Title: Cutaneous Melanoma Patients Have Normal Repair Kinetics of Ultraviolet-Induced DNA Repair in Skin In Situ
Abstract: The DNA lesions induced by ultraviolet radiation include cyclobutane pyrimidine dimers and 6–4 photoproducts. We investigated whether cutaneous melanoma patients have an impaired ability to repair their ultraviolet-induced photolesions. Seventeen patients with melanoma and 13 healthy controls took part in this study. Both groups received a dose of 40 mJ per cm2 Commission Internationale de l'Éclairage of solar simulating radiation on previously unexposed buttock skin. Skin biopsies were taken at 0 h, 24 h, and 48 h after ultraviolet exposure. A 32P-postlabeling method was used to measure both cyclobutane pyrimidine dimers and 6–4 photoproducts in skin. Cyclobutane pyrimidine dimers and 6–4 photoproduct levels did not differ in the melanoma patients from those in the control group at any time point post-ultraviolet radiation. The repair rate of cyclobutane dimer TT=C was faster than that for TT=T both at 24 h and 48 h postirradiation in both groups, providing evidence of site-specific repair (p < 0.05). We conclude that patients with melanoma have a normal ultraviolet-induced DNA repair capacity in skin in situ. The DNA lesions induced by ultraviolet radiation include cyclobutane pyrimidine dimers and 6–4 photoproducts. We investigated whether cutaneous melanoma patients have an impaired ability to repair their ultraviolet-induced photolesions. Seventeen patients with melanoma and 13 healthy controls took part in this study. Both groups received a dose of 40 mJ per cm2 Commission Internationale de l'Éclairage of solar simulating radiation on previously unexposed buttock skin. Skin biopsies were taken at 0 h, 24 h, and 48 h after ultraviolet exposure. A 32P-postlabeling method was used to measure both cyclobutane pyrimidine dimers and 6–4 photoproducts in skin. Cyclobutane pyrimidine dimers and 6–4 photoproduct levels did not differ in the melanoma patients from those in the control group at any time point post-ultraviolet radiation. The repair rate of cyclobutane dimer TT=C was faster than that for TT=T both at 24 h and 48 h postirradiation in both groups, providing evidence of site-specific repair (p < 0.05). We conclude that patients with melanoma have a normal ultraviolet-induced DNA repair capacity in skin in situ. cutaneous melanoma cyclobutane pyrimidine dimer solar simulating radiation cyclobutane pyrimidine dimers 6–4-[pyrimidine-2′-one] pyrimidine photoproducts The incidence of cutaneous melanoma (CMM) in fair-skinned people has been rising more than that of any other malignancy tumors in the past decades (International Agency for Research on Cancer., 1992International Agency for Research on Cancer IARC Monograph on the Evaluation of Carcinogenic Risk to Human: Solar and Ultraviolet Radiation, 55. IARC, Lyon1992Google Scholar). The factors underlying the rapid increase in the incidence of CMM are incompletely understood, but increased total sun exposure and altered patterns of exposure are strongly implicated. A meta-analysis of CMM case–control studies has shown a significant positive association [odds ratio (OR) = 1.71] between intermittent sun exposure and risk of CMM (Elwood and Jopsonn, 1997Elwood J.M. Jopsonn J. Melanoma sun exposure: an overview of published studies.Int J Cancer. 1997; 73: 198-203https://doi.org/10.1002/(sici)1097-0215(19971009)73:2`198::aid-ijc6b3.0.co;2-rCrossref PubMed Scopus (0) Google Scholar). There are two main kinds of DNA damages in skin after ultraviolet radiation (UVR) exposure, i.e., cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts. A number of studies have indicated a causal relationship between induction of photoproducts and UV carcinogenesis (for reviews seeAnanthaswamy and Pierceal, 1990Ananthaswamy H.N. Pierceal W.E. Molecular mechanisms of ultraviolet radiation carcinogenesis.Photochem Photobiol. 