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 cisplatin 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 mechanism 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,

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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. The derivation cDNA Reverse Transcription Kit (Applied Biosystems), and of MM200, IgR3, Me4405, Mel-RM, and Sk-mel-28 melanoma a 1:20 dilution of the resultant cDNA was used in triplicate cell lines has been described previously (28–31). Melanocytes for each sample. Relative gene expression was measured in were purchased from Cascade Biologics at the commence- triplicate and normalized to β-actin (ΔCt) using TaqMan ment of this study. DNA for cell line authentication was ex- gene expression assays (Applied Biosystems) and the 7500 tracted from all the cell lines while cultured for this study. real-time PCR system (Applied Biosystems) for the following Individual cell line authentication was confirmed using the gene transcripts: DDB2 (XPE; Hs00172068_m1), DDB1 AmpFlSTR Identifiler PCR Amplification Kit from Applied (Hs_00172410_m1), XPC (Hs01104206_m1), ERCC8 (CSA; Biosystems and GeneMarker V1.91 software. A panel of 16 Hs01122123_m1), ERCC6 (CSB; Hs00972920_m1), XPA markers were tested, and each cell line had a distinct individ- (Hs00166045_m1), RPA1 (Hs00161419_m1), RPA2 ual set of markers present. (Hs00358315_m1), ERCC3 (XPB; Hs01554450_m1), ERCC2 All of the melanoma cell lines were cultured in DMEM (5% (XPD; Hs00361161), ERCC5 (XPG; Hs00164482), ERCC4 FCS) and the melanocytes were cultured in Medium 154 (XPF; Hs00193342), ERCC1 (Hs01012161_m1), p53 (Cascade Biologics). All cell lines were maintained in expo- (Hs00153340_m1), and p21 (Hs00355782_m1). To ensure nential growth at 37°C and 5% CO2. Cells were treated with that β-actin itself did not change between cell lines and/

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or treatments, the ratio of β-actin to a second reference gene, GAPDH, was measured. The average β-actin/GAPDH ratio was 1.02 ± 0.04 across all the individual cell lines and treatment time points.

Statistical analysis −Δ Relative gene expression was calculated using 2 Ct, and unpaired two-tailed t tests (P < 0.05) were used to identify significantly altered expression in melanoma compared with melanocytes as described previously (35).

Western blot analysis Protein extraction, separation by SDS-PAGE, and Western blot analysis of cell lines were done as described previously (28). The mouse monoclonal antibody Bp53-12, used for the detection of p53, was purchased from Upstate. The mouse monoclonal antibody for the housekeeping gene GAPDH was purchased from Ambion.

Results

Global genome repair The relative expression (RE) of the GGR gene transcripts XPC, DDB1,andDDB2 (XPE) was measured at 0, 6, and 24 hours after cisplatin treatment in melanoma and melanocyte cell lines. The RE of all three GGR transcripts was higher at the basal level and at 6 hours after treatment in the melanoma cell lines, although not significantly different when compared with melanocytes (Fig. 1). The expression of these transcripts significantly increased in a time-dependent manner following cisplatin treatment in melanocytes. No cisplatin-dependent increase of these transcripts was observed in melanoma cell lines (Fig. 1). The induction of XPC, DDB1,andDDB2 (XPE)relativeto the basal expression level was measured at 6 and 24 hours after treatment. At 24 hours after cisplatin treatment, there was a 38.77-fold (P = 0.0003), 12.27-fold (P = 0.0005), and 81.46-fold (P = 0.0002) increase in RE in the melanocytes, respectively (Fig. 2). In comparison, a 1.33-fold decrease in expression was observed for DDB1 in the melanoma cell lines and only a small fold increase in RE was observed for XPC (3.68) and DDB2 (1.84), all of which were not significant (Fig. 2). Clearly, a significantly lower RE level of the GGR genes was observed in the melanoma cell lines 24 hours after cisplatin treatment (Fig. 1). In addition, a significant increase in induction of these transcripts was observed in the melanocytes but Figure 1. Relative expression of the GGR genes XPC, DDB1, and DDB2 not in the melanoma cell lines (Fig. 2). (XPE) at 0, 6, and 24 h after cisplatin treatment in melanoma and melanocyte cell lines. The RE of all three GGR transcripts was higher at Transcription-coupled repair the basal level and at 6 h after treatment in the melanoma cell lines, ERCC6 CSB although not significant. Twenty-four hours after cisplatin treatment, the The RE of the TCR gene transcripts ( )and RE of all three GGR transcripts was significantly lower in the melanoma ERCC8 (CSA) was very low in all of the melanoma and mela- cell lines compared with the melanocyte cell line. Points, mean of triplicates nocyte cell lines at all time points. However, the RE for of two independent experiments; bars, SE. **, P < 0.005. ERCC6 (CSB) was significantly lower (P = 0.005) in the melanoma cell lines (1.34) at the 24-hour time point compared point (P = 0.04). No significant differences in RE between with the melanocyte cell line (6.52). For ERCC8 (CSA), RE the melanoma and melanocyte cell lines were observed for was significantly higher in the melanoma cell lines (0.99) ERCC6 (CSB)orERCC8 (CSA) at all other time points. The than in the melanocyte cell line (0.14) at the 6-hour time relatively low level of expression of all the TCR transcripts

