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Nucleotide Excision Repair Gene Expression After Cisplatin Treatment in Melanoma

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Nucleotide Excision Repair Gene Expression After Cisplatin Treatment in Melanoma Published OnlineFirst August 31, 2010; DOI: 10.1158/0008-5472.CAN-10-0161 Molecular and Cellular Pathobiology Cancer Research Nucleotide Excision Repair Gene Expression after Cisplatin Treatment in Melanoma Nikola A. Bowden1,2,3, Katie A. Ashton1,2,3, Kelly A. Avery-Kiejda4, Xu Dong Zhang4, Peter Hersey4, and Rodney J. Scott1,2,3,5 Abstract Two of the hallmark features of melanoma are its development as a result of chronic UV radiation exposure and the limited efficacy of cisplatin in the disease treatment. Both of these DNA-damaging agents result in large helix-distorting DNA damage that is recognized and repaired by nucleotide excision repair (NER). The aim of this study was to examine the expression of NER gene transcripts, p53, and p21 in melanoma cell lines treated with cisplatin compared with melanocytes. Basal expression of all genes was greater in the melanoma cell lines compared with melanocytes. Global genome repair (GGR) transcripts showed significantly decreased relative expression (RE) in melanoma cell lines 24 hours after cisplatin treatment. The basal RE of p53 was significantly higher in the melanoma cell lines compared with the melanocytes. However, induction of p53 was only significant in the melanocytes at 6 and 24 hours after cisplatin treatment. Inhibition of p53 expression significantly decreased the expression of all the GGR transcripts in melanocytes at 6 and 24 hours after cis- platin treatment. Although the RE levels were lower with p53 inhibition, the induction of the GGR genes was very similar to that in the control melanocytes and increased significantly across the time points. The findings from this study revealed reduced GGR transcript levels in melanoma cells 24 hours after cisplatin treatment. Our findings suggest a possible mechanistic explanation for the limited efficacy of cisplatin treatment and the possible role of UV light in melanoma. Cancer Res; 70(20); 7918–26. ©2010 AACR. Introduction somal recessive disease xeroderma pigmentosum (XP). Patients with XP have a drastically diminished NER capacity; Two of the hallmark features of melanoma are its develop- therefore, DNA damage accumulates rapidly after UV light ment as a result of chronic repeated exposure to UV radia- exposure, resulting in up to a 1,000-fold increase in develop- tion (1, 2) and the limited efficacy of the DNA-damaging ment of skin cancers on sunlight-exposed areas of skin (7). agent cisplatin in the disease treatment (3). Both of these Despite this, the role of NER in the development of melanoma agents result in large helix-distorting DNA damage that is in the general population as a result of UV radiation has not recognized and repaired by the DNA repair pathway nucleo- been thoroughly investigated. To date, only weak evidence tide excision repair (NER). NER is a versatile DNA repair sys- has been reported on the genetic association of NER-related tem that eradicates UV light–induced lesions, such as genes with melanoma (8–11). cyclobutane pyrimidine dimers and pyrimidine (4–6) pyrim- Another example of DNA damage recognized and repaired idine photoproducts, as well as lesions induced by many by NER is intra- and inter-strand cross-links induced by chemical compounds including cisplatin (4–6). The impor- DNA-damaging or cross-linking agents. The most commonly tance of the NER pathway is very evident in the rare auto- used example of a DNA-damaging agent is cisplatin. Despite the high level of efficacy of cisplatin treatment for many types of malignancies, several factors such as cell resistance Authors' Affiliations: 1Centre for Information Based Medicine, University and adverse drug reactions have undermined its therapeutic of Newcastle; 2Discipline of Medical Genetics, School of Biomedical Sciences, Faculty of Health, University of Newcastle; 3Hunter Medical potential. In melanoma, the efficacy of cisplatin is limited Research Institute; 4Oncology and Immunology Unit, Calvary Mater (12), which is in contrast to the case of most germ-cell tu- Newcastle Hospital; and 5Division of Genetics, Hunter Area Pathology mors having cure rates of more than 90% (13). This contrast Service, John Hunter Hospital, Newcastle, New South Wales, Australia in efficacy is yet to be explained, and in recent years, many Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). studies have focused on elucidating the anticancer mecha- nism of cisplatin. NER is now known to be a vital component N.A. Bowden and K.A. Ashton contributed equally to this work. of this process; however, studies investigating the role of NER Corresponding Author: Nikola A. Bowden, Hunter Medical Research Institute, John Hunter Hospital, Lookout Road, New Lambton Heights, in cisplatin efficacy and resistance are yet to be conducted in 2305, NSW, Australia. Phone: 