Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden,
1 Department of Dermatology, Päijät-Häme Central Hospital, 15850 Lahti, Finland and
2 Department of Dermatology, University of Turku, 20520 Turku, Finland
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Abstract |
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Abbreviations: CPDs, cyclobutane pyrimidine dimers; NER, nucleotide excision repair; SNPs, single nucleotide polymorphisms; SSR, solar simulating radiation; UVR, UV radiation; XPD, xeroderma pigmentosum complementation group D.
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Introduction |
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Data on DNA repair rates in humans are derived from cultured cells, because methods have not been available for quantitative assay of DNA repair rates in humans in situ(14). Semi-quantitative immunofluorescence techniques have been used and have the advantage of being able to localize the damage (5,6). A DNA repair test was recently introduced based on quantification of specific UV radiation (UVR)-induced photoproducts in human skin biopsies by the 32P post-labeling technique (7). For the determination of repair rate a single dose of UVR was administered to the buttocks of volunteers and the removal of damage was followed in biopsies taken at various times after UV exposure. UV damage was quantified as specific radioactive fractions by HPLC. Synthetic standards were available for identification of two cyclobutane pyrimidine dimers (CPDs) and two 64 photoproducts. The results obtained showed that repair kinetics of CPDs and 64 photoproducts encompassed fast and slow components, probably relating to the genomic heterogeneity of nucleotide excision repair (NER) (1,8). CPDs were removed considerably slower than 64 photoproducts, with 50% removal times of ~15 and 5 h, respectively. Dimers at TT sites were repaired slower than those at TC sites (8). A 20-fold inter-individual variation in repair rates was observed, but age, basal cell carcinoma and melanoma did not appear to affect repair rates when measured up to 48 h (911). Variation due to the post-labeling method is only 30% in repeated samples; even in samples taken from the same individuals 3 weeks apart the correlation coefficients were from 0.7 to 0.85 (12).
Polymorphisms in DNA repair genes are currently being actively investigated because of possible effects on susceptibility to cancer (13). NER is a complex process in which many proteins take part (1,3). The xeroderma pigmentosum complementation group D (XPD) protein is involved in the unwinding of DNA and forms a complex with transcription factor IIH during DNA repair. Mutations in XPD cause a severe but variable depression of NER (3). Several non-synonymous SNPs have been described in the XPD gene, including those at codons 312 (exon 10 GA, Asp
Asn) and 751 (exon 23 A
C, Lys
Gln) (13). The frequencies of these variant alleles have been estimated to be ~30% in two US studies (13,14). It is not known whether these polymorphisms have functional effects. However, the more common AA genotype at exon 23 has been suggested to be a risk factor for basal cell carcinoma (15) and susceptibility to X-ray-induced chromatid aberrations (14), while no effect has been noted on lymphocyte sister chromatid exchanges (16). However, a recent study on head and neck cancers suggested that the exon 23 C allele was a risk factor and the effect was stronger among old subjects (17).
In the present study we have used the recently developed in situ DNA repair assay to functionally test the effects of the two XPD polymorphisms on the rate of repair of two specific CPDs. The human sampling was carried out in a dermatology clinic in Lahti, Finland and the 32P post-labeling analysis of UV damage in Sweden, in the context of three earlier studies (911). Genotyping analysis was carried out specifically for the present study.
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Materials and methods |
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Sampling of skin biopsies
A broadband Philips (HP 411/A) solar simulating lamp was used as the solar simulating radiation (SSR) source. The spectral distribution of the SSR resembled the spectrum of noon summer sunlight in Helsinki (911). All participants were irradiated with an erythemally weighted UV dose of 40 mJ/cm2 CIE (Commission Internationale de l'Éclairage) (18) of SSR on a buttock skin site. One skin biopsy was taken immediately after irradiation (within 5 min) from each subject and put on dry ice at once, then kept in a 20°C freezer until DNA extraction. At 24 and 48 h another two skin biopsies were taken at the exposure sites.
DNA extraction and CPD measurement
DNA extraction from epidermis was performed as described previously (7,8). The levels of SSR-induced CPDs were quantified using the 32P post-labeling method, which has been described elsewhere (10). CPDs (T=C and T=T) were assayed as trinucleotides with an unmodified nucleotide (thymine) on the 5'-side, i.e. TT=C and TT=T. For each 32P post-labeling assay 3 µg DNA was digested and labeled. Each sample was measured twice. After labeling, 10 µl of water was added to each sample and then injected into a HPLC system for analysis. The products were detected with an online Beckman radioisotope detector. For calculation of CPD levels the HPLC peaks of interest were integrated with Beckman System Gold software and the external synthetic standards of TT=C and TT=T were used for quantification.
The repair of UV photoproducts is complex in human skin, apparently composed of many components (8). In the present study we determined the rate of repair at 24 h because at this time somewhat >50% of TT=C and somewhat <50% of TT=T is repaired (811). The time required for 50% repair of these dimers is also independent of the initial level of damage (8). Thus the variation in individual rates is most optimally gauged at 24 h.
XPD exon 10 genotyping
Analysis of the XPD Asp312Asn (GA) polymorphism in the end of exon 10 was performed by restriction analysis of a 188 bp genomic PCR product. A forward primer with a C
T mismatch (D10f, 5'-ACC TGG CCA ACC CCG TGC TGC TC-3') was designed in order to create a restriction site for TaqI (TVCGA) in the PCR product of the G allele. The reverse primer was designed according to the wild-type sequence within exon 11 (D11r, 5'-ACT TCA CGT ACT CCA GCA G-3').