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Nucleotide Excision Repair after Cisplatin in Melanoma

at all time points suggests limited involvement of TCR in response to cisplatin-induced DNA damage.

Nucleotide excision repair The NER-related genes XPA, RPA1, RPA2, ERCC1, ERCC2 (XPD), ERCC3 (XPB), ERCC4 (XPF), and ERCC5 (XPG) showed an overall trend of higher basal expression in melanoma cell lines compared with the melanocyte cell line (Table 1). Spe- cifically, the basal RE of RPA1, RPA2, ERCC2 (XPD), ERCC3 (XPB), and ERCC4 (XPF) was significantly greater in the melanoma cell lines. In addition, RPA1 and RPA2 RE levels were significantly greater in the melanoma cell lines compared with the melanocyte cell line 6 hours after cisplatin treatment, but the expression levels were similar at 24 hours. In contrast, ERCC3 (XPB), ERCC4 (XPF), and ERCC5 (XPG) RE levels were significantly lower in the melanoma cell lines than in the melanocyte cell line 24 hours after cisplatin treatment. In the melanoma cell lines, ERCC2 (XPD) and XPA were slightly increased in RE from 0 to 24 hours after cisplatin treatment; however, this was not significantly different from the RE observed in the melanocytes at the 24-hour time point (Table 1). p53 p53 is currently thought to transactivate expression of the GGR genes DDB2 and XPC in response to DNA damage. The RE of p53 was measured in all the cell lines after 0, 6, and 24 hours of cisplatin treatment. The RE of p53 was significantly higher in the melanoma cell lines compared with the melanocytes at 0 and 6 hours after cisplatin treatment but not at 24 hours (Fig. 3). Although the transcript levels of p53 were higher in the melanoma cell lines at 0 and 6 hours, the induction of p53 was of borderline significance at 24 hours (3.91-fold increase, P = 0.05). Conversely, induction of p53 was significant in the melanocytes at 6 hours (1.6-fold increase, P = 0.0001) and 24 hours (19.57-fold increase, P = 0.007) after cisplatin treatment (Fig. 3). p21 Increased expression of p21 is indicative of p53 activation in response to DNA damage. The RE of p21 was measured in all cell lines after 0, 6, and 24 hours of cisplatin treatment. The RE of p21 seemed to be higher in melanoma cell lines at the 0- and 6-hour time points, but it was not statistically significant. Conversely, the RE of p21 was significantly lower in the melanoma cell lines compared with the melanocyte cell line after 24 hours of cisplatin treatment (Fig. 3). Although transcript levels of p21 were higher in the melanoma cell lines at 0 and 6 hours, the induction of p21 was not significantly elevated by 24 hours compared with the melanocyte cell line. Induction of p21 was observed in the melanocyte cell line at 6 hours (9.85-fold increase, P =0.00004) P and 24 hours (142.44-fold increase, = 0.002) after cisplatin Figure 2. Effect of cisplatin treatment on the induction of GGR genes, treatment (Fig. 3). XPC, DDB1, and DDB2 (XPE), in melanoma and melanocyte cell lines at 6 and 24 h after cisplatin treatment. After 6 and 24 h, induction of the GGR transcript expression with shRNA inhibition GGR genes was significant in the melanocyte cell line, but not significant in the melanoma cell lines. Results are expressed as the normalized of p53 fold induction of mRNA RE relative to the RE at 0 h (which has been To determine if the GGR transcript level changes in the set to 1). Points, mean of triplicates of two independent experiments; melanoma cell lines were related to the absence of p53, bars, SE. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

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Table 1. Relative expression of NER gene transcripts in melanoma and melanocyte cell lines at 0, 6, and 24 h after cisplatin treatment

Time (h) Cell type XPA RPA1 RPA2 ERCC3 (XPB) ERCC2 (XPD) ERCC5 (XPG) ERCC4 (XPF) ERCC1

