Nucleotide excision repair gene expression after Cisplatin treatment in melanoma.

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.


Introduction
Two of the hallmark features of melanoma are the development of disease as a result of chronic repeated exposure to ultraviolet (UV) radiation (1,2); and the limited efficacy of the DNAdamaging agent, cisplatin in disease treatment (3). Both of these agents result in large helix distorting DNA damage that is recognised and repaired by the DNA repair pathway, nucleotide excision repair (NER). NER is a versatile DNA repair system that eradicates UV-light induced lesions such as cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidine photoproducts (6-4PP) as well as lesions induced by many chemical compounds including cisplatin (4)(5)(6). The importance of the NER pathway is very evident in the rare autosomal recessive disease xeroderma pigmentosum (XP). Patients with XP have a drastically diminished NER capacity, therefore DNA damage accumulates rapidly after UV-light exposure, resulting in up to a 1000 fold increase in development of skin cancers on sunlight exposed areas of skin (7). Despite this, the role of NER in the development of melanoma in the general population as a result of UV radiation has not been thoroughly investigated. To date, only weak evidence of a genetic association of NER related genes with melanoma have been reported (8)(9)(10)(11).
Another example of DNA damage recognised and repaired by NER is intra-and inter-strand crosslinks induced by DNA damaging or cross-linking agents. The most commonly 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 and adverse drug reactions have undermined its therapeutic potential. In melanoma, the efficacy of cisplatin is limited (12) which is in contrast to most germ-cell tumours with cure rates of over 90% (13). This contrast in efficacy is yet to be explained and in recent years many studies have focused on elucidating the anti-cancer mechanism of cisplatin. NER is now known to be a vital component of this process however,
Melanocytes were purchased from Cascade Biologics at the commencement of this study. DNA for cell line authentication was extracted from all the cell lines whilst cultured for this study. Individual cell line authentication was confirmed using the AmpFlSTR Identifiler PCR Amplification kit from Applied Biosystems and GeneMarker V1.91 software. A panel of 16 markers were tested and each cell line had a distinct individual set of markers present.
All of the melanoma cell lines were cultured in DMEM (5% FCS) and the melanocytes were cultured in Medium 154 (Cascade Biologics). All cell lines were maintained in exponential growth at 37°C and 5% CO 2 . Cells were treated with 10μg/mL cisplatin (Pharmacia Upjohn) as previously described (28) and were harvested before treatment and 6 and 24 hours after treatment for gene expression analysis.

Inhibition of p53 by shRNA:
Constructs Short hairpin RNA (shRNA) sequences to p53 or a control were expressed in the pSIH1-H1-copGFP (Copepod green fluorescent protein) shRNA expression vector (Systems Biosciences, Mountain View, CA, USA) The p53-directed shRNA sequence corresponds to nucleotides 1026-1044 (Genebank accession number NM_000546) (32). The control shRNA sequence 5'-TTAGAGGCGAGCAAGACTA-3' showed no homology to any known human transcript. Lentiviruses were produced in HEK293T cells using the pSIH1-H1-copGFP (Copepod green fluorescent protein) shRNA expression vector (Systems Biosciences, Mountain View, CA) encased in viral capsid encoded by three packaging plasmids as described previously (33). Viruses were concentrated as described previously (34). Viral titres were determined using 1 x 10 5 U2OS cells/well in 6-well plates, transduced with serial dilutions of the concentrated viral stocks in the presence of Polybrene (8µg/ml; Sigma, Castle Hill, NSW, Australia). Cells were harvested 48 hours post-transduction, analysed by flow cytometry for copGFP expression and viral titre calculated.

Stable transduction of melanoma cell lines
To generate a p53 silenced stable melanocyte cell line, melanocytes were transduced at an MOI of 10 with either a virus encoding p53 shRNA or a control shRNA that has no homology to any human gene. Cells were transduced twice with three days in between each transduction. The efficiency of transduction was monitored with co-expression of copGFP and was consistently over 95%. All cell lines tested negative for the presence of replicative competent virus using the Retrotek HIV-1 p24 antigen ELISA kit (ZeptoMetrix Corporation; Buffalo, NY, USA).

