Resistance of plateau-phase human normal and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light

Clonogenic survival response to 254-nm ultraviolet light was measured in 2 strains of repair-proficient normal human fibroblasts and 4 strains of xeroderma pigmentosum (XP) fibroblasts belonging to complementation groups A, C, D and variant. In all strains except XPA, cells irradiated in plateau phase and subcultured immediately were much more resistant to the lethal effect of UV than cells irradiated in the exponential phase of growth. Typically, 10-20% of plateau-phase cells were extremely resistant. When the cultures were held in plateau phase for 24 h after irradiation and before subculture, there was a further enhance of survival. By use of a UV-specific endonuclease assay, no difference was found in the number of DNA lesions induced in exponentially growing and plateau cultures by the same dose of UV light. Thus plateau-phase cells appear to be more efficient in their DNA-repair capability than cells in exponential growth. XP group A cells were uniquely found to be deficient in the processes which lead to plateau-phase resistance. Since plateau-phase repair was not lacking in XP groups C, D and variant, it may be related to a DNA-repair process different from that which is responsible for the overall UV sensitivity of these cells.     

Bystander normal human fibroblasts reduce damage response in radiation targeted cancer cells through intercellular ROS level modulation

The radiation-induced bystander effect is a well-established phenomenon which results in damage in non-irradiated cells in response to signaling from irradiated cells. Since communication between irradiated and bystander cells could be reciprocal, we examined the mutual bystander response between irradiated cells and co-cultured with them non-irradiated recipients. Using a transwell culture system, irradiated human melanoma (Me45) cells were co-cultured with non-irradiated Me45 cells or normal human dermal fibroblasts (NHDF) and vice versa. The frequency of micronuclei and of apoptosis, ROS level, and mitochondrial membrane potential were used as the endpoints. Irradiated Me45 and NHDF cells induced conventional bystander effects detected as modest increases of the frequency of micronuclei and apoptosis in both recipient neighbors; the increase of apoptosis was especially high in NHDF cells co-cultured with irradiated Me45 cells. However, the frequencies of micronuclei and apoptosis in irradiated Me45 cells co-cultured with NHDF cells were significantly reduced in comparison with those cultured alone. This protective effect was not observed when irradiated melanomas were co-cultured with non-irradiated cells of the same line, or when irradiated NHDF fibroblasts were co-cultured with bystander melanomas. The increase of micronuclei and apoptosis in irradiated Me45 cells was paralleled by an increase in the level of intracellular reactive oxygen species (ROS), which was reduced significantly when they were co-cultured for 24h with NHDF cells. A small but significant elevation of ROS level in NHDF cells shortly after irradiation was also reduced by co-culture with non-irradiated NHDF cells. We propose that in response to signals from irradiated cells, non-irradiated NHDF cells trigger rescue signals, whose nature remains to be elucidated, which modify the redox status in irradiated cells. This inverse bystander effect may potentially have implications in clinical radiotherapy.     

Host cell reactivation by excision repair is error-free in human cells

Do host cell repair processes affect the mutagenesis of UV-irradiated virus in human cells? The answer was obtained by investigating the mutagenesis of UV-irradiated herpes simplex virus after the irradiated virus was grown in human cells that possess normal repair capacity (normal) or lack excision repair (XPA) or post-replication repair (XP var). Evidence is presented which indicate that XPA cells express no host cell reactivation, while XP var cells express the normal level. Viral mutagenesis was measured as the fraction of the progeny of the surviving virus capable of plaque formation in the presence of iododeoxycytidine. In the normal and XPA cells mutagenesis of the irradiated virus increased linearly with UV exposure. The UV exposure needed to yield a given mutagenesis level for virus grown in XPA cells was much lower than that for virus grown in normal cells. However, when the mutation frequencies were compared at similar virus survival levels, the data from virus grown in normal cells and in XPA cells were indistinguishable. Mutagenesis in XP var cells increased as dose squared and was similar in magnitude to that in normal cells. Thus the excision repair of normal cells which provided host cell reactivation by removing lethal UV damage also removed mutagenic lesions from the virus with the same efficiency, while the repair deficiency of XP var cells had a minor role in host cell reactivation and in mutagenesis. This demonstrates that in human cells host cell reactivation by excision repair is primarily an error-free process.     

XPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells

Nucleotide excision repair (NER) acts on a variety of DNA lesions, including damage induced by many chemotherapeutic drugs. Cancer therapy with such drugs might be improved by reducing the NER capacity of tumors. It is not known, however to what extent any individual NER protein is rate-limiting for any step of the repair reaction. We studied sensitivity to UV radiation and repair of DNA damage with regard to XPA, one of the core factors in the NER incision complex. About 150,000-200,000 molecules of XPA protein are present in NER proficient human cell lines, and no XPA protein in the XP-A cell line XP12RO. Transfected XP12RO cell lines expressing 50,000 or more XPA molecules/cell showed UV resistance similar to normal cells. Suppression of XPA protein to approximately 10,000 molecules/cell in a Tet-regulatable system modestly but significantly increased sensitivity to UV irradiation. No removal of cyclobutane pyrimidine dimers was detected in the SV40 immortalized cell lines tested. Repair proficient WI38-VA fibroblasts and transfected XP-A cells expressing 150,000 molecules of XPA/cell removed (6-4) photoproducts from the genome with a half-life of 1h. Cells in which XPA protein was reduced to about 10,000 molecules/cell removed (6-4) photoproducts more slowly, with a half-life of 3h. A reduced rate of repair of (6-4) photoproducts thus results in increased cellular sensitivity towards UV irradiation. These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.     

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 compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels.

These data suggest that the repair of potentíally 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.     

Mutagenesis and transformation of C3H 10T12 cells treated with ethyl methanesulfonate

Survival, mutagenesis and transformation were measured in mouse embryo C3H $\text{10}\text{T}\text{1}\text{2}$ cells following treatment with ethyl methanesulfonate (EMS). Ouabain-resistant cells and transformed cells were isolated, and reconstruction experiments were carried out to determine the optimum conditions for the measurement of mutation and transformation frequencies. Survival was measured by plating efficiency; mutagenesis was measured in terms of the induction of cells able to form colonies in the presence of ouabain; and transformation was measured by the induction of cells forming either morphologically altered colonies on a monolayer of contact-inhibited cells or of cells capable of forming colonies in semi-solid media. When confluent monolayers were incubated for 4 h after treatment with EMS, to allow excision repair before the resumption of DNA synthesis, survival as well as the frequencies of both mutation and transformation increased. When this repair (or holding) period was extended to 24 h, the frequencies of mutation and transformation both decreased as compared to the 4-h holding period. Thus, the holding periods affect the frequencies of EMS-induced mutagenesis and transformation similarly.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.     

Decreased UV sensitivity, mismatch repair activity and abnormal cell cycle checkpoints in skin cancer cell lines derived from UVB-irradiated XPA-deficient mice

Xeroderma pigmentosum group A gene (XPA)-deficient mice are defective in nucleotide excision repair (NER) and are therefore highly sensitive to ultraviolet (UV)-induced skin carcinogenesis. We established cell lines from skin cancers of UVB-irradiated XPA-deficient mice to investigate the phenotypic changes occurring during skin carcinogenesis. As anticipated, the skin cancer cell lines were devoid of NER activity but were less sensitive to killing by UV-irradiation than the XPA(-/-) fibroblast cell line. The lines were also more resistant to 6-thioguanine (6-TG) than XPA(-/-) and XPA(+/+) fibroblasts, which was suggestive of a mismatch repair (MMR) defect. Indeed, in vitro mismatch binding and MMR activity were impaired in several of these cell lines. Moreover, these cell lines displayed cell cycle checkpoint derangements following UV-irradiation and 6-TG exposure. The above findings suggest that MMR downregulation may help cells escape killing by UVB, as was seen previously for methylating agents and cisplatin, and thus that MMR deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells.     

Suppression of UV mutagenicity by human interferon

Effects of human interferon (HuIFN)-alpha on UV mutagenicity were examined in a human cell strain, RSa, and xeroderma pigmentosum (XP)-derived fibroblasts (XP1KY). The frequency of ouabain-resistance mutation in UV-irradiated RSa cells was unusually high (Suzuki et al., 1985), but that in cells pretreated with HuIFN-alpha before irradiation was reduced. 6-Thioguanine-resistance mutation was also depressed in XP1KY cells treated with HuIFN-alpha before irradiation. However, the depression of UV mutagenicity by HuIFN-alpha was lessened by treatment with cycloheximide immediately after UV irradiation. The relationship between HuIFN-depressed UV mutagenicity and HuIFN-affected DNA-repair and repair-related functions is discussed.     

