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Psoriasis patients with basal cell carcinoma have more repair-mediated DNA strand-breaks after UVC damage in lymphocytes than psoriasis patients without basal cell carcinoma
Cancer Letters 151 (2000) 187±192 www.elsevier.com/locate/canlet
Psoriasis patients with basal cell carcinoma have more repairmediated DNA strand-breaks after UVC damage in lymphocytes than psoriasis patients without basal cell carcinoma q,qq Peter Mùller a,*, HaÊkan Wallin a, Marianne Dybdahl a, Gerda Frentz b, Bjùrn A. Nexù a b
a National Institute of Occupational Health, DK-2100 Copenhagen, Denmark Institute of Preventive Medicine, Kommunehospitalet, DK-1399 Copenhagen K, Denmark
Received 13 July 1999; received in revised form 19 November 1999; accepted 20 November 1999
Abstract We have investigated the formation of strand-breaks following UVC irradiation in lymphocytes from psoriasis patients with or without basal cell carcinoma (BCC). Isolated lymphocytes were irradiated with UVC light at a dose of 3.6 J/m 2, and the level of DNA strand-breaks were measured 25 min after the irradiation by the alkaline comet assay. The generation of strand-breaks following UVC irradiation indicates DNA-repair-mediated incisions, as UVC light does not generate strand-breaks per se. We found that psoriasis patients with BCC had more DNA-repair incisions than non-cancer patients. The incision level correlated to two polymorphisms of the XPD gene. At present, it is not clear if the association is a primary effect that is related to differences of the XPD protein. Genes encoding for other repair proteins, namely XRCC1, ERCC1, and LIG1 are located close to the XPD gene, and it is possible that the association is due to a cosegregation with a polymorphism in one of these genes. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basal cell carcinoma; Comet assay; DNA-repair activity; Psoriasis; XPD gene
1. Introduction The DNA-repair machinery is essential in protectq This paper is dedicated to the memory of Gerda Frentz who died 9th November, 1998. qq The contribution of the dermatologists in private practice all over Denmark, the departments of dermatology of the ®ve Danish university hospitals, and the collaboration of dermato-pathologists are gratefully acknowledged. A special list of these participants is available on request, addressed to Jette Petersen, Institute of Preventive Medicine, Kommunehospitalet, DK-1399 Copenhagen K, Denmark. * Corresponding author. Tel.: 145-35-327-657; fax: 145-35327-610. E-mail address: [email protected] (P. Mùller)
ing the genetic code from mutagenic exposure from sources such as radiation or chemicals in the environment. It comprises a complex set of repair proteins that are grouped into several more or less separate pathways [9]. The NER 3 pathway involves some 25 different proteins, which in a stepwise manner recognize DNA damage, incise the strand, excise the DNA damage, and restore the DNA strand by the incorporation of new nucleotides and ligation. It is commonly accepted that reduced DNA-repair activity can render a higher risk of developing cancer. At the extreme end of low repair activity, several cancer-prone disorders among humans have been linked to genes that are involved in DNA repair. XP
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00414-0
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P. Mùller et al. / Cancer Letters 151 (2000) 187±192
patients constitute the most severe form of genetic inherited DNA-repair disorder in the NER pathway, where some patients display DNA-repair activity as low as 2% of the population mean [4]. Heterogeneity of DNA-repair activity in the general population has also been described [13]. We have undertaken a study of the role of DNA repair in the development of basal cell carcinoma (BCC) among patients suffering from psoriasis. The affections seen on the skin of psoriasis patients are due to an increased epidermal proliferation, and the treatment regime is often directed toward minimizing the proliferation. The drugs are often topically applied to the skin, and include carcinogenic agents such as coal tar, PUVA, and Grenz rays (ultrasoft Xrays) [12]. An epidemiological study among Danish psoriasis patients has thus indicated a 2.5-fold increase of the risk of developing skin cancer [16]. It is likely that some psoriasis patients develop BCC because of an unfortunate combination of the treatment with DNA-damaging agents and a low DNArepair activity. Thus, we have previously reported that psoriasis patients with BCC had reduced DNArepair activity. This was evidenced by both UVCinduced UDS [14] and HCR [7]. Our data are in agreement with other studies showing that patients with BCC had a reduced DNA-repair activity compared to non-cancer controls [20,21]. The genetic basis for individual variation in DNA-repair activity is still in the early stage. Several genetic polymorphisms in NER genes are described in DNA material from presumed healthy individuals [18]. Although the polymorphisms are known, the phenotypic outcome is not described. We have previously reported two polymorphisms in the XPD gene located in exon 23 (A ! C that gives rise to a Lys ! Gln amino acid substitution) and exon 6 (A ! C that is silent) [8]. In both polymorphisms, individuals with the AA genotype appeared to have an increased risk of BCC, although the risk did not reach statistical signi®cance. The mean age at onset of BCC was also lower for patients with the A alleles [8]. These ®ndings suggest that the polymorphisms in the XPD gene are associated with a risk of BCC. In this study of psoriasis patients with and without BCC, we measured the maximal incision rate of the repair system in DNA after UVC irradiation of lymphocytes by the alkaline comet assay.