1990; 52: 1119-1136Crossref PubMed Scopus (322) Google Scholar;Black et al., 1997Black H.S. deGruijl F.R. Forbes P.D. et al.Photocarcinogenesis: an overview.J Photochem Photobiol B. 1997; 40: 29-47https://doi.org/10.1016/s1011-1344(97)00021-3Crossref PubMed Scopus (0) Google Scholar;Langley and Sober, 1997Langley R.G.B. Sober A.J. A clinical review of the evidence for the role of ultraviolet radiation in the cutaneous melanoma.Cancer Invest. 1997; 15: 561-567Crossref PubMed Scopus (38) Google Scholar). Mammalian cells have the ability to repair DNA damage induced chemically or physically; UV-induced photoproducts are repaired by the nucleotide excision repair system (Bohr, 1995Bohr V.A. DnA repair fine structure and its relations to genomic instability.Carcinogenesis. 1995; 16: 2885-2892Crossref PubMed Scopus (94) Google Scholar;Lehman, 1995Lehman A.R. Nucleotide excision repair and the link with transcription.TIBS. 1995; 20: 402-405PubMed Google Scholar). The defect in excision repair mechanisms in xeroderma pigmentosum results in a 1000-fold increased incidence of nonmelanoma skin cancer and melanoma (Kraemer et al., 1994Kraemer K.H. Lee M.M. Andrews A.D. Lambert W.C. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer. The xeroderma pigmentosum paradigm.Arch Dermatol. 1994; 130: 1018-1021Crossref PubMed Scopus (444) Google Scholar;Kraemer, 1997Kraemer K.H. Sunlight and skin cancer: another link revealed.Proc Natl Acad Sci USA. 1997; 94: 11-14Crossref PubMed Scopus (329) Google Scholar). For melanoma patients, repair of UVR-induced photoproducts in melanocytes is probably the main issue. Because melanocytes only make up 5%-10% of the cell population only immunohistochemical techniques would allow a direct assessment of DNA damage and repair in melanocytes in situ (Young et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten C.S. The in situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 1307-1313Crossref PubMed Scopus (127) Google Scholar, Young et al., 1998Young A.R. Potten C.S. Nikaido O. Parsons P.G. Boenders J. Ramsden J.M. Chadwick C.A. Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers.J Invest Dermatol. 1998; 111: 936-940Crossref PubMed Scopus (105) Google Scholar). Instead, cultured melanocytes have been used (Gilchrest et al., 1999Gilchrest B.A. Eller M.S. Geller A. Yaar M. The pathogenesis of melanoma induced by ultraviolet radiation.N Engl J Med. 1999; 340: 1341-1348Crossref PubMed Scopus (627) Google Scholar) or DNA repair has been measured in leukocytes from melanoma patients (Ringborg et al., 1980Ringborg U. Lagerlof B. Lambert B. Normal UV-induced DNA repair synthesis in peripheral leukocytes from patients with malignant melanoma of the skin.J Invest Dermatol. 1980; 74: 72-73Crossref PubMed Scopus (10) Google Scholar) or in human melanoma cell lines (Hatton et al., 1995Hatton D.H. Mitchell D.L. Strickland P.T. Johnson R.T. Enhanced photoproduct repair: its role in the DNA damage-resistance phenotype of human malignant melanoma cells.Cancer Res. 1995; 55: 181-189PubMed Google Scholar). The recently developed 32P-postlabeling method can directly measure DNA damage and DNA repair in human skin in situ with a small amount of DNA, and it has been successfully used in studies on healthy subjects (Bykov et al., 1998aBykov V.J. Lindgren C. Tobin D. Hemminki K. 32P-HPLC technique shows base sequence dependent difference in photolesion repair in human keratinocytes.Chem–Biol Interacts. 