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.     

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.     

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.     

PSORALEN PLUS ULTRAVIOLET RADIATION-INDUCED INHIBITION OF DNA SYNTHESIS AND VIABILITY IN HUMAN LYMPHOID CELLS IN VITRO*

Abstract— 8-Methoxypsoralen (8-MOP) plus high intensity long wavelength ultraviolet radiation (UV-A) is presently being used to induce remissions of psoriasis and mycosis fungoides. Previous studies demonstrated inhibition of DNA synthesis in circulating leukocytes from some patients during this therapy. The present study is designed to determine whether conditions of 8-MOP concentration and UV-A exposure attained during therapy might be sufficient to result directly in decreased lymphoid cell DNA synthesis and viability in vitro. Tritiated thymidine (³HTdR) incorporation and cell growth in suspension culture following UV-A exposure alone or with therapeutic concentrations of 8-MOP was examined in peripheral blood lymphocytes and in Ebstein-Barr virus transformed human lymphoblas-toid cell lines. UV-A exposure alone induced a dose-dependent inhibition of HTdR incorporation in both types of lymphoid cells (3000 J/m² resulted in 77% of control ³HTdR incorporation). Pre-incubation with 0.1 μg/m/ 8-MOP before UV-A exposure induced a significantly greater inhibition of ³HTdR incorporation (3000 J/m² resulted in 61% of control ³HTdR incorporation). Greater inhibition of ³HTdR incorporation was observed by preincubation of the lymphoblastoid cells with 1.0μg/mC 8-MOP (3000 J/m² resulted in 41% of control) but not in the lymphocytes (3000 J/m² resulted in 63% of control). The concentration of viable lymphoblastoid cells did not decrease below the original concentration after the highest dose of UV-A alone (29,000 J/m²) but preincubation with 0.1 μg/mC 8-MOP resulted in 40% survival after 3000 J/m² (D₃₇ approximately 3000 J/m²) and preincubation with 1.0 μg/ 8-MOP resulted in 0.6% survival after 3000 J/,² (D₃₇ approximately 800 J/m²). This study suggests that the low doses of 8-MOP and UV-A received by patients' lymphocytes may be sufficient to explain the decreased DNA synthesis found in their circulating leucocytes. The long term consequences of such damage remain to be determined.     

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.     

RECOVERY FROM INHIBITION BY UV-IRRADIATION OF ORNITHINE DECARBOXYLASE INDUCTION IN HUMAN CELLS: IMPLICATION OF EXCISION REPAIR

Abstract— Exposure of stationary-phase human breast carcinoma(T–47D) cells to far-UV light (254 nm) inhibited the appearance of induced ornithine decarboxylase (ODC) activity. The fluence response curve had a shoulder (D₄= 2 J m_-2) followed by an exponential decline (D₀= 4.2 Jm_-2). The cells could recover from this inhibition when the stimulus of induction of ODC was delayed for20–24 h after irradiation. Hydroxyurea (HU) when present at 3 mM during the recovery period eliminated completely the ability of the cells to recover. This effect of HU on ODC induction was partially reversed by 50 nM of the four deoxyribonucleosides required for DNA synthesis. Neither HU nor the deoxyribonucleosides by themselves affected ODC induction in unirradiated cells. Since HU inhibited the recovery from potentially lethal UV damage and is a known inhibitor of excision repair, we interpret the above results to mean that recovery from UV-induced inhibition of ODC induction depends on excision-repair of DNA damage. This interpretation is strongly supported by the finding that specific photolysis of 5-bromodeoxyuridine, incorporated into DNA during the recovery period, inhibited recovery of ODC induction from inhibition by UV light.     

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.     