2. Materials and methods 2.1. Study persons A detailed description of the study persons involved in this project have been published elsewhere [7,14]. Brie¯y, 20 subjects in each group were matched for age and gender. None of the patients were treated with genotoxic anti-psoriatic drugs or ointments (UVradiation, PUVA, coal tar and soft X-rays) in a 3-month period before sampling. This part of the study involved 16 patients with psoriasis and 18 patients with psoriasis and BCC (we did not have blood samples left for the six people). 2.2. Comet assay The procedure of comet assay has been described previously in [14]. 2.3. Statistics We have used the two-sided Student's t-test to compare the differences between groups, and for the evaluation of the correlation between the comet assay and polymorphism in the XPD gene. The polymorphisms were classi®ed as CC and AA/AC genotypes for statistical comparison. 3. Results 3.1. Optimization of UVC-induced DNA damage In order to optimize the procedure of UVC irradiation, we initially investigated the formation of strandbreaks following UVC irradiation in lymphocytes obtained from buffy coats from healthy individuals (kindly donated by Bispebjerg Hospital, Copenhagen, Denmark). In general, there were considerable variations in the basal level of strand-breaks from one preparation of buffy coat to another (evidenced by the values at time zero in Fig. 1A±C). A dose± response relationship was found in the interval of 0±3.6 J/m 2. In the dose range of 3.6±10 J/m 2, the level of strand-breaks was almost the same as that for the lower dose (Fig. 1A). This is possibly due to saturation of the comet assay. The time-curves seen by doses of 3.6 J/m 2 (Fig. 1B) and 6 J/m 2 (Fig. 1C)
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strand-breaks, which was on the middle of the initial linear part of the time-curve. We thus, measured the initial rate of strand-break formation by the repair machinery, which operated at, or close to, substrate saturation. 3.2. Patients The UVC-induced DNA-repair formation of strandbreaks in lymphocytes of psoriasis patients with and without BCC is outlined in Fig. 2. There was a signi®cantly higher formation of strand-breaks in lymphocytes obtained from psoriasis patients with BCC compared to psoriasis patients without BCC (P , 0:02, Student's t-test). In order to investigate heterogeneity of the UVC-mediated DNA-repair incisions at a single cell level, we classi®ed all the cells, which were measured for each person, into six classes of DNA damage. The grade of DNA damage was arbitrarily chosen according to the value of the tail moment (I, ,10 mm; II, 10±19.9 mm; III, 20±29.9 mm; IV, 30± 39.9 mm; V, 40±40.9 mm; VI, .50 mm). It can be seen in Fig. 3 that the psoriasis patients with BCC had the
Fig. 1. Formation of DNA-repair-mediated strand-breaks following UVC irradiation of human lymphocytes. (A) Dose±response relationship of UVC after 30 min of incubation; (B), time-course of formation of strand-breaks following UVC irradiation at 3.6 J/m 2; (C), time-course of formation of strand-breaks following UVC irradiation at 6 J/m 2. Symbols represent a mean and SEM of two±three experiments. (W) UV exposed; (X), unexposed.
revealed similar time courses of strand-break formation. Maximal amplitude was reached 1 h after irradiation (Fig. 1B,C). A plateau of strand-breaks was observed in the period of 1±5 h of incubation (Fig. 1B,C), and the level of strand-breaks did not return to baseline even after 24 h (data not shown). Based on data obtained on lymphocytes from buffy coats, we decided to investigate the effect of UVC on blood samples from the patients at a dose of 3.6 J/m 2. We found that 25 min of incubation resulted in a level of
Fig. 2. The distribution of UVC-induced DNA-repair formation of strand-breaks 25 min after irradiation at a dose of 3.6 J/m 2.