1998; 110: 71-84Crossref PubMed Scopus (7) Google Scholar,Bykov et al., 1998bBykov V.J. Jansen C.T. Hemminki K. High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation.Cancer Epidemiol Biomarkers Prev. 1998; 7: 199-202PubMed Google Scholar,Bykov et al., 1998cBykov V.J. Marcusson J.A. Hemminki K. Ultraviolet B-induced DNA damage in human skin and its modulation by a sunscreen.Cancer Res. 1998; 58: 2961-2964PubMed Google Scholar,Bykov et al., 1999Bykov V.J. Sheehan J.M. Hemminki K. Young A.R. In situ repair of cyclobutane pyrimidine dimers and 6–4 photoproducts in human skin exposed to solar simulating radiation.Invest Dermatol. 1999; 112: 326-331Crossref PubMed Scopus (81) Google Scholar). We applied this technique here to investigate the UV-induced DNA global repair in skin of CMM patients in situ compared with a number of healthy controls. Seventeen CMM patients and 13 healthy controls were included in the study. This study was approved by the Medical Ethics Committee of Päijät-Häme Central Hospital, Finland. All the participants gave their informed consent. Data of the volunteers are shown in Table 1. Among CMM patients, one patient had four separate melanomas during 11 y from 1983 to 1994. Two of the cases had relatives with melanoma. The mean duration from the diagnosis to the excision of melanoma was 18.3 ± 15.3 mo (range 3–65 mo) in 16 CMM patients with one melanoma. All CMM cases were evaluated and classified histologically according either to the Clark (Clark et al., 1975Clark W.H. Ainsworth A.M. Bernadino E.A. Yang C.-H. Mihm M.C. Reed R.J. The developmental biology of primary malignant melanoma.Semin Oncol. 1975; 2: 83-103PubMed Google Scholar) or Breslow method, ranging Clark II–V and Breslow 0.3–7.1. The skin types were classified according to the system ofFitzpatrick, 1975Fitzpatrick T.B. Sodeil et peau.J Med Esthet. 1975; 2: 33-34Google Scholar.Table 1Volunteers' profile in DNA repair studyNo. of controlNo. of casep valuebStudent's t test for age; Mantel–Haenszel Chi-square test for skin type and gender.Age (yaAge expressed as mean ± SD.)13 (48.4 ± 10.03)17 (52.8 ± 14.26)0.35Skin typeI + II710III + IV670.79GenderMale10130.98Female34a Age expressed as mean ± SD.b Student's t test for age; Mantel–Haenszel Chi-square test for skin type and gender. Open table in a new tab All subjects were exposed to an erythemally weighted UV dose of 40 mJ per cm2 CIE (Commission Internationale de l'Éclairage,McKinlay and Diffey, 1987McKinlay A.F. Diffey B.L. A reference action spectrum for ultraviolet induced erythema in human skin.CIE-J Res Note. 1987; 6: 17-22Google Scholar) on previously unexposed buttock skin using equipment detailed earlier (Snellman et al., 1995Snellman E. Jansen C.T. Leszczynski K. Visuri R. Milan T. Jokela K. Ultraviolet erythema sensitivity in anamnestic (I-IV) and phototested (1–4) Caucasian skin phototypes: the need for a new classification system.Photochem Photobiol. 1995; 62: 769-772Crossref PubMed Scopus (48) Google Scholar). This dose is equivalent to an unweighted dose of 22.14 J per cm2 of UVA plus 0.57 J per cm2 of UVB. Within 24 h such a UV dose induces moderate to strong erythema in the exposed skin of photosensitive skin types I–II and light or no erythema in phototolerant skin types III–IV (Snellman et al., 1995Snellman E. Jansen C.T. Leszczynski K. Visuri R. Milan T. Jokela K. Ultraviolet erythema sensitivity in anamnestic (I-IV) and phototested (1–4) Caucasian skin phototypes: the need for a new classification system.Photochem Photobiol. 