Effects of cisplatin and gamma-irradiation on cell survival, the induction of chromosomal aberrations and apoptosis in SW-1573 cells

PURPOSE: Cisplatin was found to radiosensitize SW-1573 cells by inhibition of PLDR. Therefore, it was investigated whether cisplatin combined with gamma-radiation leads to an increase in the number of chromosomal aberrations or apoptotic cells compared with radiation alone. METHODS: Confluent cultures of the human lung carcinoma cell line SW-1573 were treated with 1 microM cisplatin for 1 h, 4 Gy gamma-radiation, or a combination of both. Cell survival was studied by the clonogenic assay. Aberrations were analysed by FISH in prematurely condensed chromosomes (PCC) and the induction of apoptosis by counting fragmented nuclei. RESULTS: A radiosensitizing effect of cisplatin on cell survival was observed if time for PLDR was allowed. An increased number of chromosomal fragments were observed immediately after irradiation compared with 24 h after irradiation whereas color junctions are only formed 24 h after irradiation. No increase in chromosomal aberrations was found after combined treatment, but a significantly enhanced number of fragmented nuclei were observed when confluent cultures were replated after allowing PLDR. CONCLUSION: The inhibition of PLDR by cisplatin in delayed plated SW-1573 cells did not increase chromosomal aberrations, but increased the induction of apoptosis.     

Mutation induction by different dose rates of gamma rays in radiation-sensitive mutants of mouse leukemia cells

Induction of cell killing and mutation to 6-thioguanine resistance was examined in a radiation-sensitive mutant strain LX830 of mouse leukemia cells following gamma irradiation at dose rates of 30 Gy/h (acute), 20 cGy/h (low dose rate), and 6.2 mGy/h (very low dose rate). LX830 cells were hypersensitive to killing by acute gamma rays. A slight but significant increase was observed in cell survival with decreasing dose rate down to 6.2 mGy/h, where the survival leveled off above certain total doses. The cells were also hypersensitive to mutation induction compared to the wild type. The mutation frequency increased linearly with increasing dose for all dose rates. No significant difference was observed in the frequency of induced mutations versus total dose at the three different dose rates so that the mutation frequency in LX830 cells at 6.2 mGy/h was not significantly different from that for moderate or acute irradiation.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Manganese superoxide dismutase protects the proliferative capacity of confluent normal human fibroblasts

We tested the hypothesis that manganese superoxide dismutase (MnSOD), an antioxidant enzyme, regulates the proliferative potential of confluent human fibroblasts. Normal human skin (AG01522) and lung (WI38, CCL-75) fibroblasts kept in confluence (>95% G(0)/G(1)) showed a significant decrease in their capacity to re-enter the proliferation cycle after 40-60 days. The inhibition of re-entry was accompanied with the age-dependent increase of p16 protein levels in the confluent culture. Adenoviral mediated overexpression of MnSOD during confluent growth suppressed p16, enhanced p21 protein accumulation, and protected fibroblasts against the loss of proliferation potential. Increases in p21 protein levels in MnSOD overexpressing confluent fibroblasts were independent of p53 protein levels. p53 protein levels did not change in control, replication-defective adenovirus containing an insertless vector (AdBgl II), or AdMnSOD-infected confluent cells cultured for 20 and 60 days. In addition, MnSOD-induced protection of the proliferation capacity of confluent fibroblasts was independent of their telomerase activity. However, telomerase-transformed fibroblasts showed increased MnSOD expression in confluent growth, maintaining their capacity to re-enter the proliferation cycle. Although inactivation of the retinoblastoma protein in cells subcultured from the 60-day confluent control, AdBgl II-, and AdMnSOD-infected fibroblasts was identical, only MnSOD-overexpressing cells showed a higher percentage of S-phase. These results support the hypothesis that a redox-sensitive checkpoint regulated the progression of fibroblasts from G(0)/G(1) to S-phase.     