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Fig. 3. The number of cells with different grades of DNA damage. The bars represent the mean and SD for all patients. (*P , 0:05, Student's t-test)
whole distribution of cells shifted towards a higher level of strand-breaks, and that a small population of cells with a high level of strands did not contribute the effect breaks. We have previously reported that two polymorphisms, in exons 6 and 23, of the XPD gene are associated, although not signi®cantly, with risk of BCC in these patients [8]. Here we found that high level of UVC-induced formation of strand-breaks was associated with AA and AC genotypes both in exon 6 (P 0:02, Student's t-test) and in exon 23 (P 0:04, Student's t-test) (Fig. 4). As the XPD genotype-association with BCC seems to be conferred by the A allele, we combined the group of patients with AA and AC genotype for the statistical analysis.
4. Discussion In this study we have utilized the comet assay to measure the formation of DNA strand-breaks in resting lymphocytes after a short pulse of UVC light. DNA strand-breaks began accumulating immediately after the exposure, and the number of breaks had a
Fig. 4. The correlation between XPD polymorphism and UVCinduced DNA-repair formation of strand-breaks. (O) psoriasis patients; (V), psoriasis patients with BCC. The line represents the mean tail moment.
suitable dose±response relationship with the UVdose. All breaks were formed in the incubation period after irradiation, i.e. there was no sign of direct strandbreak formation by the UV-light. The number of breaks reached a plateau after approximately 1 h. These observations are in accordance with other reports [3,10,11]. We found little reduction of strand-breaks after 1 h (Fig. 1). However, an incubation time of 5 h showed a tendency of a lower level of strand-breaks in the cell culture which had been exposed to 3.6 J/m 2. It has been reported that aphidicolin, which inhibits progression of the post-incision steps of the repair process, does not increase the accumulation of strand-breaks in UVC irradiated lymphocytes [5]. Both these observations suggest that the strand-breaks, which are generated by the repair machinery following UV-irradiation, can not be readily removed in resting lymphocytes. In the following experiments, we chose an UV-dose high enough to essentially saturate the repair machinery and stopped the incubation of the cells at a time when accumulation of breaks still proceeded in a linear fashion. As a consequence, we expect the assay to measure maximal incision rate. We then proceeded to determine whether the maximal incision rate was related to the risk of developing basal cell carcinoma. For this purpose, we used a set of lymphocyte samples derived from psoriasis patients with or without BCC. We found that patients with BCC had a higher maximal incision rate than the controls (Fig. 2). This is surprising as previous studies showed that BCC patients have a lower DNA-repair activity [7,14,20,21]. It also seems to be at odds with the observation that ®broblastic cell lines and lymphocytes obtained from XPD patients, and fetal ®broblasts from XPC patients, have a lower than normal level of strand-break formation following UVC irradiation [1,2,11]. It is possible that the higher level of UVC-mediated incision could be due to an effect of apoptosis, i.e. the UVC irradiation may produce apoptosis in the group of psoriasis patients with BCC. Cells that are apoptotic are usually described as cells with very long tails [15]. In Fig. 3, it can be seen that the whole population of cells had more strand-breaks in psoriasis patients with BCC. This indicates that the mean tail moment for each patient with BCC was not due to a contribution of a small population of cells with a high level of
P. Mùller et al. / Cancer Letters 151 (2000) 187±192
strand-breaks. Also, the UVC dose which we used for the comet assay, was also lower than the dose used for the UVC-induced UDS (6 J/m 2) [14]. In previous studies of XP patients, higher doses of UVC than 3.6 J/m 2 were used without observing cells from XP patients having a higher level of incisions [1,2,11]. Therefore, apoptosis is not likely to be a major contributor to our result. We have previously reported that two polymorphisms in exons 6 and 23 of the XPD gene displayed some association with the development of BCC [8]. The polymorphisms are nucleotide substitutions (A ! C); however, though there was a higher odds ratio for patients with A alleles, this was not signi®cant. There are no data on the functional signi®cance of the XPD polymorphisms, but they serve as convenient markers of cancer susceptibility. Interestingly, the maximal incision rate correlated with two polymorphisms of the XPD gene (Fig. 4). It is therefore possible that the higher rate of formation of strandbreaks in the BCC patients is related to the function of the XPD protein, which is a helicase [19]. However, the XPD gene is located on chromosome 19q13.2±13.3 in a cluster of DNA-repair genes, which is arranged as XRCC1-ERCC1-XPD-LIG1 [6]. It is possible that the XPD polymorphisms cosegregate with differences in these other genes. The close proximity to the ERCC1 gene is particularly interesting, since the ERCC1 protein, in complex with the XPF protein, is an endonuclease in the nucleotide excision pathway [17].