0 Melanocytes 1.1 3.32 1.02 2.61 1.04 1.5 1.04 6.72 Melanoma 1.6 24.50* 10.91* 7.54* 4.34* 2.89 1.68* 25 6 Melanocytes 0.64 4.43 2.42 3.64 1.41 3.54 2.34 16.98 Melanoma 2.26 26.99* 14.27* 11.49 5.52 4.67 2.3 58.56 24 Melanocytes 2.78 19.08 15.76 24.75 6.71 30.24 15.93 29.32 Melanoma 5.57 20.59 13.55 11.03† 8.21 3.42‡ 2.07† 37.64

*P < 0.05. †P < 0.001. ‡P < 0.0001.

knockdown experiments were undertaken using the melano- shRNA inhibition of p53 and its effect on p21 transcript cyte cell line. expression The transcript level of all the GGR genes, XPC, DDB1, and shRNA was used to inhibit the transcript expression of p53 DDB2, was significantly lower in the p53 shRNA melanocyte in the melanocyte cell line. To confirm inhibition of p53 tran- cell line compared with the control melanocyte cell line at 6 scription, p53 and p21 transcript expression was measured in and 24 hours after cisplatin treatment. The basal transcript the control shRNA melanocyte cell line and the p53 shRNA levels, however, were not significantly different (Fig. 4). Al- melanocyte cell line. The RE of p53 was 8.12-fold lower (P = though the RE levels were lower in the p53 shRNA melano- 0.01) in the p53 shRNA melanocyte cell line than in the con- cyte cell line, the induction of the GGR genes was very similar trol melanocyte cell line. Similarly, the RE of p21 was 2.33- to that in the control melanocyte cell line and increased sig- fold lower (P = 0.07) in the p53 shRNA melanocyte cell line nificantly across the time points (Fig. 5). than in the control melanocyte cell line. In addition, Western

Figure 3. Relative expression and induction of p53 and p21 at 0, 6, and 24 h after cisplatin treatment in melanoma and melanocyte cell lines. A, the RE of p53 transcript was significantly higher at the basal level and at 6 h after treatment in the melanoma cell lines. Twenty-four hours after cisplatin treatment, the RE of p21 transcript was significantly lower in the melanoma cell lines compared with the melanocyte cell line. B, induction of p53 was highly significant (P < 0.005) in the melanocyte cell line after 6 and 24 h, but only significant in the melanoma cell lines at 24 h. Similarly, after 6 and 24 h, induction of p21 was highly significant (P < 0.0005) in the melanocyte cell line, but not significant in the melanoma cell lines. Results are expressed as the normalized fold induction of mRNA RE relative to the RE at 0 h (which has been set to 1). Points, mean of triplicates of two independent experiments; bars, SE. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005.

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blots were used to confirm the inhibition of p53 protein expression in the p53 shRNA melanocyte cell line (Supplemen- tary Data).

Discussion

Despite overwhelming evidence that one of the primary causal agents of melanoma is chronic repeated exposure to UV radiation, its exact relationship with the disease remains to be fully elucidated. Cisplatin is a common DNA-damaging agent that is used in the treatment of many types of cancer; the efficacy of cisplatin in the treatment of melanoma, however, is very limited (12). Cisplatin binds to DNA, forming helix-distorting intra- and inter-strand cross-links (36, 37), which must be removed before either transcription or repli- cation. The removal and repair of large helix-distorting DNA damage induced by both UV light and DNA-damaging agents such as cisplatin is orchestrated by NER. The aim of the present study was to examine the changes in gene expression of the key constituents of the NER pathway in melanoma cell lines treated with cisplatin compared with a control melanocyte cell line and to examine the relationship to the cell cycle checkpoint control gene p53. GGR operates throughout the entire genome and is a cru- cial step in the initial recognition of DNA damage (38). The intra- and inter-strand cross-links caused by cisplatin are recognized by the GGR component XPC; then, the DDB1/ DDB2 complex is recruited to bind specifically to the large helix-distorting DNA adducts (38). Thereafter, the repair process proceeds through the rest of the NER pathway. Previous studies have identified a strong correlation between reduced XPC mRNA and protein levels and increased resistance of cancer cells to cisplatin treatment (39–41). In this study, the genes involved in the GGR pathway, XPC, DDB1,and DDB2 (XPE),wereshowntohavenoincreaseinREinthe melanoma cell lines after cisplatin treatment. In contrast, a significant increase in RE 24 hours after cisplatin treatment was seen in the melanocyte cell line. These results show that following cisplatin treatment, the GGR genes are poorly induced in melanoma cell lines compared with melanocytes. The most important mechanism for the anticancer action of cisplatin is recognition of cisplatin-induced DNA damage, which triggers cell death. This process is known as DNA damage–mediated apoptotic cell death (40). The cisplatin treatment–mediated p53 response and activation of caspase- 3, both key mechanisms involved in triggering apoptosis, are significantly reduced in XPC-defective cell lines, which suggests that XPC plays a critical role in initiating apoptosis mediated by cisplatin-induced DNA damage (40, 41). In addition to the key role that XPC plays in DNA damage– mediated apoptosis after cisplatin treatment, DDB2 also has Figure 4. Relative expression of GGR gene transcripts in melanocytes a role in this process. DDB2-deficient cells exhibit enhanced and p53 shRNA melanocyte cell lines after 0, 6, and 24 h of cisplatin resistance to cell growth inhibition and apoptosis induced by treatment. The RE of all three GGR transcripts was significantly lower cisplatin (42, 43), and DDB2 expression in cisplatin-resistant at 6 and 24 h after cisplatin treatment in the p53 shRNA melanocyte cell line compared with the melanocyte cell line. Columns, mean of ovarian cancer cell lines is lower than that in their cisplatin- triplicates of two independent experiments; bars, SE. *, P < 0.05; sensitive parental cells (42). Overexpression of DDB2 sensi- **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005. tizes cells to cisplatin-induced cytotoxicity and apoptosis

