Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Cancer Research (Impact Factor: 9.33). 06/2010; 70(12):4922-30. DOI: 10.1158/0008-5472.CAN-10-0095
Source: PubMed


Sunlight UV exposure produces DNA photoproducts in skin that are repaired solely by nucleotide excision repair in humans. A significant fraction of melanomas are thought to result from UV-induced DNA damage that escapes repair; however, little evidence is available about the functional capacity of normal human melanocytes, malignant melanoma cells, and metastatic melanoma cells to repair UV-induced photoproducts in DNA. In this study, we measured nucleotide excision repair in both normal melanocytes and a panel of melanoma cell lines. Our results show that in 11 of 12 melanoma cell lines tested, UV photoproduct repair occurred as efficiently as in primary melanocytes. Importantly, repair capacity was not affected by mutation in the N-RAS or B-RAF oncogenes, nor was a difference observed between a highly metastatic melanoma cell line (A375SM) or its parental line (A375P). Lastly, we found that although p53 status contributed to photoproduct removal efficiency, its role did not seem to be mediated by enhanced expression or activity of DNA binding protein DDB2. We concluded that melanoma cells retain capacity for nucleotide excision repair, the loss of which probably does not commonly contribute to melanoma progression.

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Available from: Michael G Kemp, Dec 16, 2013
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    • "Although epidemiological associations between XP-C, -D, and -F polymorphisms and increased melanoma risk have been reported [7], [8], [9], [10], no firm evidence to our knowledge supports a major role for NER pathway gene mutations in either familial or sporadic melanoma. Concerning regulatory mechanisms upstream of NER, it has been clearly shown that a functional p53 tumour suppressor pathway, triggered following genotoxic insult to regulate apoptosis and growth arrest, is also required for efficient CPD removal in various cell types including melanoma [11], [12]; nonetheless mutational inactivation of p53 appears rare in melanoma [13]. On the other hand the PTEN tumour suppressor, frequently downregulated by genetic or epigenetic means in melanoma [14], has recently been implicated in protection against UVB-induced nonmelanoma skin cancer by positively regulating NER [15]. "
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    ABSTRACT: It is well established that efficient removal of highly-promutagenic UV-induced dipyrimidine photoproducts via nucleotide excision repair (NER) is required for protection against sunlight-associated malignant melanoma. Nonetheless, the extent to which reduced NER capacity might contribute to individual melanoma susceptibility in the general population remains unclear. Here we show that among a panel of 14 human melanoma strains, 11 exhibit significant inhibition of DNA photoproduct removal during S phase relative to G0/G1 or G2/M. Evidence is presented that this cell cycle-specific NER defect correlates with enhanced apoptosis and reduced clonogenic survival following UV irradiation. In addition, melanoma strains deficient in S phase-specific DNA photoproduct removal manifest significantly lower levels of phosphorylated histone H2AX at 1 h post-UV, suggesting diminished activation of ataxia telangiectasia and Rad 3-related (ATR) kinase, i.e., a primary orchestrator of the cellular response to UV-induced DNA replication stress. Consistently, in the case of DNA photoproduct excision-proficient melanoma cells, siRNA-mediated depletion of ATR (but not of its immediate downstream effector kinase Chk1) engenders deficient NER specifically during S. On the other hand simultaneous siRNA-mediated depletion of ataxia telangiectasia mutated kinase (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exerts no significant effect on either phosphorylation of H2AX at 1 h post-UV or the efficiency of DNA photoproduct removal. Our data suggest that defective NER exclusively during S phase, possibly associated with decreased ATR signaling, may constitute an heretofore unrecognized determinant in melanoma pathogenesis.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "As shown in Figure 4B, both the anti-CPD and anti-(6–4)PP antibodies were capable of immunoprecipitating the excised oligomers following UV irradiation of HeLa cells. Consistent with the ability of cells to recognize and repair (6–4)PPs more rapidly than CPDs (10,18,19), we observed significantly more (6–4)PP-containing oligonucleotides than CPD-containing oligonucleotides at early time points following irradiation (Figure 4B and C). Furthermore, the lengths of the (6-4)PP-containing oligonucleotides showed a strong time-dependent change, such that the primary full-length excision products were exclusively observed at the early 15 min time point, and only degraded oligomers were observed after 2 h (Figure 4B, Supplementary Figure S2). "
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    ABSTRACT: The nucleotide excision repair pathway removes ultraviolet (UV) photoproducts from the human genome in the form of short oligonucleotides ∼30 nt in length. Because there are limitations to many of the currently available methods for investigating UV photoproduct repair in vivo, we developed a convenient non-radioisotopic method to directly detect DNA excision repair events in human cells. The approach involves extraction of oligonucleotides from UV-irradiated cells, DNA end-labeling with biotin and streptavidin-mediated chemiluminescent detection of the excised UV photoproduct-containing oligonucleotides that are released from the genome during excision repair. Our novel approach is robust, with essentially no signal in the absence of UV or a functional excision repair system. Furthermore, our non-radioisotopic methodology allows for the sensitive detection of excision products within minutes following UV irradiation and does not require additional enrichment steps such as immunoprecipitation. Finally, this technique allows for quantitative measurements of excision repair in human cells. We suggest that the new techniques presented here will be a useful and powerful approach for studying the mechanism of human nucleotide excision repair in vivo.
    Full-text · Article · Nov 2013 · Nucleic Acids Research
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    • "Further, genome-wide investigation found a higher prevalence of the UVR mutational signature in lowly transcribed genes, suggesting that reduced activity of the GGR component of the NER pathway is predominantly responsible for the accumulation of the UVR mutational signature in melanoma. Despite this growing body of evidence, a recent study reported no difference in overall NER capacity between melanocytes and melanoma cell lines after UV-irradiation [12]. This seemingly contradictory finding may be due to melanocytes having a lower than normal NER capacity [13], which may indeed be the reason they are susceptible to malignant transformation after UV-irradiation. "
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    ABSTRACT: Nucleotide excision repair (NER) orchestrates the repair of helix distorting DNA damage, induced by both ultraviolet radiation (UVR) and cisplatin. There is evidence that the global genome repair (GGR) arm of NER is dysfunctional in melanoma and it is known to have limited induction in melanoma cell lines after cisplatin treatment. The aims of this study were to examine mRNA transcript levels of regulators of GGR and to investigate the downstream effect on global transcript expression in melanoma cell lines after cisplatin treatment and in melanoma tumours. The GGR regulators, BRCA1 and PCNA, were induced in melanocytes after cisplatin, but not in melanoma cell lines. Transcripts associated with BRCA1, BRCA2, ATM and CHEK2 showed altered expression in melanoma cell lines after cisplatin treatment. In melanoma tumour tissue BRCA1 transcript expression correlated with poor survival and XPB expression correlated with solar elastosis levels. Taken together, these findings provide evidence of the mechanisms underlying NER deficiency in melanoma.
    Full-text · Article · Aug 2013 · PLoS ONE
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