DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (less than 290 nm) and UV-B irradiation (290-320 nm) in the 2'-deoxy-[3H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol. 52, 519-524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10-20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract
I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (< 290 nm) and UV-B irradiation (290–320 nm) in the 2'-deoxy-[³H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m^-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol . 52 , 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10–20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

ULTRAVIOLET LIGHT IRRADIATION OF DEFINED-SEQUENCE DNA UNDER CONDITIONS OF CHEMICAL PHOTOSENSITIZATION

Abstract— The formation of cyclobutane pyrimidine dimers and UV light-induced (6-4) products was examined under conditions of triplet state photosensitization. DNA fragments of defined sequence were irradiated with 313 nm light in the presence of either acetone qr silver ion. UV irradiation in the presence of both silver ion and acetone enhanced the formation of TT cyclobutane dimers, yet no (6-4) photoproducts were formed at appreciable levels. When photoproduct formation was also measured in pyrimidine dinucleotides, only cyclobutane dimers were formed when the dinucleotides were exposed to 313 nm light in the presence of photosensitizer. The relative distribution of each type of cyclobutane dimer formed was compared for DNA fragments that were irradiated with 254, 313, or 313 nm UV light in the presence of acetone. The dimer distribution for DNA irradiated with 254 and 313 nm UV light were very similar, whereas the distribution for DNA irradiated with 313 nm light in the presence of acetone favored TT dimers. Alkaline labile lesions at guanine sites were also seen when DNA was irradiated with 313 nm light in the presence of acetone.     

Involvement of human small fragment nuclease in the resistance of human cells to UV-C-induced cell death

Human small fragment nuclease (Sfn) is one of the cellular proteins that were reported to degrade small, single-stranded DNA and RNA. However, the biological role of Sfn in cellular response to various stressors such as UV-C (mainly 254 nm wavelength ultraviolet ray) remains unclear. We have examined whether modulation of human SFN gene expression affects cell survival capacity against UV-C-induced cell death, analyzing colony survival ability in UV-C-sensitive human RSa cells treated with short double-stranded RNA (siRNA) specific for SFN messenger RNA (mRNA). The expression levels of SFN mRNA in the siRNA-treated RSa cells decreased to about 15% compared with those in the control siRNA-treated cells. The siRNA-treated RSa cells showed lower colony survival and higher activity of caspase-3 after UV-C irradiation than the control siRNA-treated RSa cells. Furthermore, the removal capacity of cyclobutane pyrimidine dimers (CPD) in the siRNA-treated RSa cells decreased compared with the control siRNA-treated RSa cells. There was no difference in the colony survival and CPD removal capacity after UV-C irradiation between the control siRNA-treated RSa cells and mock-treated RSa cells. These results suggest that SFN expression is involved in resistance of RSa cells to UV-C-induced cell death through the roles it plays in the DNA repair process.     

RELATIVE INDUCTION OF CYCLOBUTANE DIMERS and CYTOSINE PHOTOHYDRATES IN DNA IRRADIATED in vitro and in vivo WITH ULTRAVIOLET-C and ULTRAVIOLET-B LIGHT

SV40 DNA was irradiated in vitro and in vivo with UV-C (240-280 nm) and UV-B (280-320 nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease III (endo III) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo III-sensitive sites (primarily cytosine photohydrates) induced was 1-2% of the frequency of endo V-sensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo III- and endo V-sensitive sites in DNA were induced in the same relative proportion at both UV-C and UV-B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.     

COMPARATIVE STUDIES ON THE CORRELATION BETWEEN PYRIMIDINE DIMER FORMATION and TYROSINASE ACTIVITY IN CLOUDMAN S91 MELANOMA CELLS AFTER ULTRAVIOLET-IRRADIATION

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.     

FORMATION OF PYRIMIDINE DIMERS IN SIMIAN VIRUS 40 CHROMOSOMES AND DNA in vitro: EFFECTS OF SALT

Abstract— Simian virus 40 chromosomes were used to determine whether packaging of DNA into chromatin affected the yield of cyclobutane pyrimidine dimers introduced by ultraviolet light (254 nm). SV40 chromatin and purified SV40 DNA (radioactively labeled with different isotopes) were mixed and irradiated in vitro . The proteins were extracted and pyrimidine dimers detected as sites sensitive to the UV-endonuclease encoded by bacteriophage T4. When irradiation was carried out in the presence of at least 0.05 M NaCl the same number of dimers were formed in chromatin as in free DNA. Irradiation in the absence of NaCl, however, reduced the relative yield of dimers in chromatin to 89% of that in free DNA. Different methods of chromatin preparation did not influence these results.     

