DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.     

The interplay among chromatin dynamics, cell cycle checkpoints and repair mechanisms modulates the cellular response to DNA damage

Cells are continuously under the assault of endogenous and exogenous genotoxic stress that challenges the integrity of DNA. To cope with such a formidable task cells have evolved surveillance mechanisms, known as checkpoints, and a variety of DNA repair systems responding to different types of DNA lesions. These lesions occur in the context of the chromatin structure and, as expected for all DNA transactions, the cellular response to DNA damage is going to be influenced by the chromatin enviroment. In this review, we will discuss recent studies implicating chromatin remodelling factors and histone modifications in the response to DNA double-strand breaks (DSBs) and in checkpoint activation in response to UV lesions.     

Interaction between daunorubicin and chromatin from Ehrlich ascites tumor cells.

Interaction of daunorubicin with chromatin from Ehrlich ascites tumor cells has been studied by spectrofluorimetry. Daunorubicin interacts with chromatin and displays at least two types of binding. The number of binding sites is reduced when compared to deoxyribonucleic acid. There is no difference in the overall structure of chromatins extracted from cells sensitive or resistant to daunorubicin.     

Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene

In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.     

The elimination of mutagenic lesions induced by alkylating agents and UV in V79 Chinese hamster cells

The mutagenicity of MNU, EMS, BMS and UV light was compared by analyzing the dose-response curve just before and after the replicative process of the HGPRT gene in synchronized V79 Chinese hamster cells. This system makes it possible to compare a 10-h period for repair of different mutagenic lesions with no time for repair. Additional time for repair in synchronized V79 cells resulted in a reduced response for MNU and UV, but not for EMS and BMS. This result suggests that an error-free repair process operates on mutagenic lesions in methylated DNA and on thymine dimers, but not on ethylated and butylated DNA. Based on these results, it is concluded that the repair capacity of V79 cells to remove mutagenic lesions is characterized as low for UV, moderate for MNU and not detectable for the mutagenic lesions induced by EMS and BMS.     

2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells

The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 micrograms/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture (greater than 5 micrograms/ml).     

Exposure of E. coli to nitrosocimetidine induces the adaptive response to alkylating agents

The cytotoxic and mutagenic properties of nitrosocimetidine (NC), together with its ability to induce the adaptive response DNA-repair pathway were compared with those of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) using Escherichia coli as test organism. MNNG was found to be 250-fold more cytotoxic and 500-fold more mutagenic than NC. Prior cultivation of E. coli in low concentrations of NC protected it against the cytotoxic and/or mutagenic effects of challenge with either NC or MNNG or methyl methanesulphonate (MMS). Induction of the adaptive response by prior cultivation in low concentrations of MNNG reduced the mutagenic and cytotoxic effects of subsequent NC challenge. These results lead us to conclude that although NC is a less potent mutagen than MNNG, the DNA lesions it produces are capable of not only inducing, but also of being repaired by, the adaptive response.     

The involvement of antigen-presenting cells and suppressor cells in the ultraviolet radiation-induced inhibition of secondary cytotoxic T cell sensitization

When mice were treated with ultraviolet (UV) radiation before immunization with the skin sensitizers, trinitro-chlorobenzene or dinitrofluorobenzene, they showed greatly depressed levels of priming for a secondary in vitro cytotoxic response against haptenated cells. Because the irradiation and skin painting were done on separate sites, these results suggest that the UV radiation had a systemic effect. The priming response of irradiated mice to allogeneic stimulation was normal, indicating some antigen selectivity to the inhibitory effect of UV radiation. A defect in antigen-presenting cells, previously demonstrated in UV-irradiated mice, was found to be largely responsible for the depressed priming response observed in these animals. In addition, the UV-irradiated, immunized mice possessed suppressor cells that were capable of blocking priming for cytotoxic responses against haptenated cells in normal mice.     

Mutation induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis.

Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr-, ssp-, hcr-ssp-) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotrophic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores, however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity.     

Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radiation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.     

DNA repair in a small yeast plasmid folded into chromatin.

The question of whether excision repair of yeast plasmids accurately reflects the repair of yeast genomic chromatin has yielded conflicting answers. These conflicts could have arisen from differences in the conformation of plasmid molecules used during these studies. We have examined excision repair of UV photoproducts in a small (2619 bp) autonomously replicating plasmid (YRp-TRURAP), known to be folded into chromatin with positioned nucleosomes in vivo, in the yeast Saccharomyces cerevisiae. A quantitative assay was used to measure the yield of cyclobutane pyrimidine dimers (PD) in plasmid DNA by measuring the fraction of Form I molecules resistant to T4 endonuclease V. After a UV dose of 100 J/m2, which yields 1.2 PD/plasmid in irradiated cells, radiation insensitive (wt) cells repair approximately 70% of the PD in TRURAP chromatin in 2 hr (a rate comparable to that of genomic chromatin). On the other hand, no measurable repair occurs in TRURAP chromatin in radiation sensitive cells (rad1) during the same time period. Thus, this small plasmid contains sufficient chromatin structure in vivo to reflect the incompetent repair of genomic chromatin seen in a rad mutant, while maintaining the competent repair level in wt cells.     

