Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P = 0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage.     

Differential effects of hypoxia and acidosis on p53 expression,repair of UVC-damaged DNA and viability after UVC in normal and tumor-derived human cells

Hypoxia and low pH are commonly associated with the tumor microenvironment. We have examined the effects of hypoxia alone (HA) and hypoxia coupled to low pH (HApH) on p53 expression, nucleotide excision repair (NER) and cellular sensitivity to UVC in normal human fibroblasts and human tumor cells. p53 expression was measured using Western blotting, NER using host cell reactivation (HCR) of a UV-damaged reporter gene and cell sensitivity using the MTT assay. HApH resulted in a transient increase in p53 expression in normal fibroblasts at 6 h and in tumor cells at 6–18 h. In normal fibroblasts HApH resulted in a transient increase in HCR at early times (12–24 h) and a concomitant decrease in UVC sensitivity. In contrast, for the tumor-derived cells, the increased HCR of the UVC-treated reporter gene was delayed (36–40 h) and UVC sensitivity increased or remained the same after HApH treatment. These results suggest that early upregulation of p53 and increased repair of UV-damaged DNA after HApH treatment is required for increased cell viability after UVC. HA treatment alone also resulted in a transient increase in HCR of the UVC-damaged reporter gene at early times (12–24 h) in normal fibroblasts and a delayed increase (36–40 h) in the tumor-derived cells. However, the enhanced p53 expression was less or even absent for treatment with HA alone, and HA had no significant affect on cell viability after UVC for any of the cell lines. These results indicate a different cellular response following HApH compared to HA alone.     

Host-cell reactivation of UV-irradiated adenovirus in Cockayne syndrome

Measurements of the host-cell reactivation (HCR) of mutagen-treated virus provides a very sensitive tool for detecting abnormal DNA repair. The best example of the utility of HCR studies in the examination of the DNA-repair capacity of human cells has come from studies of cells from the UV-sensitive repair-deficient xeroderma pigmentosum (XP) patients. We have examined the HCR of UV-treated adenovirus type 5 (Ad5) and type 2 (Ad2) in cells from patients with Cocayne syndrome (CS), another sun-sensitive syndrome whose cells also exhibits UV-sensitivity in culture. Comparisons with obligate heterozygotes and normal controls failed to reveal an abnromality in the HCR capacity of the CS cells. As the abnormality in DNA metabolism in CS appears to be in a late step in excision repair, a bypass mechanism may exist in these cells for circumventing the defect in the repair of viral DNA.     

Use of host cell reactivation of cisplatin-treated adenovirus 5 in human cell lines to detect repair of drug-treated DNA

This study demonstrates that whilst some DNA-repair deficiencies can be detected using host cell reactivation of cisplatin (CDDP)-treated adenovirus (Ad5), not all repair deficiencies affected replication of CDDP-treated Ad5 in human cells. A line of fibroblasts (XP25), derived from a patient with a UV-hypersensitive syndrome xeroderma pigmentosum (XP), was found, as previously reported [1], to be deficient in reactivating the treated virus when compared to the apparently repair-proficient human tumor cell lines established from bladder and ovarian carcinomas. However, a testicular teratoma cell line (SuSa), shown previously to be deficient in the repair of guanine-guanine (G-G) intrastrand crosslinks, adenine-guanine (A-G) intrastrand crosslinks and interstrand crosslinks [2], was found to reactivate the treated virus to a similar extent as the repair-proficient ovarian tumor cell line and the similarly repair-proficient RT112 cell line derived from a bladder carcinoma. Therefore, not all repair-deficient cell lines were deficient at CDDP-treated Ad5 reactivation. However, the HCR technique may still prove to be useful as a rapid screen for DNA-repair deficiencies in CDDP-sensitive cells of unknown repair capacity. A CDDP-sensitive ovarian tumor cell line (TR175) was deficient in reactivating CDDP-treated Ad5, whilst another ovarian cell line (TR170) of intermediate CDDP sensitivity reactivated the virus to a marginally higher extent than the other more CDDP-resistant repair proficient ovarian cell line (SKOV3). In addition, sublines of either the SuSa cells or the RT112 cells expressing approximately two-fold levels of resistance or increased sensitivity to CDDP, showed no change in their abilities to reactivate this CDDP-treated virus, compared to their parental lines. CDDP-treated Ad5 was also used as a lethal probe to obtain cell lines specifically deficient in DNA repair. One such deficient line (SKOV3-C3A), derived from the SKOV3 ovarian carcinoma cell line, displayed an unusual biphasic curve for reactivation of the CDDP-treated virus. Further cell lines derived in this novel manner may prove useful in analysing the genetics of CDDP-repair.     