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through activation of the caspase pathway and downregula- tion of antiapoptotic Bcl-2 protein (42). What we have observed in the melanoma cell lines is a failure to upregulate key damage recognition genes, including XPC and DDB2, in response to cisplatin-induced DNA damage. The failure to elicit the appropriate response of the GGR pathway after cisplatin treatment suggests that the normal relationship between DNA damage recognition and subse- quent apoptosis is no longer functionally accomplishable in these melanoma cell lines. The low expression of XPC and DDB2 may therefore play a key role in the resistance of melanoma to cisplatin. Basal expression levels of the GGR genes XPC, DDB1, and DDB2 (XPE) seemed to be greater in the melanoma cell lines compared with the melanocytes. In addition, the NER genes RPA1, RPA2, ERCC3 (XPB), ERCC2 (XPD), and ERCC4 (XPF) showed significantly higher basal-level expression in the melanoma cell lines compared with the melanocytes. Increased basal expression of several NER genes has been previously related to cisplatin resistance (44). Specifically, ovarian tumors resistant to cisplatin treatment have shown high levels of expression of XPA, ERCC1, XPF, and ERCC3 (XPB). Con- versely, testicular cancers display low expression of XPA, ERCC1, and XPF and are highly responsive to cisplatin (re- viewed in ref. 44). Although only a subset of the NER genes previously reported to have higher basal expression in relation to cisplatin resistance were identified in this study, overall, increased basal-level expression of NER genes seems to be associated with cisplatin resistance. Taken together with the results of this study, this could provide some explanation about why cisplatin is ineffective in melanoma treatment. p53 is a critical regulator of NER both at the basal level and in the induction of GGR following UV light–induced and chemically induced DNA damage (18–21, 45). The role of p53 in regulating GGR seems to be mediated, in part, by its ability to transactivate gene transcripts encoding the GGR proteins DDB2 and XPC. In this study, a key downstream p53 target gene, p21,showedasimilartrendtop53, indicating that altered expression of p53 was indeed affecting downstream signaling. We confirmed that induced p53 and GGR gene transcript levels occur in concert in melanocytes 24 hours after cisplatin treatment. In melanoma cell lines studied herein, the relationship is not as clear. The basal level of p53 is significantly higher in melanoma cell lines relative to the basal levels of the majority of the GGR genes. Further- more, the RE of p53 after 24 hours of cisplatin treatment is remarkably similar in melanoma and melanocyte cell lines, but the RE of the GGR genes in the melanoma cell lines is very low. The most likely explanation for this occurrence is the difference in the induction of p53 after cisplatin treatment in the melanoma cell lines compared with the melanocytes. There is a highly significant increase in p53 induction Figure 5. Effect of cisplatin treatment on the induction of GGR gene at the 6- and 24-hour time points for the melanocytes, but transcripts in melanocytes with p53 inhibition. The induction of XPC, the melanoma cell lines fail to respond as rapidly and p53 DDB1, and DDB2 (XPE), represented as fold change, increased is only differentially expressed after 24 hours (P = 0.05). This significantly (P < 0.0005) from 6 to 24 h after cisplatin treatment in the control and p53 shRNA melanocytes. Points, mean of triplicates of suggests that it is the induction of p53 that is required for two independent experiments; bars, SE. *, P < 0.05; ***, P < 0.0005; GGR activation, rather than just a high constitutive level of ****, P < 0.00005. p53 expression.

