Ultraviolet (UV) light from the sun causes a variety of lesions in the genome that distort the structure of DNA, resulting in blocks to gene transcription and DNA replication. Epidemiological evidence strongly indicates that UV-induced DNA damage is a primary cause of skin cancer, including melanoma (1
), an aggressive form of skin cancer which arises from specialized pigmented cells called melanocytes. Though comprising only 5-10% of human skin, melanocytes synthesize the pigment melanin, which provides skin tone, hair color, and protection from UV radiation. The significant increase in melanoma cases in the past half-century and the poor survival rates among patients with metastatic melanoma (3
) therefore merits thorough investigations of the underlying causes of melanoma initiation and progression.
Among the lesions induced by UV, cyclobutane pyrimidine dimers (CPDs; 80-90%) and [6-4] pyrimidine-pyrimidone photoproducts ([6-4] PPs; 10-20%) are most abundant, though both can be accurately removed from the genome by nucleotide excision repair (henceforth termed “excision repair”). This well-characterized repair system responds to a variety of environmental and chemotherapeutic agents that form bulky adducts in DNA, and in humans is the sole mechanism for removal of CPDs and [6-4] PPs from DNA (5
). Importantly, reconstitution of excision repair in vitro with the six essential factors (XPA, RPA, XPC, TFIIH, XPG, and XPF-ERCC1) has provided a significant understanding of the individual steps of repair and allowed a clear determination of the minimal set of factors necessary and sufficient for the complete removal of UV photoproducts from DNA (6
). The importance of the excision repair system to human health is most obvious in xeroderma pigmentosum (XP), a disease in which most patients lack one of the essential excision repair factors (8
). One consequence of this loss is a 2000-fold higher incidence of metastatic melanoma compared to normal individuals (9
Melanomas display complex genetic profiles but often show activating mutations in the oncogenes B-Raf
(50-75%) and N-Ras
), resulting in enhanced cell growth through signaling of the mitogenic ERK1/2 pathway (12
). Similarly, though the tumor suppressor p53
is disrupted in nearly half of all cancers and is known to promote repair of UV photoproducts in many cell types (14
), studies indicate that p53
mutations are rare in primary melanoma (less than 1%) (15
) but do increase in frequency in metastatic melanoma (5%) (16
). Though the p53
gene is not commonly altered in melanoma, disruption of the tumor suppressor ARF, which regulates p53 protein stability, is a common occurrence in metastatic melanoma through genetic deletion of the CDKN2A locus (17
). Therefore alterations of p53-dependent pathways have the potential to influence melanoma progression.
In contrast, though there is some evidence linking a polymorphism in the excision repair gene XPD
and susceptibility to cutaneous melanoma (20
), there is little available data indicating that altered expression of excision repair genes contributes to melanoma, and indeed a recent microarray analysis of mRNA expression profiles in metastatic melanomas did not find changes in excision repair genes (21
). Though analyses of mRNA transcript and protein expression profiles have the potential to be informative, it may be more relevant to test for functional excision repair capacity in order to make proper correlations of DNA repair and carcinogenesis. Along these lines, though early work initially indicated that melanoma cells did not show enhanced repair rates (22
), other work suggested that sub-clones of a metastatic melanoma line did indeed show elevated repair rates in comparison to non-melanoma cells, and this repair correlated with increased survival after UV (23
). An additional study similarly concluded that DNA repair capacity in mouse melanoma cell lines correlated with metastatic potential (24
). However, more recent in situ
work indicated that cutaneous melanoma patients show normal repair kinetics (25
). It is therefore unclear whether excision repair capacity is altered in melanoma cells relative to normal melanocytes, or whether genetic background (B-Raf/N-Ras/p53 status) or metastatic potential are directly correlated with excision repair capacity.
In this study, we used normal human melanocytes (NHMs) and a variety of melanoma cell lines to characterize excision repair capacity as a function of genetic and metastatic states. Our results show that in nearly all melanoma cell lines tested, excision repair occurred as efficiently as in NHMs, irrespective of mutations in the N-Ras and B-Raf oncogenes. In addition, we found no change in excision repair capacity in a highly metastatic melanoma cell line (A375SM) compared to its parental melanoma cell line (A375P), which has a low metastatic potential. Lastly, we observed that melanoma cell lines containing functional p53 repair UV photoproducts more efficiently than lines with inactive p53, but that this difference appears to be not due to enhanced levels of the UV photoproduct binding protein DDB2.