Gamma-ray-enhanced reactivation of UV-irradiated adenovirus in normal human fibroblasts.

An enhanced reactivation of UV-irradiated adenovirus type 2 (Ad 2) was detected following irradiation of the host cells with γ-rays prior to infection. Non-irradiated and γ-irradiated normal human fibroblasts were infected immediately after irradiation with either non-irradiated or UV-irradiated Ad 2. At 48h after infection, cultures were examined by indirect immunofluorescence to determine the number cells in which the viral function of viral structural antigen (Vag) was expressed. Pre-irradiation of cells with 1 krad resulted in a 2–3-fold increase in the survival of this viral function following different UV doses to the virus up to 1.75 × 10³ J/m². For a fixed UV dose of 1.0 × 10³ J/m² to the virus this enhancement increased with preirradiation dose to the cells up to a maximum factor of 2–3 for a dose of 1 krad. An examination of Vag expression at various times after infection indicates that pre-irradiation of the cells with γ-rays prior to infection with UV-irradiated virus leads to an earlier onset and/or increased rate of Vag synthesis. This enhancement of Vag production from a UV-damaged template may result from an inducible DNA-repair mechanism in human fibroblasts which may or may not be error-prone.     

Acetylation regulates DNA repair mechanisms in human cells

The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.     

A deficiency in the repair of UV and γ-ray damaged DNA in fibroblasts from Cockayne's syndrome

The host-cell reactivation of V antigen production for irradiated adenovirus was examined in fibroblasts from 5 unrelated patients with Cockayne's syndrome (CS) and 2 CS heterozygotes. The fibroblast cultures were infected with either irradiated or non-irradiated adenovirus and subsequently examined for the presence of viral structural antigens using immunofluorescent staining. All CS-homozygous strains showed a reduced host-cell reactivation (HCR) of this viral function for both UV- and γ-irradiated virus. For UV-irradiation of the virus, D₃₇ values expressed as a percentage of that obtained on normal strains, ranged from 14 to 35%. For γ-irradiation of the virus these values ranged from 61 to 80%. These results indicate some defect in the repair of both UV- and γ-ray-induced DNA damage for CS. 1 CS-heterozygote strain tested also showed a reduced HCR for UV-irradiated adenovirus intermediate between that of the patient strain and normal, whereas another CS-heterozygote strain showed an apparently normal HCR level.     

Evaluation of recombination repair pathways in thermal radiosensitization

Thermal radiosensitization has been shown to cause inhibition of repair of sublethal and potentially lethal damage and DNA DSBs. In this study we assessed thermal radiosensitization in mutants deficient in homologous recombinational (HR) repair and nonhomologous end joining repair (NHEJ). Using cells of the mouse wild-type embryo fibroblast cell line MEF and its Ku80(-/-) derivative that is deficient in NHEJ, we showed that thermal radiosensitization is the same in both cell lines. Further studies with cells of the wild-type CHO-AA8 cell line and its derivative IRS(ISF), which is deficient in HR, also showed comparable thermal radiosensitization in both cell lines. Further experiments using cells of chicken DT40 cell lines also showed comparable thermal radiosensitization between the wild-type HR mutant Rad54, the NHEJ mutant Ku70, and the double mutant Rad 54-Ku70. These results indicate that the HR and NHEJ pathways may not be targets for thermal radiosensitization.