Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.     

Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

Abstract— The repair of UV-irradiated DNA of plasmid Yep13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, radl,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endo-nuclease increased repair. We concluded that all the mutations of yeast were probably limited only to the nuclear DNA.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.     

Flow cytometry analysis of early DNA content changes in human and monkey cells following infection with Simian Virus 40.

Simian virus 40 (SV40) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfecta (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (approximately 60%) of the cells were blocked in the G2 + M phase of the cell cycle. At later time intervals, an increase in the G1 population was demonstrated. The two monkey cell lines responded to SV40 virus with an accumulation of cells in the G2 + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV40 virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transforming) cells infected with SV40 virus.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

USE OF METABOLIC INHIBITORS TO INVESTIGATE THE EXCISION REPAIR OF PYRIMIDINE DIMERS AND NON-DIMER DNA DAMAGES INDUCED IN HUMAN AND ICR 2A FROG CELLS BY SOLAR ULTRAVIOLET RADIATION

Abstract— ICR 2A frog and normal human skin fibroblasts were exposed to either 5 J/m² of 254 nm UV or 50 kJ/m² of the Mylar-filtered solar UV wavelengths produced by a fluorescent sunlamp. Following these approximately equitoxic treatments, cells were incubated in medium containing the DNA synthesis inhibitors hydroxyurea (HU) and 1–β-D-arabinofuranosyl cytosine (ara C) for 0–20 min (human fibroblasts) or 0–4 h (frog cells) to accumulate DNA breaks resulting from enzymatic incision during excision repair. It was found that breaks were formed in human cells at about a 200-f-old higher rate compared with the ICR 2A cells indicating a relatively low capacity for excision repair in the frog cells. In addition, the rate of DNA break formation in solar UV-irradiated cells was only one-third of the level detected in 254 nm-irradiated cells. This result is consistent with the conclusion that the pathway(s) involved in the repair of solar UV-induced DNA damages differs from the repair of lesions produced in cells exposed to 254 nm UV.     

Partial complementation of the DNA repair defects in cells from xeroderma pigmentosum groups A, C, D and F but not G by the denV gene from bacteriophage T4

Endonuclease V (denV) from bacteriophage T4 was examined for its ability to complement the DNA repair defect in xeroderma pigmentosum (XP) cells from complementation groups A, C, D, F and G. The denV gene was introduced into SV40-transformed normal and XP cells using a retroviral vector. Expression of denV resulted in partial correction of UV sensitivity and increased host cell reactivation (HCR) of a UV-damaged reporter gene for XP cells from groups A, C and D, but not those from group G. Expression of denV in XP-F cells resulted in enhanced HCR of a UV-damaged reporter but did not affect UV sensitivity. The observed partial complementation is thought to reflect denV-mediated repair of cyclobutane-pyrimidine dimers (CPD), and is incomplete as denV does not recognize other UV-induced lesions, and may not even efficiently remove all CPD. As XP-F cells are believed to retain near-normal levels of CPD repair in the bulk of the genome, we believe that the disparity in the ability of denV to complement the repair deficiency in these cells results from an increased rate, but not level, of CPD repair. Furthermore, we suggest that the lack of correction in the XP-G cells examined results from an inability to process denV-incised CPD by the base excision repair pathway, as has been suggested for cells from the related genetic disorder, Cockayne syndrome. Expression of denV in repair proficient normal cells also resulted in increased HCR of the UV-damaged reporter construct, possibly arising from an increased rate of CPD repair in these cells.     

Deficient Nucleotide Excision Repair in Squamous Cell Carcinoma Cells

Squamous cell carcinomas (SCCs) are associated with ultraviolet radiation and multiple genetic changes, but the mechanisms leading to genetic instability are unclear. SCC cell lines were compared to normal keratinocytes for sensitivity to ultraviolet radiation, DNA repair kinetics and DNA repair protein expression. Relative to normal keratinocytes, four SCC cell lines were all variably sensitive to ultraviolet radiation and, except for the SCC25 cell line, were deficient in global repair of cyclobutane pyrimidine dimers, although not 6‐4 photoproducts. Impaired DNA repair of cyclobutane pyrimidine dimers was associated with reduced mRNA expression from XPC but not DDB2 genes which each encode key DNA damage recognition proteins. However, levels of XPC or DDB2 proteins or both were variably reduced in repair‐deficient SCC cell lines. p53 levels did not correlate with DNA repair activity or with XPC and DDB2 levels, but p63 levels were deficient in cell lines with reduced global repair. Repair‐proficient SCC25 cells depleted of p63 lost XPC expression, early global DNA repair activity and UV resistance. These results demonstrate that some SCC cell lines are deficient in global nucleotide excision repair and support a role for p63 as a regulator of nucleotide excision repair in SCCs.     

DNA DAMAGE AND REPAIR IN HUMAN CELLS EXPOSED TO SUNLIGHT

Cultured human cells were treated with direct sunlight under conditions which minimised the hypertonic, hyperthermic and fixative effects of solar radiation. Sunlight produced similar levels of DNA strand breaks as equitoxic 254 nm UV in two fibroblast strains and a melanoma cell line, but DNA repair synthesis and inhibition of semiconservative DNA synthesis and of DNA chain elongation were significantly less for sunlight-exposed cells. DNA breaks induced by sunlight were removed more rapidly. Thus, the repair of solar damage differs considerably from 254 nm UV repair. Glass-filtered sunlight (> 320 nm) was not toxic to cells and did not induce repair synthesis but gave a low level of short-lived DNA breaks and some inhibition of DNA chain elongation; thymidine uptake was enhanced. Filtered sunlight slightly enhanced UV-induced repair synthesis and UV toxicity; photoreactivation of UV damage was not found. Attempts to transform human fibroblasts using sunlight, with or without phorbol ester, were unsuccessful.     

