) 1267-1276.]. Cells derived from these patients show impaired recovery of RNA synthesis (RRS) after UV-irradiation irrespective of having a normal level of unscheduled DNA synthesis (UDS). These characteristics are reminiscent of Cockayne syndrome (CS) cells. By comparing sensitivity of the UV-induced p53 response in cells with different types of defects in nucleotide excision repair, we hypothesized that the UV-induced p53 response is triggered by inhibition of RNA synthesis [M. Yamaizumi, T. Sugano, UV-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle, Oncogene 9 (1994) 2775-2784.]. To test this hypothesis, we determined sensitivity of the p53 response in UVs S cells by immunostaining, Western blotting, and FACScan analysis. Maximal nuclear accumulation of p53 in the UVs S cells was observed with a one-third UV dose required for that in normal cells, while almost identical p53 responses were observed in UVs S and normal cells following treatment with heat or alpha-amanitin. Recovery of RNA synthesis after a low dose of UV-irradiation was impaired in UVs S cells to the same extent as seen in CS cells. These results provide further evidence to support our previous hypothesis regarding the mechanism of the p53 response induced by DNA damage.     

Problems in the estimation of mutation rates and in the detection of induced mutations in man

The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stiationary phase haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the “petite” recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of “petites” in stationary phase cells (increase of the frequency of “petites” during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.     

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.     

Aberrant DNA repair and enhanced mutagenesis following mutagen treatment of Chinese hamster Ade-C cells in a state of purine deprivation

Ade-C is a Chinese hamster ovary cell line auxotrophic for purines because of a mutation in the de novo synthetic pathway. We now show that, in the absence of exogenous hypoxanthine, replicative DNA synthesis is rapidly shut down. Various aspects of DNA repair have been studied in purine-starved cells. Incision, the first step of excision repair of UV damage, appears normal, as do the later steps, repair synthesis (demonstrated following chemical damage as well as UV-irradiation) and ligation. However, removal of UV-induced pyrimidine dimers is not detected, and it seems that the repair that occurs is aberrant. This behaviour is associated with an increase in cell killing by UV light, and a several-fold increase in the frequency of mutations induced by UV.     

A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line

When cells of a human clonal cell line, RSa, with high sensitivity to UV lethality, were treated with the mutagen, ethyl methanesulfonate, a variant cell strain, UVr-1, was established as a mutant resistant to 254-nm far-ultraviolet radiation (UV). Cell proliferation studies showed that UVr-1 cells survived and actively proliferated at doses of UV-irradiation that greatly suppressed the proliferation of RSa cells. Colony-formation assays also confirmed the increased resistance of UVr-1 cells to UV. The recovery from a UV-induced inhibition in DNA synthesis, as [methyl-3H]thymidine uptake into cellular DNA, was more pronounced in UVr-1 cells than in RSa cells. Nevertheless, there was no significant difference in the activity of UV-induced DNA repair synthesis in either cell line, as estimated by the extent of unscheduled DNA synthesis and DNA repair replication. UVr-1 cells were also more refractory to 4-nitroquinoline 1-oxide (4NQO), but the activity of DNA repair synthesis induced by 4NQO in UVr-1 cells was much the same as in the RSa cells. Both N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) sensitivity and MNNG-induced DNA repair synthesis activity in UVr-1 cells were similar to that of RSa cells. These characteristics of UVr-1 cells are discussed in the light of a previously reported UV-resistant variant, UVr-10, which had an increased DNA repair synthesis activity.     

Inhibitors of DNA synthesis (aphidicolin and araC/HU) prevent the recovery of RNA synthesis after UV-irradiation

UV-irradiation causes an immediate depression in the rate of RNA synthesis in human skin fibroblasts. RNA synthesis rates recover to greater than or equal to 90% of unirradiated levels within 90 min in normal cells. This recovery can be prevented by incubating the cells after irradiation with araC/HU or aphidicolin, potent inhibitors of DNA replication and excision repair. The effect of these inhibitors on the recovery of RNA synthesis can also be observed in non-dividing cells; it is thus independent of their effects on DNA replication.     

Aberrant DNA repair and enhanced mutagenesis following mutagen treatment of Chinese hamster Ade−C cells in a state of purine deprivation

Ade⁻C is a Chinese hamster ovary cell line auxotrophic for purines because of a mutation in the de novo synthetic pathway. We now show that, in the absence of exogenous hypoxanthine, replicative DNA synthesis is rapidly shut down. Various aspects of DNA repair have been studied in purine-starved cells. Incision, the first step of excision repair of UV damage, appears normal, as do the later steps, repair synthesis (demonstrated following chemical damage as well as UV-irradiation) and ligation. However, removal of UV-induced pyrimidine dimers is not detected, and it seems that the repair that occurs is aberrant. This behaviour is associated with an increase in cell killing by UV light, and a several-fold increase in the frequency of mutations induced by UV.     