Approximately 50 ng DNA was used in a 20 µl PCR reaction containing 20 mM TrisHCl, pH 9.0, 100 mM KCl, 0.2% Triton X-100, 10% DMSO, 1.5 mM MgCl2, 0.25 mM dNTP and 0.5 µM each primer. After a 5 min hot start at 95°C, 1.5 U Taq polymerase (Promega, Madison, WI) was added (at 85°C) to each tube. The DNA was then amplified by 35 cycles of denaturation (94°C for 30 s), annealing (56°C for 30 s) and elongation (72°C for 30 s, with the last cycle extended by 7 min) in a thermocycler (DNA-Engine; MJ Research, Watertown, USA).
Ten microliters of the PCR product was then digested with 10 U TaqI (Fermentas, Vilnius, Lithuania) for 4 h at 65°C in a total volume of 20 µl. The digested products were separated by electrophoresis in a 6% polyacrylamide gel. The G allele was represented by a 166 bp fragment and the A allele by a 188 bp band.
XPD exon 23 genotyping
The method for genotyping exon 23 polymorphism (Lys751Gln, AC) was modified from Dybdahl et al. (15). A 322 bp PCR product including the 84 bp exon 23 was amplified using two intronic primers flanking exon 23 (D23f, 5'-ATC CTG TCC CTA CTG GCC ATT C-3', and D23r, 5'-TGG ACG TGA CAG TGA GAA AT-3'). Approximately 50 ng DNA was used in a 25 µl PCR reaction containing 10 mM TrisHCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 0.25 mM dNTP and 1 µM each primer. The DNA was amplified by adding 1 U Taq polymerase after a 5 min hot start, followed by 35 cycles of denaturation (94°C for 15 s), annealing (60°C for 15 s) and elongation (72°C for 15 s, with the last cycle extended by 7 min).
Five microliters of the PCR product was then digested with 10 U PstI (Promega, Madison, WI) for 4 h at 37°C in a total volume of 15 µl, followed by electrophoresis in a 5% polyacrylamide gel. The A allele was cut into two fragments (104 and 218 bp) while the C allele was cut into three fragments (104, 155 and 63 bp).
Statistical methods
The disease status had no effect on the rate of DNA repair measured at 24 and 48 h (911). Thus there was no need to stratify the subjects by disease status in the present study. The repair rates were calculated as percent CPDs repaired at 24 h compared with the CPD level at 0 h, immediately after UV exposure. The relationship between repair of the two CPD types was quantified using Pearson's correlation coefficient. Differences in allelic distribution were tested by 2 analysis. Differences in repair rate between the two genotype groups were assessed using the Wilcoxon rank sum test (two-sided). The non-parametric trend test (two-sided) was used to evaluate any possible trend in repair rate across groups with increasing number of variant alleles.
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Results |
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For one individual with an exon 10 GG and exon 23 AC genotype only TT=C repair was measured (Table I), whereas for another individual with an exon 10 GA and exon 23 AC genotype only TT=T repair was measured (Table II
). Thus the effects of the exon 10 and exon 23 polymorphisms on repair rate were analyzed in all but one individual for either of the CPD types.
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A similar tabulation is shown for the rate of TT=T repair (Table II). Again, only the four double homozygotes (exon 10 AA and exon 23 CC) differed from the others (P = 0.09). These four had repair rates below the overall median of 34%. The mean rate for these individuals (15.8%) was <50% of the overall mean for subjects with other genotypes (36.2%). Two of the double homozygotes were below age 50 years and two were older, with mean repair rates of 27.8 and 3.8%, respectively. The uniform findings on the age effect in Tables I and II
lend support to each other, as the repair of TT=T was significantly correlated with the repair of TT=C (r = 0.65, P < 0.0001). There was, however, no apparent effect of the exon 10 polymorphism on TT=T.
The data on repair rates was then stratified by age (Table III). Among the 43 subjects analyzed in each repair assay 18 were younger than 50 years at the time of sampling. Age did not modify the effect of the exon 10 polymorphism on repair rate. The repair rate, however, decreased systematically with presence of the exon 23 C allele among those who were at least 50 years old (P = 0.05 for TT=C and P = 0.02 for TT=T, trend test). Within the group of exon 23 CC homozygotes the lowest repair rates for both TT=C and TT=T were shown by three of the four older subjects. Two of them were the same subjects as the double homozygotes with depressed repair in Tables I and II
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Discussion |
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The present results showed no association between DNA repair rate and individual genotype among all subjects. The combined exon 10 AA and exon 23 CC genotype was associated with depressed repair, ~50% of that for the other genotypes. However, this decrease was only observed among those who were 50 years or older, suggesting that age was a modifying factor. The decrease in repair was observed for both TT=C and TT=T, showing technical consistency. Older subjects turned out to have depressed repair of both TT=C and TT=T if they had the exon 23 CC genotype. This genotype-mediated age effect has no obvious biological explanation and remains tentative. On the other hand, it may suggest that the exon 23 C allele is associated with depressed repair when combined with greater age. This is supported by the significant trend of decreasing repair rate for both TT=C and TT=T with increasing numbers of the variant allele in exon 23 among older subjects. The present results are not in accord with previous studies on basal cell carcinoma and chromatid aberration in vitro, which associated the common exon 23 A allele with risk, however, the end-points of all these studies were different (14,15). However, the results are in line with a recent study associating the exon 23 C allele with risk of head and neck cancer (17). Interestingly, greater age was associated with the increased risk, exactly as in our study.
In summary, our data do not show consistent XPD genotype-specific differences in DNA repair rates among all subjects, but the exon 23 C allele may be associated with depressed repair among older subjects. This finding, however, needs to be confirmed in larger studies.
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Notes |
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Acknowledgments |
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References |
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