RESISTANCE OF PIGMENTED HUMAN CELLS TO KILLING BY SUNLIGHT AND OXYGEN RADICALS

Solar irradiation of a panel of human cell lines revealed three phenomena relevant to understanding the biological role of melanin; a heavily melanised melanoma line (MM418) was considerably more resistant to solar killing compared with HeLa and amelanotic melanoma cells of similar size and DNA content; MM418 cells were also resistant to killing by artificial UVB and by hydrogen peroxide generated in situ with extracellular glucose oxidase; and no difference in survival between the cell lines was found using 254 nm UV or gamma radiation. MM418 cells were resistant to sunlight when irradiated as attached monolayers but not when irradiated in suspension. Further studies showed that resistance to solar radiation in MM418 cells was not due to less DNA damage, as judged by inhibition of semiconservative DNA synthesis, or to enhanced constitutive or induced repair determined by reactivation of irradiated adenovirus. These results indicate that melanisation protects human cells from solar UVB in vitro and that the mechanism is associated with protection from hydrogen peroxide-type damage rather than direct shielding of DNA.     

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.     

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.     

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.     

ALKALINE ELUTION STUDIES OF HEMATOPORPHYRIN-DERIVATIVE PHOTOSENSITIZED DNA DAMAGE AND REPAIR IN CHINESE HAMSTER OVARY CELLS

Abstract We have used alkaline elution to study DNA damage produced by the photosensitizer hematoporphyrin derivative (HPD) in cultured Chinese hamster cells. Dosimetry was performed by measuring fluence and calculating photon absorption by intracellular HPD. HPD photosensitization causes DNA strand breakage. These breaks are repaired by the cell, although their fractional rate of repair is smaller than that for X-ray induced strand breaks at equivalent levels of strand breakage. The combined DNA polymerase inhibitors cytosine arabinoside and hydroxyurea suppress the repair of HPD-photosensitized breaks more strongly than they suppress repair of X-ray induced breaks. Addition of novobiocin to the aforementioned inhibitors causes almost total suppression of photosensitized break repair. A nucleotide excision repair system with inhibitor susceptibility similar to that of the system which removes pyrimidine dimers thus does not act upon HPD-photosensitized damage. The repair rate and inhibitor sensitivity findings together suggest biologically important differences in the chemical nature of X-ray induced and HPD-photosensitized strand breaks. In addition to strand breaks, HPD photosensitization produces covalent DNA-protein crosslinks, some of which persist through at least 90 min incubation, but which are repaired within 180 min.     

EFFECTS OF UV, SUNLIGHT AND X-RAY RADIATION ON QUIESCENT HUMAN CELLS IN CULTURE

Nondividing human diploid fibroblasts (HDF) in culture have been used to study the effect on cell lethality of ultraviolet light, natural sunlight and X-rays. A lethal effect is defined as cellular degeneration, loss from the culture and inability to exclude vital strains. Far-and mid-UV have a readily observable lethal effect (cell loss), with DNA and DNA damage as the critical target and critical damage respectively. In part, natural sunlight kills cells by a similar mechanism but has an additional lethal effect at longer exposure times. This additional effect is expressed by the retention of the dead cells in culture, in contrast to the UV-induced promotion of cell degeneration and loss. Relatively large doses of X-rays that destroy proliferative capacity, have no detectable lethal effect on the maintenance of nondividing cells. The biological response of nondividing HDF to radiations from different parts of the electromagnetic spectrum is dissimilar.     

ULTRAVIOLET LIGHT-INDUCED INHIBITION OF CELL DIVISION AND DNA SYNTHESIS IN AXENICALLY GROWN REPAIR MUTANTS OF DICTYOSTELIUM DISCOIDEUM