Acknowledgements The authors would like to thank Dr Margrethe Gade, Dr Birgit Albrectsen, and Dr E.A. Knudsen for their help with examination of the study persons. Jette Petersen and Dr Lisbeth E. Knudsen are thanked for organizing the recruitment of study persons, Lourdes Petersen, Birgitte Korsholm, and AnneKarin Jensen for assistance with the laboratory work. The study was supported by the Danish Medical Research Council (grant 9600259), the Danish Research Academy, Hofbuntmager Aage Bang's Foundation, and the Psoriasis Research Foundation.
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References [1] C. Alapetite, A. Benoit, E. Moustacchi, A. Sarasin, The comet assay as a repair test for prenatal diagnosis of xeroderma pigmentosum and trichothiodystrophy, J. Invest. Dermatol. 108 (1997) 154±159. [2] C. Alapetite, T. Wachter, E. Sage, E. Moustacchi, Use of the alkaline comet assay to detect DNA repair de®ciencies in human ®broblasts exposed to UVC, UVB, UVA and g-rays, Int. J. Radiat. Biol. 69 (1996) 359±369. [3] C.F. Arlett, J.E. Lowe, S.A. Harcourt, A.P. Waugh, J. Cole, L. Roza, B.L. Diffey, T. Mori, O. Nikaido, M.H. Green, Hypersensitivity of human lymphocytes to UV-B and solar irradiation, Cancer Res. 53 (1993) 609±614. [4] J.E. Cleaver, K.H. Kraemer, Xeroderma pigmentosum, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic Basis of Inherited Disease, McGraw±Hill, New York, 1989, pp. 2949±2971. [5] A.R. Collins, M. Ai-guo, S.J. Duthie, The kinetics of repair of oxidative DNA damage (strand-breaks and oxidised pyrymidines) in human cells, Mutat. Res. 366 (1995) 69±77. [6] P. Deloukas, G.D. Schuler, G. Gyapay, E.M. Soderlund, P. RodriguesTomeÂ, L. Hui, T.C. Matise, K.B. McKusich, J.S. Beckman, S. Bentolila, M.-T. Bihoreau, B.B. Birren, J. Browne, A. Butler, A.B. Castle, N. Chiannikulchai, C. Clee, P.J.R. Day, A. Dehejia, T. Dibling, N. Drouot, S. Duprat, C. Fizames, S. Fox, S. Gelling, L. Green, P. Harrison, R. Hocking, E. Holloway, S. Hunt, P. Lijnzaad, C. Louis-Dit-Sully, J. Ma, A. Mendis, J. Miller, J. Morissette, D. Muselet, H.C. Nusbaum, A. Peck, S. Rozen, D. Simon, D.K. Slonim, R. Staples, L.D. Stein, E.A. Steward, M.A. Suchard, T. Thangarajah, N. Vega-Czarny, C. Webber, X. Wu, J. Hudson, C. Auffray, N. Nomura, J.M. Sikela, M.H. Polmeropoulos, M.R. James, E.S. Lander, T.J. Hudson, R.M. Myers, D.R. Cox, J. Weissenbach, M.S. Boguski, D.R. Bentley, A physical map of 30 000 human genes, Science 282 (1998) 744±746. [7] M. Dybdahl, G. Frentz, U. Vogel, H. Wallin, B.A. Nexù, Low DNA repair is a risk factor in skin carcinogenesis: a study of basal cell carcinoma in psoriasis patients, Mutat. Res. 