LACK OF INHIBITION OF ULTRAVIOLET RADIATION-INDUCED ORNITHINE DECARBOXYLASE ACTIVITY BY RETINOIC ACID

Abstract— Normally present at low levels, ornithine decarboxylase (ODC) activity is induced to higher levels in animal skin by such disparate agents as tumor promoter 12–0-tetradecanoylphorbol-13-acetate (TPA), UV radiation, and hair plucking. Retinoids are known to inhibit induction by TPA. Repeated applications of retinoic acid (RA) in acetone have also been reported to inhibit UV-induced ODC in hairless mice. As a preliminary study, it was of interest to know whether RA in a cream vehicle would have the same effect. Groups of Skh-hairless-1 albino mice were irradiated once with Westinghouse FS-20 lamps (0.045 Joules/cm^/). Immediately post-irradiation, RA was applied to the dorsum in different concentrations (0.001%, 0.002%, 0.02%), vehicles (cream and acetone) and on various time schedules (1–5 times). Sacrifice was by cervical dislocation 24 h later. Epidermis was obtained by mild heat separation and two epidermal sheets were pooled for each extract. In all experiments, the 70-fold increase in UV-induced ODC activity was further increased by retinoic acid by a factor ∼ 1.6. Since ODC levels are usually elevated in proliferating systems, the results are in concordance with the fact that both UV radiation and RA induce epidermal hyperplasia.     

EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

TOXICITY, DNA DAMAGE AND INHIBITION OF DNA REPAIR SYNTHESIS IN HUMAN MELANOMA CELLS BY CONCENTRATED SUNLIGHT

A 1 m diameter water lens was used to focus solar radiation, giving an 8-fold concentration of the total spectrum and a cytocidal flux similar to that of laboratory UV sources. Survival curves for human melanoma cells were similar for sunlight and 254 nm UV, in that D _q, was usually larger than D _o. An xeroderma pigmentosum lymphoblastoid line was equally sensitive to both agents and human cell lines sensitive to ionizing radiation (lymphoblastoid lines), crosslinking agents or monofunctional alkylating agents (melanoma lines) had the same 254 nm UV and solar survival responses as appropriate control lines. Two melanoma sublines derived separately by 16 cycles of treatment with sunlight or 254 nm UV were crossresistant to both agents. In one melanoma cell line used for further studies, DNA strand breaks and DNA-protein crosslinking were induced in melanoma cells by sunlight but pyrimidine dimers (paper chromatography) and DNA interstrand crosslinking (alkaline elution) could not be detected. The solar fiuence response of DNA repair synthesis was much less than that from equitoxic 254 nm UV, reaching a maximum near the D _o value and then declining; semiconservative DNA synthesis on the other hand remained high. These effects were not due to changes in thymidine pool sizes. Solar exposure did not have a major effect on 254 nm UV-induced repair synthesis.     

GERMICIDAL ULTRAVIOLET LIGHT INDUCES ORNITHINE DECARBOXYLASE IN MOUSE EPIDERMAL CELLS AND MODIFIES THE INDUCTION CAUSED BY PHORBOL ESTER TUMOR PROMOTERS

Germicidal ultraviolet light (UVC. 8–10 J/m²) induces ornithine decarboxylase (ODC) in mouse epidermal cells in vitro in a biphasic manner with maxima of 2–3 fold induction at 4–6 h and of 10–20 fold induction at 15–18 h after irradiation. At this dose of UVC overall protein synthesis is inhibited by 10–30% and RNA synthesis by 40–50%. Induction of both ODC peaks is prevented by actinomycin D or cycloheximide. Similar culture factors appear to influence the extent of ODC induction by UVC and by the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), since the ratio of peak activities is approximately constant at 2, whereas absolute values vary considerably between experiments. If cells are irradiated with UVC and then exposed to TPA, the effects are additive at 10 J/m², less than additive at higher and enhanced at lower doses of UVC.     

EXCISION REPAIR IN TETRAHYMENA: EVENTS FOLLOWING REFEEDING OF STARVED UV-IRRADIATED CELLS

Abstract —Starvation of early-log-phase Tetrahymena pyriformis in non-nutrient phosphate buffer for 24 h results in a 40 per cent increase in cell number, as well as a complete cessation of DNA synthesis. Low levels of DNA synthesis are detectable between 1 and 2h after starved cells are transferred to a nutrient medium. Larger amounts of DNA synthesis are detected after the first 2 h of refeeding, and one round of replication is complete 4.5 h after refeeding. Damage, caused by sublethal doses of UV radiation (254 nm) administered just prior to refeeding, to the DNA of starved Tetrahymena appears to be corrected by an excision-repair process after refeeding of starved, irradiated cells. Changes in buoyant density of DNA synthesized, rate of DNA synthesis, and the chromatographic distribution of photoproducts were investigated following refeeding of starved, irradiated cells. Excision repair begins 1 h after refeeding and appears to be essentially complete within 7 h. During this time, thymine dimers produced by irradiation are removed. Semiconservative DNA synthesis commences 2–3 h after the first appearance of excision repair. In addition, between 3 and 8 h after refeeding, the rate of DNA synthesis in irradiated, refed cultures is much lower than the rate of DNA synthesis in unirradiated, refed cultures. Also, the specific activity in vitro of DNA polymerase from irradiated refed cells is very much greater than that of polymerase from unirradiated, refed cells.     