UV-irradiation induces oxidative damage to mitochondrial DNA primarily through hydrogen peroxide: analysis of 8-oxodGuo by HPLC

Roles of reactive oxygen species (ROS) in damage to mitochondrial DNA (mtDNA) following ultraviolet (UV)-irradiation were investigated in the human hepatoma cell line SK-HEP-1. We altered the intracellular status of ROS by the overexpression of manganese superoxide dismutase (MnSOD) and/or catalase. Using HPLC, we analyzed 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), known as a marker of damage to DNA molecules. UV-irradiation resulted in the accumulation of 8-oxodGuo in these cells. The overexpression of MnSOD enhanced the accumulation of 8-oxodGuo by UV. The co-overexpression of catalase inhibited the accumulation of 8-oxodGuo by UV in MnSOD-transfectants. The overexpression of MnSOD reduced the colony forming capacity in SK-HEP-1 cells and the co-overexpression of catalase with MnSOD stimulated the capacity compared to control. UV-irradiation inhibited the colony forming capacity in these cells; no difference was observed among the capacities of control, MnSOD- and catalase-transfectants. However, the overexpression of MnSOD/catalase significantly rescued the reduction of colony forming capacity by UV-irradiation. Our results suggest that the accumulation of hydrogen peroxide plays a key role in the oxidative damage to mtDNA of UV-irradiated cells, and also that the overexpression of both MnSOD and catalase reduces the mtDNA damage and blocks the growth inhibition by UV. Our results also indicate that the increased activity of MnSOD may lead to a toxic effect on mtDNA by UV-irradiation.     

UV-irradiation induces oxidative damage to mitochondrial DNA primarily through hydrogen peroxide: Analysis of 8-oxodGuo by HPLC

Roles of reactive oxygen species (ROS) in damage to mitochondrial DNA (mtDNA) following ultraviolet (UV)-irradiation were investigated in the human hepatoma cell line SK-HEP-1. We altered the intracellular status of ROS by the overexpression of manganese superoxide dismutase (MnSOD) and/or catalase. Using HPLC, we analyzed 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), known as a marker of damage to DNA molecules. UV-irradiation resulted in the accumulation of 8-oxodGuo in these cells. The overexpression of MnSOD enhanced the accumulation of 8-oxodGuo by UV. The co-overexpression of catalase inhibited the accumulation of 8-oxodGuo by UV in MnSOD-transfectants. The overexpression of MnSOD reduced the colony forming capacity in SK-HEP-1 cells and the co-overexpression of catalase with MnSOD stimulated the capacity compared to control. UV-irradiation inhibited the colony forming capacity in these cells; no difference was observed among the capacities of control, MnSOD- and catalase-transfectants. However, the overexpression of MnSOD/catalase significantly rescued the reduction of colony forming capacity by UV-irradiation. Our results suggest that the accumulation of hydrogen peroxide plays a key role in the oxidative damage to mtDNA of UV-irradiated cells, and also that the overexpression of both MnSOD and catalase reduces the mtDNA damage and blocks the growth inhibition by UV. Our results also indicate that the increased activity of MnSOD may lead to a toxic effect on mtDNA by UV-irradiation.     

Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient.

Accumulated evidence indicates that proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and forms tight association with DNA replication sites during DNA replication or DNA repair synthesis. In this study, such PCNA complex formation was investigated by the indirect immunofluorescence method, using both normal human fibroblasts and those derived from a xeroderma pigmentosum group A (XP-A) patient. XP-A fibroblasts in both proliferating and quiescent states did not show any differences from normal fibroblasts in the properties of PCNA-staining in the untreated conditions. The PCNA complex formation was induced in quiescent normal fibroblasts by both ultraviolet light (UV)- and X-irradiation, whereas in XP-A fibroblasts it was induced by X-irradiation, but not by UV-irradiation. However, PCNA complex was induced in quiescent XP-A fibroblasts by UV-irradiation when the cells had previously incorporated 5-bromodeoxyuridine (BrdU). These observations indicate a close correlation of PCNA complex formation and unscheduled DNA synthesis (UDS). Thus, it was concluded that PCNA complex formation was commonly induced in at least three conditions to produce UDS in spite of different types of DNA damages and DNA repair mechanisms.     

Dependence of DNA dark repair on protein synthesis in Escherichia coli.