Cell division and DNA synthesis were studied during axenic growth following 254 nm ultraviolet light (UV) irradiation of a repair-proficient parental strain ( rad⁺ , D₁₀ colony formation = 195 J/m²) and two repair mutants ( rad C. D₁₀= 50 J/m²; rad B. D₁₀= 5 J/m²) of Dictyostelium discoideum. Isopycnic CsCI gradients were used to distinguish uptake of labeled precursors into nuclear (n) and mitochondrial (m) DNA, using Netropsin to enhance the density resolution. In all strains, m-DNA synthesis was inhibited to a lesser extent than was n-DNA synthesis. For rad C, which has been shown in other experiments to be slow in incision and dimer removal, the UV-induced lags in division and n-DNA synthesis were longer than for rad⁺. However, rad B showed a more complex response. Although brief division lags were observed for < 10 J/m², little immediate division lag was detected at greater fluences. Instead, a brief period of cell multiplication of up to but not exceeding two-fold occurred, followed by a cessation of division, and then by lysis. Fluences that yielded extensive lags in n-DNA synthesis in rad^- and rad C resulted in little detectable immediate postirradiation lag in n-DNA synthesis in rad B. However, later in the postirradiation period, when DNA synthesis had resumed in rad⁺ and rad C. it gradually declined to near zero in rad B. We conclude: (1) that the more extended lag in division and n-DNA synthesis in rad C is consistent with its slower rate of excision repair, and (2) that rad B contains a defect resulting in less initial blockage of DNA replication by UV lesions.     

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.     

INHIBITION OF DNA SYNTHESIS BY PSORALEN-INDUCED LESIONS IN XERODERMA PIGMENTOSUM AND FANCONI''S ANEMIA FIBROBLASTS

Abstract— Exposure of human cells to psoralens and near-UV light produces a mixture of monoadducts and crosslinks in DNA, which inhibit DNA synthesis by blocking replicon initiation and chain elongation. 8-Methoxypsoralen (8-MOP) has a greater effect than angelicin in normal, xeroderma pigmentosum, and Fanconi's anemia cells. Recovery of DNA synthesis is not detectable up to 8 h after exposure. The average distance between lesions that block replication in individual replicons was measured by means of bromodeoxyuridine photolysis. After exposure to 10 μg/mℓ of 8-MOP and 7500 J/m² of near-UV light, blocks were formed every 20 μm. Replicon initiation was inhibited by exposure to near-UV light alone in normal and xeroderma pigmentosum. Exposure to low concentrations of angelicin or 8-MOP plus near-UV light inhibited replicon initiation in normal and Fanconi's anemia cells, but not in xeroderma pigmentosum cells. Inhibition of initiation was not obvious after treatment with high concentrations of 8-MOP or angelicin because of the dominant effect of crosslinks in blocking chain elongation.     

DNA SYNTHESIS INHIBITION OF NITROXIDE RADICALS ON LEUKEMIA CELLS

4-isothiocyanate-2,2,6,6-tetramethyl-piperidine-l-oxyl has the strongest effects on the DNA synthesis and viability of isolated leukemia 7712 cells. This compound is a very potent inhibitor of DNA synthesis with 50% inhibition of cell DNA synthesis occurring at 2.2 μg/ml, while the LD50 for white mice is 277 mg/kg. During the concentration of 50 μg/ml, the inhibition of DNA synthesis is 99.7%, which is unable to damage the DNA replicate templet. The inhibition of this compound, a combination of nitroxide and isothiocyanate, appears to be stronger than that of the sum of the two separate actions. Much more moderate effects are seen for all the other nitroxide radicals. After reducing nitroxide to hydroxyla-mine, there is no inhibition for DNA synthesis. The free radical mechanism of carcinogene-sis and antitumor action has been discussed.     

POTENCY AND SELECTIVE TOXICITY OF TETRA(HYDROXYPHENYL)- AND TETRAKIS(DIHYDROXYPHENYL)PORPHYRINS IN HUMAN MELANOMA CELLS, WITH AND WITHOUT EXPOSURE TO RED LIGHT