REDUCED REPAIR OF NON-DIMER PHOTOPRODUCTS IN A GENE TRANSFECTED INTO XERODERMA PIGMENTOSUM CELLS

Abstract— 4ells from patients with the sun sensitive cancer-prone disease, xeroderma pigmentosum (XP) have defective repair of UV damaged DNA with reduced excision of the major photoproduct, the cyclobutane type pyrimidine dimer. Other (non-dimer) photoproducts, have recently been implicated in UV mutagenesis. Utilizing an expression vector host cell reactivation assay, we studied UV damaged transfecting DNA that was treated by in vitro photoreactivation to reverse pyrimidine dimers while not altering other photoproducts. We found that the reduced expression of a UV damaged transfecting plasmid in XP complementation group A cells is only partially reversed by photoreactivation. E. coli photolyase treatment of pSV2catSVgpt exposed to 100 or 200 J m⁻² of 254 nm radiation removed 99% of the T4 endonuclease V sensitive sites. Transfection of XP12BE(SV40) cells with photoreactivated pSV2catSVgpt showed residual inhibition corresponding to 25 to 37% of the lethal hits to the cat gene. This residual inhibition corresponds to the fraction of non-dimer photoproducts induced by UV. This result implies that XP12BE(SV40) cells do not repair most of the non-dimer photoproducts in DNA.     

Pyrimidine dimer formation and oxidative damage in M13 bacteriophage inactivation by ultraviolet C irradiation

The mechanism by which UV-C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South-Western blotting for pyrimidine dimers. The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single-stranded circular genomic DNA (sc-DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc-DNA and linear-form DNA. However, the amount of sc-DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6-4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 8-Oxo-7,8-dihydro-2'-deoxyguanosine did not form in the M13 genome after UV-C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV-C irradiation under these conditions. These results indicated that UV-C irradiation induced not only CPD and (6-4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV-C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV-C.     

GENETIC EFFECTS OF UV-B: MUTAGENICITY OF 308 nm LIGHT IN CHINESE HAMSTER V79 CELLS

Abstract —Chinese hamster V79 cells were irradiated with 254 nm (UV-C) and 308 nm (UV-B) light, emitted by a germicidal lamp and an excimer laser, respectively. Induction of mutations at two distinct genetic loci was measured by selecting colonies resistant to 6-thioguanine or to ouabain. Unlike 6-thioguanine resistance which can be presumed to be due to many different types of genetic damage, mutation to ouabain resistance seems to result from base-pair substitution events only. Much higher doses of 308 than of 254 nm radiation are required to induce equivalent numbers of mutants. However, induction of cell inactivation and 6-thioguanine resistant mutations with the two UV sources appears to be correlated, suggesting that a common mechanism, perhaps involving the induction of pyrimidine-containing dimers, is involved. The frequency of ouabain resistant mutants per lethal event is on the other hand much higher after irradiation with the 308 nm light. This latter finding further defines a part of the UV-B spectral region which seems to induce a unique kind of DNA damage which specifically results in base-pair substitution events. Action spectra studies therefore appear necessary in the definition of the mutagenic effects of UV-B radiations in mammalian cells.     

Effects of cytosine methylation on pyrimidine dimer formation in DNA

The relative induction of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4)pyrimidone photoproducts ([6-4]PD) was quantified in the duplex homopolymers polydeoxyadenosine:polydeoxythymidine, polydeoxyguanosine:polydeoxycytidine and polydeoxyguanosine:polydeoxy-5-methylcytidine irradiated with UVC or UVB radiation. Cytosine methylation significantly increased the yield of cytosine (6-4)PD after irradiation with UVC light and of cytosine CPD and (6-4)PD after irradiation with UVB light. The data suggest that CPD and (6-4)PD are preferentially induced at 5-methylcytosine bases in DNA of cells exposed to sunlight and comprise a major component of the mutation spectrum leading to the initiation of sunlight-induced skin cancer.     

CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B _8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.     

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.     

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.     

UNSCHEDULED DNA SYNTHESIS: A QUANTITATIVE INDICATOR OF RESIDUAL IMMUNODETECTABLE PYRIMIDINE DIMERS IN HUMAN FIBROBLASTS AFTER ULTRAVIOLET-B IRRADIATION

We have addressed the question whether the level of UV-B induced DNA damage can be accurately assessed by the measurement of the rate of unscheduled DNA synthesis (UDS). Cultured human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 at 313 nm, and 70 kJ/m2 at 365 nm. The analysis of UDS measurements indicate complete arrest of repair processes within 24 h after irradiation, irrespective of the dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between the disappearance of the antibody-binding determinants and the variation of the rate of UDS vs time after the end of the irradiation. Thus it is concluded that in UV-B irradiated normal cultured human fibroblasts, the lack of UDS reflects the absence of immunodetectable pyrimidine dimers.     