433 (1999) 15±22. [8] M. Dybdahl, U. Vogel, G. Frentz, H. Wallin, B.A. Nexù, Polymorphism in the DNA repair gene XPD: correlations with the risk and age at onset of basal cell carcinoma, Cancer Epidemiol. Biomarkers Prev. 8 (1999) 77±81. [9] E.C. Friedberg, G.C. Walker, W. Siede, DNA Repair and Mutagenesis, ASM Press, Washington, DC, 1995. [10] C.M. Gedik, S.W.B. Ewen, A.R. Collins, Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells, Int. J. Radiat. Biol. 62 (1992) 313±320. [11] M.H.L. Green, J.E. Lowe, H.P. Akinluyi, T. Rowe, J. Cole, A.V. Anstey, C.F. Arlett, UV-C sensitivity of unstimulated and stimulated human lymphocytes from normal and xeroderma pigmentosum donors in the comet assay: a potential diagnostic technique, Mutat. Res. 273 (1992) 137±144. [12] IARC monographs on the evaluation of carcinogenic risks to humans. Overall evaluation of carcinogenicity: An updating
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P. Mùller et al. / Cancer Letters 151 (2000) 187±192 of IARC monographs volumes 1 to 42, IARC (Suppl. 7), 1987, pp. 193-194. H.W. Mohrenweiser, I.M. Jones, Variation in DNA repair is a factor in cancer susceptibility: a paradigm for the promises and perils of individual and population risk estimation? Mutat. Res. 400 (1998) 15±24. P. Mùller, L.E. Knudsen, G. Frentz, M. Dybdahl, H. Wallin, B.A. Nexù, Seasonal variation of DNA damage and repair in patients with non-melanoma skin cancer and referents with and without skin cancer, Mutat. Res. 407 (1998) 25±34. P.L. Olive, G. Frazer, J.P. Banath, Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay, Radiat. Res. 136 (1993) 130±136. J.H. Olsen, G. Frentz, H. Mùller, Malignant tumors in patients with psoriasis, J. Am. Acad. Dermatol. 27 (1992) 716±722. C.-H. Park, T. Bessho, T. Matsunaga, A. Sancar, Puri®cation and characterization of the XPF-ERCC1 complex of human
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DNA repair excision nuclease, J. Biol. Chem. 270 (1995) 22657±22660. M.R. Shen, I.M. Jones, H. Mohrenweiser, Non-conservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans, Cancer Res. 58 (1998) 604±608. P. Sung, V. Bailly, C. Weber, L.H. Thompson, L. Prakash, S. Prakash, Human xeroderma pigmentosum group D gene encodes a DNA helicase, Nature 365 (1993) 852±855. Q. Wei, G.M. Matanoski, E.R. Farmer, M.A. Hedayati, L. Grossman, DNA repair and aging in basal cell carcinoma: a molecular epidemiology study, Proc. Natl. Acad. Sci. USA 90 (1993) 1614±1618. Q. Wei, G.M. Matanoski, E.R. Farmer, M.A. Hedayati, L. Grossman, DNA repair related to multiple skin cancers and drug use, Cancer Res. 54 (1994) 437±440.