Summary We investigated the influence of aminoacidless treatments applied prior and after UV irradiation on survival, dimer excision, postirradiation DNA degradation, DNA synthesis and sedimentation profiles of parental DNA ofE. coli B/r Hcr⁺ cells. In dependence on the treatment applied, the fluence 50 J/m² yielded distinctly different fractions of survivors within 0,03–85%. In all cases dimers were completely excised. The rate of DNA degradation was similar during a 30–40 min period after UV during which the bulk of dimers was excised. Degradation ceased, however, earlier in the prestarved cells than in exponentially growing ones; it was prolonged by aminoacidless postincubation. Sedimentation profiles of parental DNA did not differ during the whole period of dimer excision. In cells DNA synthesis was not restored for several hours after addition of amino acids. In cells addition of amino acids resulted in a fast resumption of DNA synthesis. We conclude that removal of dimers and repair of gaps were similar in all cases. We believe that aminoacidless treatments influence production and repair of damage to the sites of DNA replication. The treatment appears to prevent this damage when applied before UV irradiation, but interferes with its restoration when applied after UV irradiation. Consequently, the former treatment increases survival of cells while the latter produces an opposite effects.     

DNA repair replication,DNA breaks and sister-chromatid exchange in human cells treated with adriamycin in vitro

The effects of adriamycin (AM) on DNA repair replication, the frequency of sister-chromatid exchange (SCE), the rate of cell proliferation and the frequency of DNA strand breaks were studied in human cells in vitro. No repair replication was observed in lymphocytes exposed to AM in concentrations up to 10^?3 moles/1. DNA repair replication induced by UV and alkylating agents was not affected by a concentration of AM that completely inhibited cell proliferation (10^?6 moles/1).Fibroblasts exposed to AM at 10^?4 moles/1 in the presence of hydroxyurea showed an increase of strand breaks and cross-links in DNA. When AM was added to UV-irradiated fibroblasts, there was an increase of DNA strand breaks in addition to the breaks caused by UV alone. Similar effects were observed in lymphocytes.A dose-dependent increase of SCE was observed in lymphocytes exposed to low concentrations of AM (<10^?7 moles/1). At higher concentrations the increase of SCE levelled off, and cell proliferation became severely inhibited. There was no evidence of removal of SCE-inducing damage in cells exposed to AM during G₀ or G₁. The level of SCE induced in the third cell cycle after treatment with AM was not different from that induced during the first two cell cycles.These results suggest that the various genotoxic and cytotoxic effects of AM are caused by different types of cellular damage. Moreover, AM-induced DNA damage persists for several cell cycles in human cells in vitro and seems to be resistant to repair activity.     

Qualitative differences between replicative and repair synthesis of DNA in normal and transformed mouse cells as measured by precursor discrimination

Inhibitors of DNA polymerase alpha such as aphidicolin (APC) or 1-beta-D-arabinofuranosyl-cytosine (araC) cause DNA-strand breaks to accumulate after UV-irradiation, at sites where repair resynthesis is inhibited. Transformed cells accumulate fewer such breaks than normal cells do; this may be due to differences in the extent, or the nature, of excision-repair synthesis in transformed and in normal cells. We have looked for differences in the nature of repair synthesis, comparing the labelling of DNA by deoxycytidine (dC) and araC through UV-induced repair in normal and transformed mouse cells. We have made parallel determinations of precursor discrimination in replicative synthesis, and find that normal cells discriminate better against araC in replicative synthesis than do transformed cells. But repair synthesis discriminates against araC less than normal replicative synthesis does, to a similar extent in both cell types. Thus, there are qualitative differences between the DNA polymerases engaged in UV excision repair and replication in normal and transformed mouse cells; but there is no evidence for a predominantly araC-insensitive repair synthesis in transformed cells, such as might account for the difference in break accumulation.     

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.     

Relationship between pyrimidine dimers, 6-4 photoproducts, repair synthesis and cell survival: studies using cells from patients with trichothiodystrophy

Trichothiodystrophy is a genetic disease which in the majority of cases studied is associated with a deficiency in the ability to repair UV damage in cellular DNA. Three categories of UV response have been identified. In type 1 the response is completely normal, whereas type 2 cells are deficient in excision-repair, with properties indistinguishable from those of XP complementation group D. Type 3 cells have normal survival following UV-irradiation and normal rates of removal of cyclobutane pyrimidine dimer sites. Nevertheless repair synthesis is reduced by 50% in these cell strains and this is associated with a marked reduction in the repair of 6-4 photoproducts from cellular DNA. The present results show that 50% or more of repair synthesis at early times after irradiation of normal primary human fibroblasts is attributable to repair of 6-4 products. They also suggest that repair of cyclobutane dimers is crucial for cell survival.