1995; 62: 769-772Crossref PubMed Scopus (48) Google Scholar). The irradiance of the lamp (Philips HP 411/A) was measured (250–400 nm) prior to the study at 30 cm using an Optronic 742 spectroradiometer with Teflon diffuser as input optics, 97.5% of the irradiance being UVA and 2.5% UVB. The lamp emitted no UVR in the waveband area below 290 nm and its spectral curves mimicked the spectrum of the sun on the earth closely (Snellman et al., 1995Snellman E. Jansen C.T. Leszczynski K. Visuri R. Milan T. Jokela K. Ultraviolet erythema sensitivity in anamnestic (I-IV) and phototested (1–4) Caucasian skin phototypes: the need for a new classification system.Photochem Photobiol. 1995; 62: 769-772Crossref PubMed Scopus (48) Google Scholar). The total irradiated site was 4 cm × 4 cm. After irradiation, altogether three punch skin biopsies (4 mm in diameter) were taken using lidocaine–epinephrine local anesthesia. The samples were taken at three different time points, i.e., the first sample within 20 min, the second at 24 h and the third at 48 h. the skin samples were immediately put in ice, frozen and stored at -20°C. The procedure of DNA extraction and photoproduct measurement were performed as described in a previous study (Bykov et al., 1998bBykov V.J. Jansen C.T. Hemminki K. High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation.Cancer Epidemiol Biomarkers Prev. 1998; 7: 199-202PubMed Google Scholar). DNA extraction from epidermis was performed using the chloroform–isoamyl alcohol (24:1) method after separation of epidermis from dermis with blunt scalpel. For each 32P-postlabeling assay 3 μg DNA was used. The HPLC conditions were similar to a previous study (Bykov et al., 1999Bykov V.J. Sheehan J.M. Hemminki K. Young A.R. In situ repair of cyclobutane pyrimidine dimers and 6–4 photoproducts in human skin exposed to solar simulating radiation.Invest Dermatol. 1999; 112: 326-331Crossref PubMed Scopus (81) Google Scholar), i.e., gradient elution was performed to separate the photoproducts with a buffer (0.5 M ammonium formate, 20 mM orthophosphoric acid, pH 4.6) that mixed with methanol. The flow rate through the analytic column was 0.2 ml per min under +40°C with the aid of the Cool Pocket (Keystone Scientific, Gene Tee, Sweden). For calculations of the photoproduct levels the high-performance liquid chromatography (HPLC) peaks areas were integrated with the Beckman System Gold software. The relevant peaks were determined by the retention times of the external standards. The signals of the relevant fractions exceeded the peak areas of the background noise levels at least two times. In this study we used no unirradiated background samples from the subjects because previous studies have shown that no peaks eluted in the fractions of the photoproducts (Bykov et al., 1998bBykov V.J. Jansen C.T. Hemminki K. High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation.Cancer Epidemiol Biomarkers Prev. 1998; 7: 199-202PubMed Google Scholar,Bykov et al., 1998cBykov V.J. Marcusson J.A. Hemminki K. Ultraviolet B-induced DNA damage in human skin and its modulation by a sunscreen.Cancer Res. 1998; 58: 2961-2964PubMed Google Scholar) The levels of photoproducts were expressed as per 106 nucleotides. All data were analyzed with the Excel 97 and Epi Info 6 programs (WHO, Geneva, Switzerland 1997). The significance level was 0.05. For DNA repair kinetics analysis, the levels of photoproducts at 0 h post-UVR were taken as 100%. The percentage of unrepaired photoproducts at 24 h and 48 h was calculated by the following formula: % of remaining photoproducts at 24 h or 48 h = the levels of photoproducts at 24 h or 48 h (100/the levels of photoproducts at 0 h. The levels of CPD and 6–4 photoproducts in skin at different time points post-UVR are shown in Table 2. No level in the CMM patients significantly differed from those in the control group at any time point after UVR. The stratified analysis of photoproduct levels by age and skin type did not show significant differences between the CMM and the control