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. 2010 Sep 16:9:248.
doi: 10.1186/1476-4598-9-248.

"VSports注册入口" Cisplatin sensitivity of testis tumour cells is due to deficiency in interstrand-crosslink repair and low ERCC1-XPF expression

Svetlana Usanova  1 , Andrea Piée-Staffa, Ulrike Sied, Jürgen Thomale, Astrid Schneider, Bernd Kaina, Beate Köberle
Affiliations

Cisplatin sensitivity of testis tumour cells is due to deficiency in interstrand-crosslink repair and low ERCC1-XPF expression

Svetlana Usanova et al. Mol Cancer. .

Abstract

Background: Cisplatin based chemotherapy cures over 80% of metastatic testicular germ cell tumours (TGCT). In contrast, almost all other solid cancers in adults are incurable once they have spread beyond the primary site. Cell lines derived from TGCTs are hypersensitive to cisplatin reflecting the clinical response. Earlier findings suggested that a reduced repair capacity might contribute to the cisplatin hypersensitivity of testis tumour cells (TTC), but the critical DNA damage has not been defined VSports手机版. This study was aimed at investigating the formation and repair of intrastrand and interstrand crosslinks (ICLs) induced by cisplatin in TTC and their contribution to TTC hypersensitivity. .

Results: We observed that repair of intrastrand crosslinks is similar in cisplatin sensitive TTC and resistant bladder cancer cells, whereas repair of ICLs was significantly reduced in TTC. γH2AX formation, which serves as a marker of DNA breaks formed in response to ICLs, persisted in cisplatin-treated TTC and correlated with sustained phosphorylation of Chk2 and enhanced PARP-1 cleavage V体育安卓版. Expression of the nucleotide excision repair factor ERCC1-XPF, which is implicated in the processing of ICLs, is reduced in TTC. To analyse the causal role of ERCC1-XPF for ICL repair and cisplatin sensitivity, we over-expressed ERCC1-XPF in TTC by transient transfection. Over-expression increased ICL repair and rendered TTC more resistant to cisplatin, which suggests that ERCC1-XPF is rate-limiting for repair of ICLs resulting in the observed cisplatin hypersensitivity of TTC. .

Conclusion: Our data indicate for the first time that the exceptional sensitivity of TTC and, therefore, very likely the curability of TGCT rests on their limited ICL repair due to low level of expression of ERCC1-XPF V体育ios版. .

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Figures

Figure 1
Figure 1
Induction and removal of cisplatin-induced DNA lesions. A) Clonogenic cell survival curves of 833 K and SuSa testis tumour cells and MGH-U1 bladder cancer cells after cisplatin treatment for 1 h. B) MGH-U1 bladder cancer cells were treated with cisplatin (7.5 or 15 μg/ml) for 1 h, DNA was isolated 0 h, 6 h or 24 h post-treatment and examined for the presence of GpG-intrastrand crosslinks. C) MGH-U1 bladder cancer cells, 833 K and SuSa testis tumour cells were treated with cisplatin (7.5 or 15 μg/ml) for 1 h. The levels of GpG-intrastrand crosslink were determined 6 h post-treatment. D) MGH-U1 bladder, 833 K and SuSa testis and XPA-deficient XP12RO cells were treated with cisplatin (7.5 or 15 μg/ml) for 1 h, GpG-intrastrand crosslink levels were determined 6 and 24 h post-treatment. The levels measured 6 h post-treatment were set to 100%. The levels measured 24 h post-treatment were corrected for dilution due to DNA synthesis during the recovery period. Each point represents the mean of 3 to 6 replicate experiments. E) MGH-U1 bladder (circles) and 833 K testis (squares) tumour cells were treated with cisplatin for 1 h and analysed for ICL cross-linking 7 h post-treatment. The results are the means of three independent experiments. F) MGH-U1 bladder, 833 K and SuSa testis and ERCC1-deficient 43-3B cells were treated with cisplatin (15 μg/ml) for 1 h, the level of ICL cross-linking was determined 7 and 24 h post-treatment. Each column represents the mean of 3 to 6 replicate experiments.
Figure 2
Figure 2
γH2AX staining in bladder and testis tumour cell lines. A) Representative examples for MGH-U1 and 833 K cells treated with cisplatin (6 μg/ml) for 1 h and stained for γH2AX at the indicated time points. C: untreated control. DNA counterstaining is with DAPI. B) Quantification of γH2AX staining of MGH-U1 bladder, 833 K and SuSa testis tumour cells after exposure to cisplatin (6 μg/ml) for 1 h, followed by a recovery period of 24, 48 or 72 h. For each data point, more than 400 nuclei/experiment were examined, the γH2AX fluorescence was determined and the % cells with a fluorescence intensity more than 500 units (control value) were plotted. Each column represents the mean of 3 replicate experiments.
Figure 3
Figure 3
Phosphorylation of Chk1 and Chk2 and PARP-1 cleavage in testis versus bladder cancer cell lines. A) Immunoblot analysis of p-Chk1Ser317, p-Chk2Thr68 and cleaved PARP-1 in 50 μg protein extract of MGH-U1 bladder, 833 K and SuSa testis tumour cells. RPA2 was used as a loading control. Cells were harvested at the indicated time points after treatment with cisplatin (6 μg/ml) for 1 h. B) Quantification of the data obtained in immunoblots.
Figure 4
Figure 4
Effect of ERCC1-XPF over-expression on ICL repair and cisplatin sensitivity. A) 833 K cells were transfected with plasmid pEF6(XPF-IRES-ERCC1). Immunoblot analysis of XPF and ERCC1 proteins in extracts harvested at the indicated time points. RPA2 served as loading control. B) 833 K cells (non-transfected, transfected with pEF6(ERCC1-IRES-XPF), transfected with vector pEF6, mock transfected) were treated with cisplatin (15 μg/ml) for 1 h, the level of interstrand crosslinking was determined 7 and 24 h post-treatment. Each column represents the mean of 3 to 6 replicate experiments. C) Cells were transfected with plasmid pEF6(XPF-IRES-ERCC1) for 24 h, treated with cisplatin (12 and 15 μg/ml) for 1 h and post-incubated for 96 h. Apoptosis was measured by flow cytometry (sub-G1 content). * represents statistical significance.
Figure 5
Figure 5
Effect of ERCC1-XPF down-regulation on cisplatin ICL damage and sensitivity. A) MGH-U1 cells were transfected with 10 nM ERCC1 siRNA and analysed 48, 72 and 96 h after transfection for ERCC1 and XPF expression. RPA2 served as loading control. B) MGH-U1 cells were transfected with 10 nM ERCC1 siRNA or control siRNA, cultivated for 96 h, treated with cisplatin (6 μg/ml) for 1 h and post-incubated for 24 or 48 h. γH2AX fluorescence was quantified in MGH-U1 parental cells and cells transfected with either ERCC1 siRNA or control siRNA. For each data point, more than 400 nuclei/experiment were examined, the γH2AX fluorescence was determined and the % cells with a fluorescence intensity more than 500 units (control value) were plotted. Each column represents the mean of 3 independent experiments. C) MGH-U1 cells were transfected with 10 nM ERCC1 siRNA or control siRNA, cultivated for 96 h, treated with cisplatin (9 μg/ml) for 1 h and post-incubated for 48 or 72 h. Apoptosis was measured by flow cytometry (sub-G1 content). * represents statistical significance.
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