Psoriasis patients with basal cell carcinoma have more repairmediated DNA strand-breaks after UVC damage in lymphocytes than psoriasis patients without basal cell carcinoma q,qq Peter Mùller a,*, HaÊkan Wallin a, Marianne Dybdahl a, Gerda Frentz b, Bjùrn A. Nexù a b
a National Institute of Occupational Health, DK-2100 Copenhagen, Denmark Institute of Preventive Medicine, Kommunehospitalet, DK-1399 Copenhagen K, Denmark
Received 13 July 1999; received in revised form 19 November 1999; accepted 20 November 1999
Abstract We have investigated the formation of strand-breaks following UVC irradiation in lymphocytes from psoriasis patients with or without basal cell carcinoma (BCC). Isolated lymphocytes were irradiated with UVC light at a dose of 3.6 J/m 2, and the level of DNA strand-breaks were measured 25 min after the irradiation by the alkaline comet assay. The generation of strand-breaks following UVC irradiation indicates DNA-repair-mediated incisions, as UVC light does not generate strand-breaks per se. We found that psoriasis patients with BCC had more DNA-repair incisions than non-cancer patients. The incision level correlated to two polymorphisms of the XPD gene. At present, it is not clear if the association is a primary effect that is related to differences of the XPD protein. Genes encoding for other repair proteins, namely XRCC1, ERCC1, and LIG1 are located close to the XPD gene, and it is possible that the association is due to a cosegregation with a polymorphism in one of these genes. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basal cell carcinoma; Comet assay; DNA-repair activity; Psoriasis; XPD gene
1. Introduction The DNA-repair machinery is essential in protectq This paper is dedicated to the memory of Gerda Frentz who died 9th November, 1998. qq The contribution of the dermatologists in private practice all over Denmark, the departments of dermatology of the ®ve Danish university hospitals, and the collaboration of dermato-pathologists are gratefully acknowledged. A special list of these participants is available on request, addressed to Jette Petersen, Institute of Preventive Medicine, Kommunehospitalet, DK-1399 Copenhagen K, Denmark. * Corresponding author. Tel.: 145-35-327-657; fax: 145-35327-610. E-mail address: [email protected] (P. Mùller)
ing the genetic code from mutagenic exposure from sources such as radiation or chemicals in the environment. It comprises a complex set of repair proteins that are grouped into several more or less separate pathways [9]. The NER 3 pathway involves some 25 different proteins, which in a stepwise manner recognize DNA damage, incise the strand, excise the DNA damage, and restore the DNA strand by the incorporation of new nucleotides and ligation. It is commonly accepted that reduced DNA-repair activity can render a higher risk of developing cancer. At the extreme end of low repair activity, several cancer-prone disorders among humans have been linked to genes that are involved in DNA repair. XP
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00414-0
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P. Mùller et al. / Cancer Letters 151 (2000) 187±192
patients constitute the most severe form of genetic inherited DNA-repair disorder in the NER pathway, where some patients display DNA-repair activity as low as 2% of the population mean [4]. Heterogeneity of DNA-repair activity in the general population has also been described [13]. We have undertaken a study of the role of DNA repair in the development of basal cell carcinoma (BCC) among patients suffering from psoriasis. The affections seen on the skin of psoriasis patients are due to an increased epidermal proliferation, and the treatment regime is often directed toward minimizing the proliferation. The drugs are often topically applied to the skin, and include carcinogenic agents such as coal tar, PUVA, and Grenz rays (ultrasoft Xrays) [12]. An epidemiological study among Danish psoriasis patients has thus indicated a 2.5-fold increase of the risk of developing skin cancer [16]. It is likely that some psoriasis patients develop BCC because of an unfortunate combination of the treatment with DNA-damaging agents and a low DNArepair activity. Thus, we have previously reported that psoriasis patients with BCC had reduced DNArepair activity. This was evidenced by both UVCinduced UDS [14] and HCR [7]. Our data are in agreement with other studies showing that patients with BCC had a reduced DNA-repair activity compared to non-cancer controls [20,21]. The genetic basis for individual variation in DNA-repair activity is still in the early stage. Several genetic polymorphisms in NER genes are described in DNA material from presumed healthy individuals [18]. Although the polymorphisms are known, the phenotypic outcome is not described. We have previously reported two polymorphisms in the XPD gene located in exon 23 (A ! C that gives rise to a Lys ! Gln amino acid substitution) and exon 6 (A ! C that is silent) [8]. In both polymorphisms, individuals with the AA genotype appeared to have an increased risk of BCC, although the risk did not reach statistical signi®cance. The mean age at onset of BCC was also lower for patients with the A alleles [8]. These ®ndings suggest that the polymorphisms in the XPD gene are associated with a risk of BCC. In this study of psoriasis patients with and without BCC, we measured the maximal incision rate of the repair system in DNA after UVC irradiation of lymphocytes by the alkaline comet assay.