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Nucleotide Excision Repair after Cisplatin in Melanoma

The current understanding of the relationship between was confirmed by studies aimed at inhibiting p53 expression p53 and GGR relies on the hypothesis that an increase in in the melanocytes, which showed a reduction in the expres- p53 results in transactivation of GGR transcript expression sion, but not induction, of the GGR transcripts. (16, 17). To investigate this notion further, shRNA was used Low mRNA (40, 41) and protein (39) expression of GGR to inhibit p53 expression in the melanocyte cell line used for genes in relation to cisplatin resistance have been previously this study. Once reduced p53 expression was confirmed by reported. Although the present study has only reported mRNA Western blot and direct measurement of p53 transcript levels expression of NER genes, the results are in accordance with the and the consequent reduction in p21 mRNA, the expression previous studies and suggest that GGR deficiency may play a of the three GGR transcripts was investigated. The RE levels role in cisplatin resistance in melanoma. Further studies con- of the three GGR genes, XPC, DDB1,andDDB2 (XPE), was firming the differences in NER expression after cisplatin treat- significantly lower in the p53 shRNA melanocytes at all time ment at the protein level would further support this finding. points after cisplatin treatment. However, the induction of These findings provide evidence of the possible biological these transcripts was not altered by the inhibition of p53, mechanisms involved in the reduced efficacy of cisplatin in as all three GGR genes showed significant fold-change in- melanoma. Notwithstanding, the DNA damage caused by cis- creases in expression at 24 hours after cisplatin treatment, platin is similar to UV radiation, as both result in large helix- thereby showing their similarity to the control melanocytes. distorting DNA damage that is recognized by the NER pathway. This result confirms that a high level of p53 expression is not Therefore, the findings of this study point toward a biological the only requirement for GGR activation. In addition, the in- mechanism involved in melanoma resistance to cisplatin and duction of GGR in the melanocytes despite the inhibition of in melanoma development as a result of UV exposure. p53 suggests that p53 may not be responsible for the lack of GGR induction in the melanoma cell lines. Disclosure of Potential Conflicts of Interest In 2006, a study by Yang and colleagues identified a number of p53 target genes that were significantly downregulated No potential conflicts of interest were disclosed. in melanomas compared with melanocytes (46). One of their major findings was the association of reduced DDB2 activity Acknowledgments and melanoma development (46). In addition, the expression of other p53 target genes was also altered, including the cell We thank Lyndee L. Scurr and Helen Rizos from Westmead Institute for cycle regulator CDKN1A (p21CIP), which was confirmed by Cancer Research, University of Sydney at Westmead Millennium Institute, NSW, Australia, for the p53 shRNA melanocyte cell lines and Amanda Croft Kaufmann and colleagues (47). Taken together, the findings for the Western blot analysis. of these studies and those of the current study reveal that reduced DDB2 activity is potentially implicated in melanoma development and/or progression and that a number of other Grant Support p53 targets may have altered expression in melanoma. The Hunter Medical Research Institute and The University of Newcastle In summary, this study has revealed that melanoma cell Centre for Information Based Medicine. N.A. Bowden is supported by the Na- lines treated with cisplatin have lower expression of the tional Health and Medical Research Council, NBN Telethon Children's Cancer XPC, DDB1 DDB2 XPE Research Group, and The Hunter Medical Research Institute. K.A. Ashton is GGR genes , and ( ) when compared with supported by Mr. and Mrs. Bogner and the Hunter Medical Research Institute. melanocytes after 24 hours. Interestingly, the basal expression The costs of publication of this article were defrayed in part by the payment levels of p53 and several NER repair genes in melanoma cell of page charges. This article must therefore be hereby marked advertisement in lines were greater than those observed in the melanocyte cell accordance with 18 U.S.C. Section 1734 solely to indicate this fact. line, yet on stimulation by the DNA-damaging agent cisplatin, Received 01/15/2010; revised 07/12/2010; accepted 07/27/2010; published only the melanocyte cell line was capable of response. This OnlineFirst 08/31/2010.

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7926 Cancer Res; 70(20) October 15, 2010 Cancer Research

Nucleotide Excision Repair Gene Expression after Cisplatin Treatment in Melanoma

Nikola A. Bowden, Katie A. Ashton, Kelly A. Avery-Kiejda, et al.

Cancer Res 2010;70:7918-7926. Published OnlineFirst August 31, 2010.

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