QUANTITATION OF PYRIMIDINE DIMERS BY IMMUNOSLOT BLOT FOLLOWING SUBLETHAL UV-IRRADIATION OF HUMAN CELLS

An immunoslot blot assay was developed to detect pyrimidine dimers induced in DNA by sublethal doses of UV (254 nm) radiation. Using this assay, one dimer could be detected in 10 megabase DNA using 200 ng or 0.5 megabase DNA using 20 ng irradiated DNA. The level of detection, as measured by dimer specific antibody binding, was proportional to the dose of UV and amount of irradiated DNA used. The repair of pyrimidine dimers was measured in human skin fibroblastic cells in culture following exposure to 0.5 to 5 J m^-2 of 254 nm UV radiation. The half-life of repair was approximately 24, 7 and 6 h in cells exposed to 0.5, 2 and 5 J m^-2 UV radiation, respectively. This immunological approach utilizing irradiated DNA immobilized to nitrocellulose should allow the direct quantitation of dimers following very low levels of irradiation in small biological samples and isolated gene fragments.     

INACTIVATION OF NUCLEOTIDE EXCISION REPAIR IN CHINESE HAMSTER OVARY CELLS BY EXPOSURE TO AN ALKYLATING AGENT

Abstract— Unscheduled DNA synthesis and excision of pyrimidine dimers in Chinese hamster ovary cells irradiated with UV light were inhibited by prior exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)(1–10 μ M ) Although the pathways for excision of pyrimidine dimers and alkylation damage are known to differ, alkylations from MNNG exposure appear to have a direct effect on the nucleotide excision repair system. These results indicate that the method of exposing cells to two DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.     

ALPHA POLYMERASE INVOLVEMENT IN EXCISION REPAIR OF DAMAGE INDUCED BY SOLAR RADIATION AT DEFINED WAVELENGTHS IN HUMAN FIBROBLASTS

Abstract Cultured fibroblasts derived from normal human skin have been irradiated at a series of monochromatic wavelengths throughout the ultraviolet region and exposed to the specific α polymerase inhibitor, aphidicolin (1 μg/m l , 2 days) prior to assay for colony forming ability. Repair of 75-80% of the lethal damage induced by UVC (254 nm) or UVB (302 nm, 313 nm) radiation is inhibited by aphidicolin suggesting that such damage is repaired by a common α polymerase dependent pathway. Exposure to aphidicolin after irradiation at longer UVA (334 nm, 365 nm) or a visible (405 nm) wavelength leads to slight protection from inactivation implying that the processing of damage induced in this wavelength region is quite distinct from that occurring at the shorter wavelengths and does not involve α polymerase.     

INDUCTION OF DNA-PROTEIN CROSSLINKS IN HUMAN CELLS BY ULTRAVIOLET and VISIBLE RADIATIONS: ACTION SPECTRUM

Abstract— DNA-protein crosslinking was induced in cultured human P3 teratocarcinoma cells by irradiation with monochromatic radiation with wavelengths in the range254–434 nm (far-UV, near-UV, and blue light). Wavelength 545 nm green light did not induce these crosslinks, using the method of alkaline elution of the DNA from membrane filters. The action spectrum for the formation of DNA-protein crosslinks revealed two maxima, one in the far-UV spectrum that closely coincided with the relative spectrum of DNA at 254 and 290 nm, and one in the visible light spectrum at 405 nm, which has no counterpart in the DNA spectrum. The primary events for the formation of DNA-protein crosslinks by such long-wavelength radiation probably involve photosensitizers. This dual mechanism for DNA-protein crosslink formation is in strong contrast to the single mechanism for pyrimidine dimer formation in DNA, which apparently has no component in the visible light spectrum.     

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.     