group at any time point (data not shown). The abundance of the photoproduct levels in the CMM group followed the same order as that in the control group: TT=T > TT=C > TT–C > TT–T at 0 h after irradiation with UV. The 6–4 photoproduct levels were about one-tenth of the corresponding CPD in the CMM and the control groups. The data on the 6–4 photoproducts may be unreliable in the 24 h and 48 h samples because the HPLC peaks were small, and it was difficult to determine the exact peak area. So, we only presented the immediate levels of 6–4 photoproducts after UVR in this study.Table 2Normal photoproduct levels and repair in melanoma patients compared with control groupAdductsaAdducts levels expressed as mean ± SD. Nt, nucleotide. (per 106 Nt)Time point (h)Control groupMelanoma groupTT=C04.30 ± 2.77 (11)bNumber of subjects.5.96 ± 3.81 (17)242.18 ± 1.46 (13)2.94 ± 2.42 (16)480.98 ± 0.84 (12)1.16 ± 1.42 (16)TT=T05.13 ± 2.94 (12)7.39 ± 4.03 (17)243.86 ± 1.92 (12)4.68 ± 2.93 (16)482.32 ± 1.06 (12)2.71 ± 2.03 (16)TT–T00.41 ± 0.32 (12)0.60 ± 0.36 (17)TT–C00.65 ± 0.37 (12)0.64 ± 0.49 (17)a Adducts levels expressed as mean ± SD. Nt, nucleotide.b Number of subjects. Open table in a new tab The immediate (0 h) photoproduct levels showed a large interindividual variation both in the CMM group and in the control group, from 7- to 15-fold depending on the type of photoproducts. For instance, 8.6-fold variation for TT=C levels (range from 1.16 to 10.01/106 Nt) and 8-fold for TT=T levels (range from 1.58 to 12.64/106 Nt) were found in the control group; 14.7-fold for TT=C levels (range from 0.92 to 13.5/106 Nt) and 13.2-fold for TT=T levels (range from 1.03 to 13.5/106 Nt) in the CMM group. There was no significant difference of the variation in the immediate photoproduct levels between the two groups (p > 0.05). Figure 1 and Figure 2 show the DNA repair kinetics of the two CPD. Neither TT=C nor TT=T in CMM group showed significantly different repair rates from those in the control group. Twenty-four hours after the UV exposure, more than 40% in the control group and almost 60% in the CMM group of the cyclobutane dimer TT=C remained unrepaired, but the difference did not reach statistical significance. At 48 h, about 20% of TT=C still existed in the both groups (Figure 1). For cyclobutane dimer TT=T, the unrepaired levels at 24 h and 48 h were similar in the CMM patients to those in the controls (Figure 2). The comparison of the repair rates of TT=C and TT=T showed that TT=C was repaired much faster than TT=T both at 24 h and 48 h time points (p<0.05). For instance, 79.6% of TT=C, but only 53.6% of TT=T, was removed after 48 h of UV exposure in the CMM group. There was a large interperson variation in the rate of DNA repair (percent of repaired photoproducts ranged from 0% to 60% at 24 h after SSR) but no significant difference of the variation was found between the two groups.Figure 2Similar repair kinetics of TT=T in the CMM patients and in the control group. Three skin biopsies were taken from each subject at 0 h, 24 h, and 48 h after 40 mJ per cm2 of UVR. TT=T levels in the DNA from the skin biopsies were measured by the HPLC–32P-postlabeling. The levels of TT=T at 0 h was assigned as 100%. The bars are the mean ± SD of percentage of unrepaired TT=T. There were 12 subjects in the control group, 16 in the CMM group. Notice that the number of subjects at 24 h and 48 h is less than given in Table 2 because percentage unrepaired photoproduct could only be calculated for those who had the 0 h result.View Large Image Figure ViewerDownload (PPT) This study shows an application of the 32P-postlabeling HPLC method to investigate in situ repair kinetics of CMM patients. Previously, we have demonstrated the feasibility of the method in healthy volunteers (Bykov et al., 1998bBykov V.J. Jansen C.T. Hemminki K. High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation.Cancer Epidemiol Biomarkers Prev. 1998; 7: 199-202PubMed Google Scholar,Bykov et al., 1999Bykov V.J. Sheehan J.M. Hemminki K. Young A.R. In situ repair of cyclobutane pyrimidine dimers and 6–4 photoproducts in human skin exposed to solar simulating radiation.Invest Dermatol. 