Activation of the stress-activated JNK and p38 MAP kinases in human cells by Photofrin-mediated photodynamic therapy

We have examined the possible role of the stress-activated JNK and p38 protein kinases in cellular sensitivity following Photofrin-mediated photodynamic therapy (PDT). Previously we reported that immortalized Li-Fraumeni syndrome (LFS) cells are more resistant to Photofrin-mediated PDT compared to normal human fibroblasts (NHF) at equivalent cellular Photofrin levels. In the current work we report that Photofrin-mediated PDT increased the activity of JNK1 and p38 within 30 min in both cell types. However, the increased activity of JNK1 and p38 was transient in the sensitive NHF cells and returned back to near basal levels by 3 h after PDT. In contrast, the resistant LFS cells exhibited a more prolonged activation of JNK and p38, which lasted for at least 11 h and 7 h after PDT, respectively. Blocking of the p38 pathway in LFS cells by transient infection with a recombinant adenovirus expressing a dominant negative mutant of p38 or in HeLa cells by stable transfection with a dominant negative mutant of p38 had no effect on cell survival following PDT. These data suggest that although Photofrin-mediated PDT is able to induce JNK1 and p38 in human cells, the p38 pathway alone does not play a major role in the sensitivity of LFS cells to Photofrin-mediated PDT.     

EXCISION OF CYCLOBUTANE DIMERS IN GENOMIC AND EPISOMAL DNA IN HUMAN CELLS

Abstract Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo . Maintaining SV40-transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed cyclobutane dimers were induced at 1.5–2-fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.     

Replication in UV-lrradiated Caenorhabditis elegans Embryos

Abstract— Replication continues in wild-type (but not rad mutant) Caenorhabditis elegans embryos even after exposure to massive fluences of UV radiation. It is of interest to elucidate the mechanism(s) for this "damage-resistant" DNA synthesis. In this study, DNA from unirradiated and UV-irradiated wild-type embryos was examined using the electron microscope. Large fluences of UV radiation (180 J m⁻²) had little effect on either replication bubble size or distances between bubbles in wild-type embryos, indicating that the damage-resistant DNA synthesis was not grossly aberrant. Conversely, UV irradiation significantly decreased center-to-center distances between bubbles in excision-repair-deficient rad-3 embryos. This suggests that the decreased DNA synthesis observed after UV irradiation in rad-3 embryos is due largely to blockage of elongation of DNA synthesis.     

INDUCIBLE POSTREPLICATION REPAIR IS RESPONSIBLE FOR MINIMAL MEDIUM RECOVERY IN UV-IRRADIATED Escherichia coliK–12

Abstract— Ultraviolet (UV)-irradiated Escherichia coli K–12 uvrA cells showed higher survival if plated on minimal growth medium rather than on rich growth medium, i.e., they showed minimal medium recovery (MMR). A 2-hour treatment of UV-irradiated cells with rifampicin inhibited the subsequent expression of MMR, and produced a large reduction in survival. We have recently isolated a new mutant ( mmrA1 ) that does not show MMR. The mmrA mutation protected UV-irradiated uvrA cells from the effect of rich growth medium on survival, but not from the effect of rifampicin on survival. DNA daughter-strand gap (DSG) repair in UV-irradiated (4 J/m²) uvrA cells was inhibited to the same degree whether rich growth medium was added immediately after irradiation or after 10 min of postirradiation incubation in minimal growth medium. However, chloramphenicol added immediately after irradiation greatly reduced this repair; there was less reduction if it was added 10 min after UV irradiation. These findings suggest that MMR is an inducible repair phenomenon, and that rich growth medium inhibits this repair process itself rather than its induction.     

Inverse relationship between increased apoptosis and decreased skin cancer in UV-irradiated CD1d-/- mice

We previously demonstrated that CD1d knockout mice were resistant to ultraviolet (UV)-induced immunosuppression. Because immune suppression is a critical factor in the development of UV-induced skin cancers, we investigated the response of wild type (WT) and CD1d-/- mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d-/- mice developed skin tumors. To investigate the mechanisms involved in the resistance of CD1d-/- mice to UV-induced carcinogenesis, we determined the time course and kinetics of keratinocyte cell death after UV irradiation. After acute UV exposure, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive keratinocytes were eliminated from the skin of WT mice by 72 h post-UV, but they still persisted until 96 h in CD1d-/- mice. The kinetics of p53 protein expression closely followed the kinetics of apoptotic cell death. Chronic UV irradiation resulted in induction of a significantly higher number of apoptotic keratinocytes in CD1d-/- than WT mice. In addition, epidermis and dermis from chronically UV-irradiated CD1d-/- mice harbored significantly fewer p53 mutations than WT mice. These results indicate that the resistance of CD1d-/- mice to UV carcinogenesis may be due to increased cell death and elimination of keratinocytes and fibroblasts containing DNA damage and p53 mutations.