Abstract A series of tetra(hydroxyphenyl)-(2-, 3- and 4-hydroxy; THPP) and tetrakis(dihydroxyphenyl)porphyrins (2,3-, 2,4-, 2,5-, 3,4-, and 3,5-dihydroxy; TDHPP) was synthesized and tested for toxicity in HeLa cells and human melanoma cell lines. Irradiation of drug-treated cells with >600 nm light greatly increased the toxicity of all drugs except the 2,5- and 3,5-TDHPP. The THPP were more toxic than TDHPP in all cell lines, with or without irradiation; of the dihydroxy derivatives, the 3,4- and 2,4-isomers were the most toxic and the 2,5-isomer was the least toxic. The MM96E melanoma cell line, shown previously to be sensitive to hydrogen peroxide and superoxide ion, was not hypersensitive to killing by any of the above agents. HeLa cells, which lacked glutathione- S -transferase activity, were sensitive to the 4- and 2,3-isomers after irradiation; similar amounts of all drugs were taken up by HeLa cells. The pigmented melanoma cell line MM418, resistant to UV-B and in situ -generated hydrogen peroxide but sensitive to glutathione (GSH) depletion, was found to be resistant to the 2,3-isomer (no irradiation) and sensitive to the 3,4-isomer. The results indicate that (1) phototoxicity in these phenylporphyrins is not mediated by superoxide ions or hydroxyl radicals, (2) toxicity is dependent on the orientation of the hydroxy groups, (3) GSH transferase and possibly GSH itself offer protection from the 4- and 3,4-derivatives, respectively, and (4) the 3,4-derivative and analogues of similar selectivity should be evaluated further for the treatment of primary melanoma.     

EFFICIENCY OF REPAIR OF PYRIMIDINE DIMERS AND PSORALEN MONOADDUCTS IN NORMAL AND XERODERMA PIGMENTOSUM HUMAN CELLS

Abstract —Repair of DNA damage produced by ultraviolet light or 5-methylisopsoralen in normal and xeroderma pigmentosum human cells involves many similar steps. Aphidicolin and cytosine arabinoside block repair of both kinds of damage with similar efficiency, indicating that DNA polymerase a has a major role in repair for these lesions. In xeroderma pigmentosum cells of various complementation groups, the relative efficiency of excision repair for both ultraviolet- and 5-methylisopsoralen-induced damage was group A < C < D, indicating a close resemblance between both kinds of lesions in relation to the repair deficiencies in these groups. At high doses, the maximum rate of repair of damage by ultraviolet light was about twice that for methylisopsoralen damage, possibly because ultraviolet-induced damage forms a substrate that is more readily recognized and excised than that of the psoralen adducts. Differences in the structural distortions to DNA caused by these kinds of damage could be detected using single strand specific nucleases which excised dimers but not 5-MIP adducts from double strand DNA.     

HAEMATOPORPHYRIN-TREATED MURINE LYMPHOCYTES: IN VITRO INHIBITION OF DNA SYNTHESIS AND LIGHT-MEDIATED INACTIVATION OF CELLS RESPONSIBLE FOR GVHR*

Abstract— It has been known that haemoatoporphyrin derivative (Hpd) has a preferential distribution in tissues with high mitotic index. Furthermore, cytocidal activity of light activated Hpd within the cells has been exploited in the therapy of experimental and human cancer. It is reported here that maximum, long lasting, although reversible, inhibition of DNA synthesis was obtained in Hpd-treated lymphocytes. However, Hpd-treated lymphoid cells did not stimulate allogeneic lymphocytes in the primary mixed lymphocyte reaction (MLR) culture. Phytohaemagglutinin (PHA) stimulated murine lymphocytes treated with Hpd and exposed to laser light have shown higher susceptibility to lysis than resting, PHA unstimulated, lymphocytes. In vitro DBA/2 stimulated C57 lymphocytes, inoculated into X-irradiated BD2F₁ mice, upon Hpd treatment followed by exposure to light, did not cause a lethal graft vs. host reaction (GVHR). Haematoporphyrin derivative was preferentially incorporated by large and metabolically active cells. inhibited the incorporation of [³H]-thymidine in the lymphocyte nucleus and could be exploited to selectively remove blast cells from resting lymphocytes.