1999; 112: 326-331Crossref PubMed Scopus (81) Google Scholar). The 50% removal times were about 15 h for CPD and somewhat over 5 h for 6–4 photoproducts. The average removal rates in this study are similar to the previous study (Bykov et al., 1998bBykov V.J. Jansen C.T. Hemminki K. High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation.Cancer Epidemiol Biomarkers Prev. 1998; 7: 199-202PubMed Google Scholar) (TT=C, 79.6% vs 85%; TT=T, 53.6% vs 68% after 48 h of SSR) and with only three sampling time points we cannot determine the 50% removal rates more exactly. The results on the CMM patients showed no difference on photoproduct levels immediately after UVR and in the later samplings, suggesting that the repair of UV lesions is normal in CMM patients. To our knowledge, DNA repair kinetics have never been assayed in the human skin among CMM patients. In cell cultures, the human melanoma cell lines isolated from metastatic melanoma display an increased resistance to killing by UVR because of an enhanced rate of postreplication recovery and on overall increase in photoproduct repair (Hatton et al., 1995Hatton D.H. Mitchell D.L. Strickland P.T. Johnson R.T. Enhanced photoproduct repair: its role in the DNA damage-resistance phenotype of human malignant melanoma cells.Cancer Res. 1995; 55: 181-189PubMed Google Scholar). The high level of DNA repair capacity is also seen in highly metastatic melanoma cells (Wei et al., 1997Wei Q. Cheng L. Xie K. Bucana C.D. Dong Z. Direct correlation between DNA repair capacity and metastatic potential of K-173 murine melanoma cells.J Invest Dermatol. 1997; 108: 3-6Crossref PubMed Scopus (18) Google Scholar). Ringborg et al., 1980Ringborg U. Lagerlof B. Lambert B. Normal UV-induced DNA repair synthesis in peripheral leukocytes from patients with malignant melanoma of the skin.J Invest Dermatol. 1980; 74: 72-73Crossref PubMed Scopus (10) Google Scholar) analyzed unscheduled DNA synthesis in leukocytes of CMM patients and found no difference to the control group. Assays on endonuclease sensitive sites (Alcalay et al., 1990Alcalay J. Freeman S.E. Goldberg L.H. Wolf J.E. Excision repair of pyrimidine dimers induced by simulated solar radiation in the skin of patients with basal cell carcinoma.J Invest Dermatol. 1990; 95: 506-509Abstract Full Text PDF PubMed Google Scholar) or lymphocytes repair capacity (Athas et al., 1991Athas W.F. Hedayatai M.A. Matanoski G.M. Farmer E.R. Grossman L. Development and field-test validation of an assay for DNA repair in circulating lymphocytes.Cancer Res. 1991; 51: 5786-5793PubMed Google Scholar;Grossman and Wei, 1995Grossman L. Wei Q. DNA repair and epidemiology of basal cell carcinoma.Clin Chem. 1995; 41: 1854-1863PubMed Google Scholar;Wei et al., 1995Wei Q. Matanoski G.M. Farmer E.R. Hedayati M.A. Grossman L. DNA repair capacity for ultraviolet light-induced damage is reduced in peripheral lymphocytes from patients with basal cell carcinoma.J Invest Dermatol. 1995; 104: 933-936Crossref PubMed Scopus (67) Google Scholar) have not been applied to CMM patients. Our data also show that UV-induced DNA repair in the CMM patients takes place in the same sequence-dependent fashion as in the control group, i.e., CPD are repaired faster in TT=C sequence than in TT=T sequence (Table 2). This sequence-dependent fashion of DNA repair has also been found in human keratinocytes in vitro (Bykov et al., 1998aBykov V.J. Lindgren C. Tobin D. Hemminki K. 32P-HPLC technique shows base sequence dependent difference in photolesion repair in human keratinocytes.Chem–Biol Interacts. 