Resistance of the genome of Escherichia coli and Listeria monocytogenes to irradiation evaluated by the induction of cyclobutane pyrimidine dimers and 6-4 photoproducts using gamma and UV-C radiations

The effect of gamma and UV-C irradiation on the production of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) in DNA was investigated to compare the natural resistance of the genome of a Gram-positive bacterium and a Gram-negative bacterium against irradiation. Solution of pure DNA and bacterial strains Listeria monocytogenes and Escherichia coli were irradiated using gamma and UV-C rays. Extracted DNA from bacteria and pure DNA samples were then analysed by ELISA using anti-CPDs and anti-6-4 PPs monoclonal antibodies. The results show that gamma rays, as well as UV-C rays, induce the formation of CPDs and 6-4 PPs in DNA. During UV-C irradiation, the three samples showed a difference in their sensitivity against formation of CPDs (P≤0.05). Pure DNA was the most sensitive while the genome of L. monocytogenes was the most resistant. Also during UV-C irradiation, the genome of L. monocytogenes was the only one to show a significant resistance against formation of 6-4 PPs (P≤0.05). During gamma irradiation, for both types of lesion, pure DNA and the genome of E. coli did not show significant difference in their sensitivity (P>0.05) while the genome of L. monocytogenes showed a resistance against formation of CPDs and 6-4 PPs.     

SIMULTANEOUS ESTABLISHMENT OF MONOCLONAL ANTIBODIES SPECIFIC FOR EITHER CYCLOBUTANE PYRIMIDINE DIMER OR (6-4)PHOTOPRODUCT FROM THE SAME MOUSE IMMUNIZED WITH ULTRAVIOLET-IRRADIATED DNA

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.     

Repair of the main UV-induced thymine dimeric lesions within Arabidopsis thaliana DNA: evidence for the major involvement of photoreactivation pathways.

The UV-B induced formation of thymine cis-syn cyclobutane dimer and related (6-4) photoproduct was monitored within DNA of cultured cells and plants of Arabidopsis thaliana. This was achieved using a sensitive and accurate HPLC-tandem mass spectrometry assay. It was found that the cyclobutane pyrimidine dimer was formed in a ninefold higher yield than the (6-4) photoproduct. The removal of the lesions was then studied by incubating irradiated cells either in the darkness, under visible light or upon exposure to UV-A radiation. Dark repair of both cyclobutane dimers and (6-4) photoproducts was found to be very ineffective. In contrast, a rapid decrease in the level of photoproducts was observed when UV-B-irradiated cells were exposed to UV-A and, to a lesser extent, to visible light. The removal of (6-4) adducts was found to occur more efficiently. These results strongly suggest that repair of UV-induced photolesions in plants is mainly mediated by photolyases.     

The effects of grape seeds polyphenols on SKH-1 mice skin irradiated with multiple doses of UV-B

The study investigated the protective activity of red grape seeds (Vitis vinifera L, Burgund Mare variety) (BM) extracts in vivo on multiple doses of ultraviolet radiation (UV)-B-induced deleterious effects in SKH-1 mice skin. Eighty 8-weeks-old female SKH-1 mice were divided into 8 groups: control, vehicle, UV-B irradiated, vehicle+UV-B irradiated, BM 2.5mg polyphenols (PF)/cm(2)+UV-B irradiated, BM 4 mg PF/cm(2)+UV-B irradiated, UV-B+BM 2.5mg PF/cm(2), UV-B+BM 4 mg PF/cm(2). The extract was applied topically before or after each UV-B exposure (240 mJ/cm(2)), for 10 days consecutively. The antioxidant activity of BM extract is higher than gallic acid (k(BM)=0.017, k(gallic acid)=0.013). Multiple doses of UV-B generated the formation of cyclobutane pyrimidine dimers (CPDs) and sunburn cells, increased glutathione peroxidase (GPx) and catalase (CAT) activities respectively glutathione (GSH) and IL-1β levels in skin. In group treated with 2.5mg PF/cm(2) before UV-B irradiation BM extract inhibited UV-B-induced sunburn cells, restored the superoxide dismutase (MnSOD) activity, increased insignificantly CAT and GPx activities and reduced IL-1β level. The BM 4.0 mg PF/cm(2) treatment decreased GSH level and reduced the percentage of CPDs positive cells in skin. Both doses of BM extract administered after UV-B irradiation increased the MnSOD and GPx activities and reduced the formation of sunburn cells in skin. Our results suggest that BM extract might be a potential chemo-preventive candidate in reducing the oxidative stress and apoptosis induced by multiple doses of UV-B in skin.