2. Materials and methods 2.1. Study persons A detailed description of the study persons involved in this project have been published elsewhere [7,14]. Brie¯y, 20 subjects in each group were matched for age and gender. None of the patients were treated with genotoxic anti-psoriatic drugs or ointments (UVradiation, PUVA, coal tar and soft X-rays) in a 3-month period before sampling. This part of the study involved 16 patients with psoriasis and 18 patients with psoriasis and BCC (we did not have blood samples left for the six people). 2.2. Comet assay The procedure of comet assay has been described previously in [14]. 2.3. Statistics We have used the two-sided Student's t-test to compare the differences between groups, and for the evaluation of the correlation between the comet assay and polymorphism in the XPD gene. The polymorphisms were classi®ed as CC and AA/AC genotypes for statistical comparison. 3. Results 3.1. Optimization of UVC-induced DNA damage In order to optimize the procedure of UVC irradiation, we initially investigated the formation of strandbreaks following UVC irradiation in lymphocytes obtained from buffy coats from healthy individuals (kindly donated by Bispebjerg Hospital, Copenhagen, Denmark). In general, there were considerable variations in the basal level of strand-breaks from one preparation of buffy coat to another (evidenced by the values at time zero in Fig. 1A±C). A dose± response relationship was found in the interval of 0±3.6 J/m 2. In the dose range of 3.6±10 J/m 2, the level of strand-breaks was almost the same as that for the lower dose (Fig. 1A). This is possibly due to saturation of the comet assay. The time-curves seen by doses of 3.6 J/m 2 (Fig. 1B) and 6 J/m 2 (Fig. 1C)
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strand-breaks, which was on the middle of the initial linear part of the time-curve. We thus, measured the initial rate of strand-break formation by the repair machinery, which operated at, or close to, substrate saturation. 3.2. Patients The UVC-induced DNA-repair formation of strandbreaks in lymphocytes of psoriasis patients with and without BCC is outlined in Fig. 2. There was a signi®cantly higher formation of strand-breaks in lymphocytes obtained from psoriasis patients with BCC compared to psoriasis patients without BCC (P , 0:02, Student's t-test). In order to investigate heterogeneity of the UVC-mediated DNA-repair incisions at a single cell level, we classi®ed all the cells, which were measured for each person, into six classes of DNA damage. The grade of DNA damage was arbitrarily chosen according to the value of the tail moment (I, ,10 mm; II, 10±19.9 mm; III, 20±29.9 mm; IV, 30± 39.9 mm; V, 40±40.9 mm; VI, .50 mm). It can be seen in Fig. 3 that the psoriasis patients with BCC had the
Fig. 1. Formation of DNA-repair-mediated strand-breaks following UVC irradiation of human lymphocytes. (A) Dose±response relationship of UVC after 30 min of incubation; (B), time-course of formation of strand-breaks following UVC irradiation at 3.6 J/m 2; (C), time-course of formation of strand-breaks following UVC irradiation at 6 J/m 2. Symbols represent a mean and SEM of two±three experiments. (W) UV exposed; (X), unexposed.
revealed similar time courses of strand-break formation. Maximal amplitude was reached 1 h after irradiation (Fig. 1B,C). A plateau of strand-breaks was observed in the period of 1±5 h of incubation (Fig. 1B,C), and the level of strand-breaks did not return to baseline even after 24 h (data not shown). Based on data obtained on lymphocytes from buffy coats, we decided to investigate the effect of UVC on blood samples from the patients at a dose of 3.6 J/m 2. We found that 25 min of incubation resulted in a level of
Fig. 2. The distribution of UVC-induced DNA-repair formation of strand-breaks 25 min after irradiation at a dose of 3.6 J/m 2.
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Fig. 3. The number of cells with different grades of DNA damage. The bars represent the mean and SD for all patients. (*P , 0:05, Student's t-test)
whole distribution of cells shifted towards a higher level of strand-breaks, and that a small population of cells with a high level of strands did not contribute the effect breaks. We have previously reported that two polymorphisms, in exons 6 and 23, of the XPD gene are associated, although not signi®cantly, with risk of BCC in these patients [8]. Here we found that high level of UVC-induced formation of strand-breaks was associated with AA and AC genotypes both in exon 6 (P 0:02, Student's t-test) and in exon 23 (P 0:04, Student's t-test) (Fig. 4). As the XPD genotype-association with BCC seems to be conferred by the A allele, we combined the group of patients with AA and AC genotype for the statistical analysis.
