XPA versus ERCC1 as chemosensitising agents to cisplatin and mitomycin C in prostate cancer cells: Role of ERCC1 in homologous recombination repair
Introduction
NER is the primary repair system for the removal of bulky DNA lesions caused by a variety of chemotherapeutic drugs, reviewed in [1], [2]. The core proteins required for NER are XPA, RPA, XPC-hR23B, TFIIH, XPG and ERCC1/XPF [3]. XPA forms part of the pre-incision complex in both global and transcription-coupled repair, and is thought to verify NER lesions and correctly position other NER factors around the lesion [1]. Cells belonging to the XP complementation group A are the most severely impaired in NER and are sensitive to UV and drugs such as cisplatin [1], [4]. The ERCC1/XPF heterodimer is a structure-specific endonuclease and its function in NER is to make the 5′-incision on the damaged strand [5], [6]. Unlike XPA, which only functions in NER, ERCC1/XPF has also been implicated in the removal of interstrand crosslinks (ICLs) caused by bifunctional alkylating agents [7], and in homologous recombination (HR) [8], [9].
Cisplatin resistance is multifactorial (reviewed in [10]), being associated with drug efflux, glutathione levels and DNA repair. The NER pathway is an important factor in cisplatin resistance, as shown by the association between expression levels of NER factors (mainly ERCC1 and XPA), NER and cisplatin resistance [11], [12], [13], [14], [15], [16], [17], [18], [19]. Thus, the NER pathway is an attractive target with which to sensitise cancer cells to cisplatin, as well as other DNA crosslinking drugs.
In this study, synthetic siRNAs targeted to XPA and ERCC1 were transfected into the human prostate cancer cell lines PC3 and DU145, and their effectiveness as chemosensitising agents to cisplatin and MMC compared. SiRNA-mediated downregulation of ERCC1 but not XPA sensitised both cell lines to MMC, presumably because of the role of ERCC1 in repairing DNA ICLs. With cisplatin, however, the response to XPA and ERCC1 downregulation differed between the two cell lines. We present evidence that the contribution of HRR to cell survival can be circumvented by ERCC1 downregulation. Our results suggest that because of its dual role in NER and HRR, ERCC1 may be of wider use as a chemosensitising target.
Section snippets
Cell lines and culture
The prostate cancer cell lines PC3 and DU145 were maintained in RPMI-1640 medium (Invitrogen), supplemented with 8% FCS and 2 mM l-glutamine (complete medium). Cells were grown at 36.5 °C in a 5% CO2 incubator.
Transfection with siRNAs
SiRNA Smartpools designed to target human ERCC1 and XPA were purchased from Dharmacon RNA Technologies, catalogue numbers M-006311-00 and M-005067-00 for ERCC1 and XPA, respectively. The catalogue numbers for the single siRNAs used in this study are D-006311-02 and -04 (ERCC1 siRNAs 2 and
SiRNA-mediated downregulation of XPA and ERCC1 in prostate cancer cell lines
PC3 and DU145 cells were transiently transfected with Smartpool siRNAs directed against ERCC1 and XPA. To assess the level of downregulation, serial dilutions of the control cell extract were included in the Western blots, for comparison with the XPA or ERCC1 siRNA transfected cells. This strategy was used because ECL signals are often not linear, making the extent of protein downregulation difficult to quantify. The XPA Smartpool siRNA was able to downregulate XPA protein by at least 90% (Fig.
Discussion
In this study we have explored the use of XPA and ERCC1 as potential therapeutic targets to sensitise prostate cancer cells to the chemotherapeutic drugs MMC and cisplatin. We found that ERCC1 downregulation sensitised both PC3 and DU145 cells to MMC, by approximately two-fold. XPA downregulation did not sensitise PC3 cells to MMC, but did slightly sensitise DU145 cells. MMC is a bifunctional DNA alkylating agent that results in ICLs and double strand breaks (DSBs). MMC ICLs are recognised and
Acknowledgements
We are very grateful to Dr. Mike Tilby, Newcastle University for his advice in setting up the cisplatin intrastrand crosslink assay, and for providing the platinated standards and ICR4 antibody. This work was supported by the Orchid Cancer Appeal.
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Present address: Molecular Epidemiology Unit, Centre for Epidemiology and Biostatistics, Faculty of Medicine and Health, LIGHT Laboratories, University of Leeds, Leeds LS2 9JT, United Kingdom.