Sensitization to UV-induced apoptosis by the histone deacetylase inhibitor trichostatin A (TSA)

UV-induced apoptosis is a protective mechanism that is primarily caused by DNA damage. Cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts are the main DNA adducts triggered by UV radiation. Because the formation of DNA lesions in the chromatin is modulated by the structure of the nucleosomes, we postulated that modification of chromatin compaction could affect the formation of the lesions and consequently apoptosis. To verify this possibility we treated human colon carcinoma RKO cells with the histone deacetylase inhibitor trichostatin A (TSA) prior to exposure to UV radiation. Our data show that pre-treatment with TSA increased UV killing efficiency by more than threefold. This effect correlated with increased formation of CPDs and consequently apoptosis. On the other hand, TSA treatment after UV exposure rather than before had no more effect than UV radiation alone. This suggests that a primed (opened) chromatin status is required to sensitize the cells. Moreover, TSA sensitization to UV-induced apoptosis is p53 dependent. p53 and acetylation of the core histones may thus contribute to UV-induced apoptosis by modulating the formation of DNA lesions on chromatin.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

The influence of the wavelength of ultraviolet radiation on survival,mutation induction and DNA repair in irradiated chinese hamster cells

Chinese hamster ovary cells were used to compare the cytotoxicity and mutagenicity of far-UV radiation emitted by a low-pressure mercury, germicidal lamp (wavelength predominantly 254 nm) with that of near-UV radiation emitted by a fluorescent lamp with a continuous spectrum (Westinghouse “Sun Lamp”), of which only the radiation with wavelengths greater than 290 nm or greater than 310 nm was transmitted to the cells. The radiation effects were compared on the basis of an equal number of pyrimidine dimers, the predominant lesion induced in DNA by far-UV, for the induction of which much more energy is needed with near-UV than with 254-nm radiation.The numbers of dimers induced were determined by a biochemical method detecting UV-endonuclease-susceptible sites. The equivalence of these sites with pyrimidine dimers was established, qualitatively and quantitatively, in studies with enzymic photoreactivation in vitro and chromatographic analysis of dimers.On the basis of induced dimers, more cells were killed by >310-nm UV than by >290-nm UV; both forms of radiation were more cytotoxic than 254-nm UV when equal numbers of dimers were induced. Moreover, 5–6 times as many mutants were induced per dimer by >310-nm UV than by >290-nm UV; the latter appeared approximately as mutagenic as 254-nm UV. The differences in lethality and mutagenicity were not caused by differences in repair of dimers: cells with an equal number of dimers induced by either 254-nm or near-UV showed the same removal of sites susceptible to a UV endonuclease specific for dimers, as well as an identical amount of repair replication.The results indicate that near-UV induces, besides pyrimidine dimers, other lesions that appear to be of high biological significance.     

Papillomaviruses: death-defying acts in skin cancer

Ultraviolet (UV) radiation is an environmental agent that has a major impact on humans, and cumulative exposure poses a serious risk in terms of developing skin cancer. Acute doses of UV induce apoptotic cell death in the skin via signalling pathways that are, in part, dependent on the p53 tumour suppressor protein. However, p53-independent mechanisms have also been described. Recent findings show that a high proportion of non-melanoma skin cancers contain human papillomavirus. The viral E6 protein effectively blocks the epidermal apoptotic response to UV and might play a key role in promoting tumour development in cooperation with the mutagenic effects of UV.     

Effects of histone acetylation and CpG methylation on the structure of nucleosomes

Nucleosomes are the fundamental packing units of the eukaryotic genome. A nucleosome core particle comprises an octameric histone core wrapped around by ~147bp DNA. Histones and DNA are targets for covalent modifications mediated by various chromatin modification enzymes. These modifications play crucial roles in various gene regulation activities. A group of common hypotheses for the mechanisms of gene regulation involves changes in the structure and structural dynamics of chromatin induced by chromatin modifications. We employed single molecule fluorescence methods to test these hypotheses by monitoring the structure and structural dynamics of nucleosomes before and after histone acetylation and DNA methylation, two of the best-conserved chromatin modifications throughout eukaryotes. Our studies revealed that these modifications induce changes in the structure and structural dynamics of nucleosomes that may contribute directly to the formation of open or repressive chromatin conformation.     

Studies of the mechanisms for the induction of in vivo tumor immunity. V. A comparison of the generation of the primary cell-mediated cytotoxic response using in vitro mixed-lymphocyte--tumor cell culture and an in vivo technique

In vivo immunization of C57BL/6 mice with FBL-3, a syngeneic Friend-virus-induced leukemia, can induce a specific, T-cell-mediated cytotoxic response, as measured by the ¹²⁵IUdR release assay. In vitro it was difficult to generate an analogous, primary cytotoxic response, using a normal spleen responder population. After modification of the splenic responders by adding normal peritoneal cells, this modified population then had the capacity to mount a primary cytotoxic response in the mixed-lymphocyte-tumor cell culture reaction to FBL-3. We have characterized the effector population as well as the helper (peritoneal) cells which were responsible for elevating the cytolytic response to FBL-3. The results indicate that there are at least two populations of cells which are essential for inducing a primary cell-mediated cytotoxic response. First, the effectors which are directly responsible for mediating the cytotoxic reactions and are derived from radiosensitive T cells and second, a helper cell population which is radioresistant and has the characteristics of macrophages.     

DNA lesion and mutagenesis induced in phageM13mp2 by UVA, UVB and UVC irradiation.

Sunlight is carcinogenic and mutagenic and its genotoxic effects are believed to be the result of UV light-induced lesions in DNA. These lesions include pyrimidine dimers and (6-4) photoproducts, but it is uncertain whether the pyrimidine modifications are the sole pre-mutagenic lesions induced by UV light. Previous studies indicate that some sunlight-induced mutations in the single-stranded DNA phage M13mp2 may not be caused by these photoproducts. In this work, purified single-stranded phage DNA was exposed to UVA, UVB and UVC and the induced mutations were analyzed. All 3 types of UV light increase the mutation frequency. The mutants were sequenced and the results suggest that UVA exposure may induce formation of a non-dipyrimidine lesion in DNA.