61-2-4985-5663; Fax: 61-2-4985-5895; melanoma. E-mail: [email protected]. NER consists of approximately 30 proteins that remove doi: 10.1158/0008-5472.CAN-10-0161 helix-distorting lesions through four steps: (a) recognition ©2010 American Association for Cancer Research. of the DNA lesion, (b) opening of a bubble around the lesion, 7918 Cancer Res; 70(20) October 15, 2010 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst August 31, 2010; DOI: 10.1158/0008-5472.CAN-10-0161 Nucleotide Excision Repair after Cisplatin in Melanoma (c) incision of the DNA upstream and downstream of the le- 10 μg/mL cisplatin (Pharmacia Upjohn) as previously de- sion by endonucleases, and (d) DNA resynthesis and ligation scribed (28) and were harvested before and at 6 and 24 hours (14). There are two damage recognition arms of the NER after treatment for gene expression analysis. pathway: global genome repair (GGR) and transcription- coupled repair (TCR). GGR encompasses the noncoding parts Inhibition of p53 by short hairpin RNA of the genome, silent genes, and the nontranscribed strand of Constructs. Short hairpin RNA (shRNA) sequences to p53 active genes (15). TCR ensures that the transcribed strand or a control were expressed in the pSIH1-H1-copGFP (Cope- of active genes is repaired with higher priority than the rest pod green fluorescent protein) shRNA expression vector of the genome by using RNA polymerase II as a lesion sensor (Systems Biosciences). The p53-directed shRNA sequence (15). Once the damage is recognized through one of these corresponds to nucleotides 1026–1044 (GenBank accession processes, the remainder of the repair process follows a con- no. NM_000546; ref. 32). The control shRNA sequence 5′- vergent pathway (7). TTAGAGGCGAGCAAGACTA-3′ showed no homology to The tumor suppressor protein p53 has recently been impli- any known human transcript. cated in the transactivation of the GGR components of the Stable transduction of melanoma cell lines. Lentiviruses NER pathway following DNA-damaging events (16, 17). p53 were produced in HEK293T cells using the pSIH1-H1-copGFP plays an important role in many cellular responses to DNA shRNA expression vector (Systems Biosciences) encased in damage, including cell cycle arrest and apoptosis. Recent viral capsid encoded by three packaging plasmids as de- evidence suggests that p53 is essential for both basal and in- scribed previously (33). Viruses were concentrated as de- duced GGR response to certain types of UV- and chemical- scribed previously (34). Viral titres were determined using induced DNA damage (18–21). The role of p53 in regulating 1×105 U2OS cells per well in six-well plates, which were GGR seems to be mediated in part by its ability to transacti- transduced with serial dilutions of the concentrated viral vate the gene transcripts encoding the GGR proteins DDB2 stocks in the presence of 8 μg/mL polybrene (Sigma). Cells and XPC. were harvested 48 hours after transduction and analyzed In contrast with other tumor types, melanoma has a rela- by flow cytometry for copGFP expression, and viral titre tively infrequent p53 mutation rate of only around 9% (re- was calculated. viewed in refs. 22, 23). In addition, p53 protein and mRNA To generate a p53-silenced stable melanocyte cell line, expression have been reported in melanoma, with reports melanocytes were transduced at a multiplicity of infection showing increases in p53 with tumor progression (24–27). of 10 with either a virus encoding p53 shRNA or a control The presence of wild-type p53 expression in melanoma indi- shRNA that has no homology to any human gene. Cells were cates that other events that bypass p53 must be occurring for transduced twice at 3-day interval. The efficiency of trans- tumor development and/or progression to occur. One candi- duction was monitored with coexpression of copGFP and date event could be the lack of transactivation of GGR in re- was consistently >95%. All cell lines tested negative for the sponse to DNA damage. Given the limited knowledge presence of replicative competent virus using the Retrotek available detailing the role of NER in melanoma, the aim of HIV-1 p24 antigen ELISA kit (ZeptoMetrix Corporation). this study was to determine the level of NER activity and its relationship with p53 in response to cisplatin-induced DNA Sample preparation and real-time PCR damage in melanocytes and melanoma cell lines. Total RNA was extracted from all of the cell lines after cisplatin treatment using the SV Total RNA Isolation Sys- Materials and Methods tem Kit (Promega). The total RNA was quantified using the Quant-iT RiboGreen RNA Assay Kit (Invitrogen) and a Cell lines and cisplatin treatment fluorometer (BMG Labtech). To ensure consistency across all One melanocyte, three primary melanoma (MM200, IgR3, the samples being tested, a standardized amount (500 ng) of and Me4405), and two metastatic melanoma (Mel-RM and total RNA was reverse transcribed using the High Capacity Sk-mel-28) cell lines were used for this study.
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