Melanocytes are deficient in repair of oxidative DNA damage and UV-induced photoproducts Hsiang-Tsui Wang1, Bongkun Choi1, and Moon-shong Tang2 Department of Environmental Medicine, Pathology and Medicine, New York University School of Medicine, Tuxedo, NY 10987 Communicated by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY, April 23, 2010 (received for review December 30, 2009) Melanomas occur mainly in sunlight-exposed skin. Xeroderma that UVA-induced oxidative stress (OS) and oxidative DNA pigmentosum (XP) patients have 1,000-fold higher incidence of damage (ODD) are the two major contributors to UVA-induced melanoma, suggesting that sunlight-induced “bulky” photoprod- melanomagenesis (18). The occurrence of mucosal melanoma in ucts are responsible for melanomagenesis. Sunlight induces a high regions that are rarely exposed to sunlight—such as genitalia, the level of reactive oxygen species in melanocytes (MCs); oxidative colon, and the nasal septum (19–21)—suggests that DNA damage DNA damage (ODD) may thus also contribute to melanomagenesis, other than photoproducts is also involved in melanomagenesis. If and XP gene products may participate in the repair of ODD. We we hypothesize that ODD is the culprit responsible for triggering examined the effects of melanin on UVA (320–400 nm) irradiation- mucosal melanomagenesis, then one has to assume MCs in these induced ODD and UV photoproducts and the repair capacity in MC tissues are undergoing endogenous OS and generating ROS, even and XP cells for ODD and UV-induced photoproducts. Our findings without sunlight irradiation. Because melanin is an effective free indicate that UVA irradiation induces a significantly higher amount radical scavenger and would presumably mitigate OS, the role of of formamidopyrimidine glycosylase-sensitive ODD in MCs than in melanin in melanomagenesis remains unclear (22). normal human skin fibroblasts (NHSFs). In contrast, UVA irradiation Previously, using host cell reactivation (HCR) and in vitro re- induces an insignificant amount of UvrABC-sensitive sites in either pair synthesis assays, we have found that OS and lipid peroxida- of these two types of cells. We also found that, compared to NHSFs, tion (LPO) byproducts—such as 4-hydroxy-2-nonenal (4-HNE), MCs have a reduced repair capacity for ODD and photoproducts; malondialdehyde (MDA), and acrolein (Acr) —inhibit NER and fi H2O2 modi ed- and UVC-irradiated DNAs induce a higher mutation enhance mutagenesis through direct modification of repair pro- frequency in MCs than in NHSFs; and, XP complementation group A teins by the carbonyl group in the aldehydes (23–25). These find- (XPA), XP complementation group C, and XP complementation ings raise the possibility that the intrinsically high OS in mela- fi group G cells are de cient in ODD repair and ODD induces a higher nocytes and OS induced by UVA irradiation may induce LPO; mutation frequency in XPA cells than in NHSFs. These results suggest LPO byproducts could then reduce the DNA repair capacity, i that: ( ) melanin sensitizes UVA in the induction of ODD but not which in turn could contribute to melanomagenesis. ii bulky UV photoproducts; ( ) the high susceptibility to UVA-induced To understand the role of melanin in UVA radiation-induced ODD and the reduced DNA repair capacity in MCs contribute to iii DNA damage formation and the mechanism that leads to high carcinogenesis; and ( ) the reduced repair capacity for ODD contrib- melanoma incidence in XP individuals, we determined the ef- utes to the high melanoma incidence in XP patients. fects of melanin on UVA-induced ODD and UV photoproduct formation; the repair capacity for ODD and UV photoproducts DNA repair and mutagenesis | UV photoproducts | ultraviolet light | in MC, XPA, XPC, and XPG cells; and the mutagenicity of melanoma | xeroderma pigmentosum ODD in these cells. Our results show that melanin plays a crucial role in UVA-induced mutagenesis and also, most likely, in t has long been recognized that sunlight exposure is the major melanomagenesis. Icause of skin cancers, including melanoma (1–3). Although evi- dence from both epidemiological and molecular studies show that Results photoproducts induced by UVB (290–320 nm) irradiation are the Oxidative DNA Damage and Bulky Photoproducts Induced by UVA major cause for nonmelanoma skin cancer (4), epidemiological Irradiation in MCs. It has been shown that melanin enhances free studies strongly implicate UVA (320–400 nm) irradiation in sun- radical generation, including reactive ROS resulting from UVA light exposure-related cutaneous melanomagenesis (5, 6). In animal irradiation (15–17). Hence, it is possible that melanin enhances models, it has been found that both UVA and UVB irradiation ODD. To test this possibility, lightly and darkly pigmented MCs can trigger melanocytic hyperplasia and melanomagenesis (7–9). and normal human skin fibroblasts (NHSFs, CRL2097) were ir- Although UVB radiation can induce