4. Discussion In this study we have utilized the comet assay to measure the formation of DNA strand-breaks in resting lymphocytes after a short pulse of UVC light. DNA strand-breaks began accumulating immediately after the exposure, and the number of breaks had a
Fig. 4. The correlation between XPD polymorphism and UVCinduced DNA-repair formation of strand-breaks. (O) psoriasis patients; (V), psoriasis patients with BCC. The line represents the mean tail moment.
suitable dose±response relationship with the UVdose. All breaks were formed in the incubation period after irradiation, i.e. there was no sign of direct strandbreak formation by the UV-light. The number of breaks reached a plateau after approximately 1 h. These observations are in accordance with other reports [3,10,11]. We found little reduction of strand-breaks after 1 h (Fig. 1). However, an incubation time of 5 h showed a tendency of a lower level of strand-breaks in the cell culture which had been exposed to 3.6 J/m 2. It has been reported that aphidicolin, which inhibits progression of the post-incision steps of the repair process, does not increase the accumulation of strand-breaks in UVC irradiated lymphocytes [5]. Both these observations suggest that the strand-breaks, which are generated by the repair machinery following UV-irradiation, can not be readily removed in resting lymphocytes. In the following experiments, we chose an UV-dose high enough to essentially saturate the repair machinery and stopped the incubation of the cells at a time when accumulation of breaks still proceeded in a linear fashion. As a consequence, we expect the assay to measure maximal incision rate. We then proceeded to determine whether the maximal incision rate was related to the risk of developing basal cell carcinoma. For this purpose, we used a set of lymphocyte samples derived from psoriasis patients with or without BCC. We found that patients with BCC had a higher maximal incision rate than the controls (Fig. 2). This is surprising as previous studies showed that BCC patients have a lower DNA-repair activity [7,14,20,21]. It also seems to be at odds with the observation that ®broblastic cell lines and lymphocytes obtained from XPD patients, and fetal ®broblasts from XPC patients, have a lower than normal level of strand-break formation following UVC irradiation [1,2,11]. It is possible that the higher level of UVC-mediated incision could be due to an effect of apoptosis, i.e. the UVC irradiation may produce apoptosis in the group of psoriasis patients with BCC. Cells that are apoptotic are usually described as cells with very long tails [15]. In Fig. 3, it can be seen that the whole population of cells had more strand-breaks in psoriasis patients with BCC. This indicates that the mean tail moment for each patient with BCC was not due to a contribution of a small population of cells with a high level of
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strand-breaks. Also, the UVC dose which we used for the comet assay, was also lower than the dose used for the UVC-induced UDS (6 J/m 2) [14]. In previous studies of XP patients, higher doses of UVC than 3.6 J/m 2 were used without observing cells from XP patients having a higher level of incisions [1,2,11]. Therefore, apoptosis is not likely to be a major contributor to our result. We have previously reported that two polymorphisms in exons 6 and 23 of the XPD gene displayed some association with the development of BCC [8]. The polymorphisms are nucleotide substitutions (A ! C); however, though there was a higher odds ratio for patients with A alleles, this was not signi®cant. There are no data on the functional signi®cance of the XPD polymorphisms, but they serve as convenient markers of cancer susceptibility. Interestingly, the maximal incision rate correlated with two polymorphisms of the XPD gene (Fig. 4). It is therefore possible that the higher rate of formation of strandbreaks in the BCC patients is related to the function of the XPD protein, which is a helicase [19]. However, the XPD gene is located on chromosome 19q13.2±13.3 in a cluster of DNA-repair genes, which is arranged as XRCC1-ERCC1-XPD-LIG1 [6]. It is possible that the XPD polymorphisms cosegregate with differences in these other genes. The close proximity to the ERCC1 gene is particularly interesting, since the ERCC1 protein, in complex with the XPF protein, is an endonuclease in the nucleotide excision pathway [17].
Acknowledgements The authors would like to thank Dr Margrethe Gade, Dr Birgit Albrectsen, and Dr E.A. Knudsen for their help with examination of the study persons. Jette Petersen and Dr Lisbeth E. Knudsen are thanked for organizing the recruitment of study persons, Lourdes Petersen, Birgitte Korsholm, and AnneKarin Jensen for assistance with the laboratory work. The study was supported by the Danish Medical Research Council (grant 9600259), the Danish Research Academy, Hofbuntmager Aage Bang's Foundation, and the Psoriasis Research Foundation.
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