SITES OF PHOTODYNAMICALLY INDUCED DNA REPAIR IN HUMAN CELLS

Abstract Human REH cells were incubated with the photosensitizers meso -tetra(4-sulfonatophenyl)porphyrin (TSPP=TPPS₄) or meso -tetra(3-hydroxyphenyl)porphyrin (3-THPP). The relatively hydrophilic TSPP was partly found in the cytoplasm and partly in the nuclei, whereas the lipophilic 3-THPP was found apparently in membranes and not inside the nuclei. After illumination, sites of DNA repair were labeled by means of a monoclonal antibody against proliferating cell nuclear antigen (PCNA) bound in the nuclei. The amount of bound PCNA in non-S-phase cells was proportional to the light dose. The bound PCNA was homogeneously distributed in the nuclei 0.5 h after photodynamic treatment (PDT) with TSPP. In contrast, for cells given PDT with 3-THPP, the periphery of the nuclei was selectively labeled, indicating that the initial DNA damage was localized close to the sensitizer at the nuclear membrane.     

DNA REPAIR IN ULTRAVIOLET LIGHT IRRADIATED HeLa CELLS AND ITS REVERSIBLE INHIBITION BY HYDROXYUREA

Abstract— –The repair of u.v. damaged DNA in HeLa cells can be detected using the alkaline sucrose gradient technique. As a result of pyrimidine dimer excision single strand breaks are produced in DNA of irradiated cells. Rejoining of these breaks occurs during an 8 hr post-irradiation incubation period and is prevented by hydroxyurea and acriflavine. The inhibition of repair by hydroxyurea can be reversed by a mixture of all 4 deoxyribonucleosides at a concentration that does not reverse the inhibition of total DNA synthesis.     

INHIBITION OF DNA REPAIR SYNTHESIS BY SUNLIGHT

Abstract— DNA repair synthesis as determined by thymidine incorporation in the presence of hydroxyurea reached a much lower maximum level after solar compared with UVC exposure in five human melanoma cell lines, in HeLa cells, and in two human fibroblast strains. This finding was confirmed by determination of unscheduled DNA synthesis where both the number of labelled nuclei and grain count per nucleus were lower in sun-exposed cells. In a cloned human melanoma line (MM253cl), glass-filtered sunlight inhibited UVC repair synthesis, and solar UVB alone induced a higher level of repair synthesis than either complete sun or solar UVA plus solar UVB. The fluence response of filtered sunlight for inhibition of UVB (sunlamp) and UVC showed that most inhibition was obtained at low fluences (5-10 min), further exposure giving a plateau at 40% of the original level. Ultraviolet C and sunlight inactivated adenovirus 5 giving F ₀ values for virus survival 40-fold higher than for cell survival. Replication of either UVC- or solar-irradiated virus was not affected by prior irradiation of cells with glass-filtered sunlight. Stathmokinetic analysis of cell cycle progression by DNA flow cytometry showed that UVC and sunlamp UVB retarded cell movement from the G1 and S phases whereas equitoxic sunlight and glass-filtered sunlight (nontoxic) had no effect. These results indicate that solar UVA at low environmental fluences partially inhibits UVB repair synthesis in a range of human cell types but does not affect the replication of a UVB- or UVC-damaged virus when applied to the genome alone or to the host cell.     

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.     

REPAIR SYNTHESIS IN HUMAN LYMPHOCYTES PROVOKED BY PHOTOLYSIS OF ETHIDIUM AZIDE

Abstract— The azide analog of ethidium was mixed with human lymphocytes and when photolyzed with visible light provoked repair synthesis as shown by incorporation of tritiated thymidine in the presence of hydroxyurea. The use of photolyzed drug, or incubation of drug-cell mixtures in the dark was without effect. These experiments should prove useful in targeting drug action sites and in studying the details of DNA repair.     

UV-INDUCED ALKALINE LABILE DNA DAMAGE IN HUMAN ADENOVIRUS AND ITS REPAIR AFTER INFECTION OF HUMAN CELLS

Abstract— UV-induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 10³ particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV-induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses studied.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

INHIBITION OF DNA REPAIR AND PRODUCTION OF SINGLE-STRAND BREAKS IN ESCHERICHIA COLI BY REDUCTONE

Abstract— Reductone (HOCH₂COCHO), a keto-aldehyde produced by thermal degradation of some sugars, at alkaline pHs, blocks the excision repair of DNA lesions in uv-irradiated wild type Escherichia coli. This probably occurs as a result of inhibition of the exonucleolytic activity of DNA polymerase I. In addition, reductone alone induces DNA single-strand breaks. Repair of this damage is mainly dependent on the polA gene products.     

INHIBITION OF PROTEIN SYNTHESIS IN CULTURED TOBACCO CELLS BY ULTRAVIOLET RADIATION

Abstract— Ultraviolet (254 nm) irradiation of liquid-cultured tobacco cells strongly and quickly inhibited their ability to incorporate labeled amino acids into protein. An incident dose of only 388 J/m² reduced incorporation to 37 per cent of the original rate. The effect on amino acid incorporation did not seem to depend on inhibition of amino acid uptake, inhibition of the supply of nucleoside triphosphates, or inhibition of the supply of messenger RNA to cytoplasmic ribosomes.