both cyclobutane pyrimidine radiated with UVA light and the formation of ODD was detected dimers (CPDs) and pyrimidine < 6–4 > pyrimidone photoproducts by the formamidopyrimidine glycosylase (Fpg) incision assay. (<6–4 > PPs), which can trigger mutagenesis and carcinogenesis Formamidopyrimidine glycosylase incises 8-oxo-deoxyguanosine (10, 11), the photoproduct yield of these types of DNA damage by (8-oxo-dG) and the imidazole ring-opened adducts of purines; UVA irradiation is two to three orders of magnitude lower than by these adducts are generated in cells under OS and also in H2O2- UVB radiation (12). Thus, exactly how UVA acts to promote treated DNA (26). The results in Fig. 1A show that UVA irradi- melanomagenesis remains controversial. It has been found that XP ation does indeed enhance Fpg-sensitive site formation in both patients have 1,000-fold higher incidence of melanoma than normal individuals (13). Xeroderma pigmentosum cells are sensitive to UV radiation but resistant to ionizing radiation, and XP gene products Author contributions: H.-T.W., B.C., and M.-s.T. designed research; H.-T.W. and B.C. per- are also crucial for CPD and <6–4 > PP repair (14). Therefore, it is formed research; H.-T.W., B.C., and M.-s.T. analyzed data; and H.-T.W., B.C., and M.-s.T. possible that UVA irradiation of melanocytes (MCs) may induce wrote the paper. “bulky” photoproducts that are repairable by a nucleotide excision The authors declare no conflict of interest. repair (NER) mechanism and these photoproducts, if not repaired, Freely available online through the PNAS open access option. trigger mutagenesis and, consequently, melanomagenesis. 1H.-T.W. and B.C. contributed equally to this work. It is well established that the major effect of UVA irradiation is 2To whom correspondence should be addressed. E-mail: [email protected]. the induction of reactive oxygen species (ROS) and that melanin This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. can greatly enhance this process (15–17). It has been proposed 1073/pnas.1005244107/-/DCSupplemental. 12180–12185 | PNAS | July 6, 2010 | vol. 107 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1005244107 Downloaded by guest on October 1, 2021 lightly and darkly pigmented MCs, but not in NHSFs, indicating product in the luciferase gene is sufficient to block the expression of that UVA irradiation induces ODD in MCs but not in NHSFs. this gene, then the results in Fig. 2A suggest that 2 photoproducts in We used the UvrABC incision method to determine the effect of the coding strand of the luciferase gene are needed to block 63% of melanin on CPD and <6–4 > PP induction by UVA. It is well luciferase gene expression in XPA cells, which indicates XPA cells established that UvrABC, the NER enzyme complex, can spe- are totally defective in CPD and <6–4 > PP repair. In contrast, 9 cifically and quantitatively incise CPDs and <6–4 > PPs (27, 28). photoproducts are needed in the luciferase gene in darkly pig- The results in Fig. 1B show that genomic DNAs isolated from mented MCs to induce the same level of reduction in luciferase gene UVA-irradiated MCs and NHSFs are only slightly, if at all, sen- expression, 15 in lightly pigmented MCs, and 34 in NHSFs. These sitive toward UvrABC nuclease, which indicates that melanin results validate the assay system for measuring the repair capacity of does not affect UVA irradiation-induced bulky photoproduct the host cells and demonstrate that MCs are partially defective in formation. It is worth noting that UvrABC does not recognize photoproduct repair. We also observed that the lightly pigmented H2O2-modified DNA and Fpg does not recognize UVC-induced MCs have a lower repair capacity for photoproducts than darkly bulky photoproducts (Fig. S1). Based on the results shown in pigmented MCs. In addition, our results show that the amount of Fig. 1, we concluded that UVA irradiation induces ODD in MCs melanin in the lightly pigmented MCs was indeed lower than the but not in NHSFs, and that the frequency of UvrABC-sensitive darkly pigmented MCs; however, the ROS level in the former was sites formed in UVA-irradiated MCs is much lower than the significantly higher than in the latter (Figs. S2 and S3). It has been frequency of Fpg-sensitive sites formed in the same cells. suggested that the ROS level in MCs depends on an interplay be- tween the melanin-enhanced generation of ROS (15–17) and its Melanocytes Have a Lower Level of HCR for H2O2-Damaged and UVC- ability to scavenge the ROS (22). The luciferase HCR assay is based Irradiated Luciferase Gene. Because melanin is able to absorb sun- on the expression level of the luciferase gene being inversely pro- light and scavenge free radicals, it shields cells from the damaging portional to the level of DNA damage in this gene (Fig. S4). It is effects of sunlight, including the induction of photoproducts and conceivable that the transcription-coupled repair (TCR) mecha- ROS (22).