Sample Preparation and Real-time PCR:
Total RNA was extracted from all of the cell lines after cisplatin treatment using the SV Total RNA (Hs01012161_m1), p53 (Hs00153340_m1) and p21(Hs00355782_m1). To ensure β-Actin itself did not change between cell lines and/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 timepoints.

Statistical Analysis:
Relative gene expression was calculated using 2 -ΔCt and unpaired, 2-tailed t-tests (p<0.05) were used to identify significantly altered expression in melanoma compared to melanocytes as described previously (35).

Western Blot Analysis:
Protein extraction, separation by SDS-PAGE, and Western blot analysis of cell lines was 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. Research.
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Copyright © 2010 American Association for Cancer Research

Global Genome Repair
The Clearly, a significantly lower RE level of the GGR genes was observed in the melanoma cell lines, 24 hours after cisplatin treatment ( Figure 1). In addition, a significant increase in induction of these transcripts was observed in the melanocytes but was not observed in the melanoma cell lines ( Figure 2).

Transcription Coupled Repair
The RE of the Transcription Coupled Repair (TCR) gene transcripts, ERCC6 (CSB) and ERCC8 were slightly increased in RE from 0 to 24 hours after cisplatin treatment however this was not significantly different to the RE observed in the melanocytes at the 24 hour time point (Table 1). GGR operates throughout the entire genome and is a crucial step in the initial recognition of DNA damage (38). The intra and interstrand crosslinks caused by cisplatin are recognised 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)(40)(41). In this study, the genes involved in the GGR pathway, XPC, DDB1 and DDB2 (XPE), were shown to have no increase in RE in the 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 demonstrate that following cisplatin treatment, the GGR genes are poorly induced in melanoma cell lines compared to melanocytes.
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 XPC plays a critical role in initiating cisplatin DNA damage-mediated apoptosis (40,41).
In addition to the key role XPC plays in DNA damage-mediated apoptosis after cisplatin treatment, DDB2 also has a role in this process. DDB2-deficient cells exhibit enhanced resistance to cell growth inhibition and apoptosis induced by cisplatin (42,43) and DDB2 expression in cisplatinresistant ovarian cancer cell lines is lower than their cisplatin-sensitive parental cells (42).

Overexpression of DDB2 sensitizes cells to cisplatin-induced cytotoxicity and apoptosis via
activation of the caspase pathway and downregulation of antiapoptotic Bcl-2 protein (42). several NER genes has been previously related to cisplatin resistance (44 and XPF and are highly responsive to cisplatin (reviewed by (44). Even though 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 appears to be associated with cisplatin resistance. Taken together with the results of this study, this could provide some explanation as to 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 UVlight and chemically induced DNA damage (18)(19)(20)(21)45). The role of p53 in regulating GGR appears 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, showed a similar trend to p53, indicating that altered expression of p53 was indeed affecting downstream signalling. 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.
Basal levels of p53 are significantly higher in melanoma cell lines relative to the basal levels of the majority of the GGR genes. Furthermore, the RE of p53 after 24 hours 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 to the melanocytes. There is a highly significant increase in p53 induction at the 6 and 24 hour time points for the melanocytes but the melanoma cell lines fail to respond as rapidly and p53 is only differentially expressed after 24 hours (p=0.05). This suggests that it is the induction of p53 that is required for GGR activation rather than just a high constitutive level of p53 expression. However, the induction of these transcripts was not altered by the inhibition of p53, as all 3 GGR genes showed significant fold change increases in expression at 24 hours after cisplatin treatment, thereby demonstrating their similarity to the control melanocytes. This result confirms that a high level of p53 expression is not the only requirement for GGR activation. In addition, the induction of GGR in the melanocytes despite p53 inhibition suggests that p53 may not be responsible for the lack of GGR induction in the melanoma cell lines.
In 2006, a study by Yang et al. identified a number of p53 target genes that were significantly down-regulated in melanomas compared to melanocytes (46). One of their major findings was the association of reduced DDB2 activity and melanoma development (Yang et al. 2006). In addition, the expression of other p53 target genes was also altered, including the cell cycle regulator