1998; 110: 71-84Crossref PubMed Scopus (7) Google Scholar) and in human skin in situ from normal population (Bykov et al., 1998bBykov V.J. Jansen C.T. Hemminki K. High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation.Cancer Epidemiol Biomarkers Prev. 1998; 7: 199-202PubMed Google Scholar,Bykov et al., 1999Bykov V.J. Sheehan J.M. Hemminki K. Young A.R. In situ repair of cyclobutane pyrimidine dimers and 6–4 photoproducts in human skin exposed to solar simulating radiation.Invest Dermatol. 1999; 112: 326-331Crossref PubMed Scopus (81) Google Scholar). Although the reason for this sequence-specificity of UV-induced DNA repair is unknown, its finding in this study indicates that melanoma patients have a similar mechanism to repair UV-induced DNA damage as the control population. In this study we observed the large interindividual variation (7–15-fold, depending on the photoproduct) in the immediate level of photoproducts found by earlier studies (Bykov et al., 1998bBykov V.J. Jansen C.T. Hemminki K. High levels of dipyrimidine dimers are induced in human skin by solar-simulating UV radiation.Cancer Epidemiol Biomarkers Prev. 1998; 7: 199-202PubMed Google Scholar,Bykov et al., 1998cBykov V.J. Marcusson J.A. Hemminki K. Ultraviolet B-induced DNA damage in human skin and its modulation by a sunscreen.Cancer Res. 1998; 58: 2961-2964PubMed Google Scholar). A small part of this variation can be explained by age and skin type as shown elsewhere (Xu et al., in pressXu G, Snellman E, Bykov VJ, Jansen CT, Hemminki K. Effects of age on the formation and repair of UV photoproducts in human skin in situ. Mutat Res, in press.Google Scholar). There was no difference between the CMM and control group, however, suggesting that the vulnerability to damage is not a direct cause of CMM. Another source of interindividual variation was that of repair rates, ranging from 0% to 60% of repaired photoproducts at 24 h. Again, however, the two groups did not differ. We have found no modulation of repair rates by age or skin type (Xu et al., in pressXu G, Snellman E, Bykov VJ, Jansen CT, Hemminki K. Effects of age on the formation and repair of UV photoproducts in human skin in situ. Mutat Res, in press.Google Scholar). As discussed in a previous study (Bykov et al., 1999Bykov V.J. Sheehan J.M. Hemminki K. Young A.R. In situ repair of cyclobutane pyrimidine dimers and 6–4 photoproducts in human skin exposed to solar simulating radiation.Invest Dermatol. 1999; 112: 326-331Crossref PubMed Scopus (81) Google Scholar), the analyzed CPD and 6–4 photoproducts represent only a fraction of the total level of these lesions in DNA. The method employed in this study provides data on the photoproducts only in defined trinucleotide sequences, where the first nucleotide can be any of the four. In this study, we measured global repair and changes in subtypes, such as transcription-coupled repair could probably not be observed. It is possible that melanoma patients have impaired transcription-coupled repair or lower ability to repair other DNA damages induced by UVR, such as DNA breaks, DNA–protein cross-links, etc., which could be important for the development of melanoma. A further qualification of these data is that the whole epidermis was used in this study, reflecting photoproduct levels in keratinocytes rather than melanocytes which constitute only 5%-10% of the cell population. This study was supported by the EU Environment Program and the Swedish Cancer Fund.