Identification of ataxia telangiectasia heterozygotes by flow cytometric analysis of X-ray damage

Flow cytometry was used to identify heterozygotes for the autosomal recessive DNA-repair deficiency disease ataxia telangiectasia (AT). Confluent G0/G1 fibroblasts from 4 homozygotes (at/at), 5 obligate heterozygotes (at/+) and 7 presumed normal controls (+/+) were X-irradiated with 200 Rad and subcultured immediately in medium containing 5-bromodeoxyuridine (BrdU). Cells were harvested 72 h later and stained with fluoresceinated anti-BrdU antibody to identify cells that had entered S phase. They were counterstained with propidium iodide to measure total DNA content. On the basis of relative release from G0/G1, the at/+ strains as a group (33 +/- 3% release) were distinguished from both the presumed +/+ strains (60 +/- 3%) and at/at strains (85 +/- 3%), although the individual values for some strains did show overlap between genotypes. When 10 cell strains were coded and analyzed in 'blind' experiments, all 4 heterozygotes were correctly assigned, although one poorly growing presumed normal line was incorrectly assigned as a heterozygote. By a similar assay in which exponentially growing cultures were pulsed briefly with BrdU 8 h after irradiation with 400 Rad and then harvested immediately, presumed +/+ cells as a group could be distinguished from at/at cells but not from at/- cells. This combination of assays assists in the identification of all 3 AT genotypes. This should be of both basic and diagnostic use, particularly in families known to segregate AT.     

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.     

Differential response to mitomycin-C- and cis-diamminedichloroplatinum(II)-induced damage in normal human fibroblasts during confluent holding

Confluent cultures of normal human fibroblasts were treated with the chemotherapeutic agents, mitomycin C (MMC) and cis-diamminedichloroplatinum(II) (cis-Pt(II]. This treatment induced a decrease in clonogenic cell survival. Recovery of the cytotoxic effect was observed in the case of cis-Pt(II)-treated cultures maintained at confluence for 1-5 days. No such recovery was observed after treatment with MMC. These data suggest that contrary to potential lethal damage induced by cis-Pt(II) which is repaired in confluent cells, DNA damage induced by MMC is not repaired in confluent cells.     

Development of a liquid-holding technique for the study of DNA-repair in human diploid fibroblasts.

Liquid-holding conditions can be obtained for human diploid skin fibroblasts by keeping confluent cultures stationary over periods of 7 days or longer by means of conditioned medium. Under this condition recovery of radiation damage induced by ultraviolet light or X-rays is observed as an increase in cloning efficiency. The amount of recovery when expressed in a dose-modifying-factor appears higher than in bacteria and yeast. The repair-deficient human cell strains XP25Ro and XP7Be (xeroderma pigmentosum from complementation groups A and D respectively) exhibit less but still discernible recovery after UV-irradiation and the same was observed for AT5Bi (ataxia telangiectasia) after X-irradiation. Experiments on mutation induction indicated that the repair which takes place during liquid holding of UV-irradiated XP7Be cells reduces the mutant frequency considerably while after liquid holding of UV-irradiated wild-type cells the same or lower mutant frequencies were found for the lower exposures and the same or higher mutant frequencies for the higher exposures.     

Low synthesis of retinoic acid due to impaired cytochrome P450 1a1 expression in mouse xeroderma pigmentosum fibroblasts

New tumor formation was suppressed by retinoic acid (RA) administration in xeroderma pigmentosum (XP) patients who have a defect in nuclear excision repair. However, the inhibition is not due to enhanced removal of UV-damaged DNA. These results prompted us to investigate whether or not RA metabolism is abnormal in XP fibroblasts and what the underlying mechanism is. Compared with wild type fibroblasts, low activities of RA synthesis were determined on HPLC in mouse fibroblasts lacking XP group A (XPA) gene and UV-induced XPA deficient cancer cells. Moreover, we observed an impaired expression of cytochrome P450 1a1 in XPA deficient fibroblasts by RT-PCR and a decreased expression of retinoic acid receptor gamma in XPA deficient cancer cells by Western blotting. Finally, pre-treatment of RA isoforms significantly protected the XPA deficient fibroblasts from UV-induced death. These results suggest that decreased structure activity of RA synthesis, resulting from impaired mRNA expression of cytochrome P450 1a1 may, at least together with UV irradiation, involve in skin carcinogenesis in XP patients.