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

To investigate cisplatin-damage repair in TTC, we used the cell lines 833 K and SuSa and compared them with MGH-U1 bladder cancer cells. Earlier studies using colony formation assays revealed a 3-fold higher cisplatin sensitivity of 833 K and SuSa cells (for comparison of sensitivity of TTC with bladder cancer cells see Figure 1A) suggesting these lines represent a useful model system for cisplatin sensitive and resistant cells, respectively [8]. DNA intrastrand crosslinks are the major DNA lesions induced by cisplatin. To investigate whether repair of cisplatin-induced intrastrand crosslinks is impaired in TTC, experiments were performed to measure their removal from genomic DNA. GpG-intrastrand crosslinks were detected using a lesion-specific antibody [9]. Time-response curves using MGH-U1 cells showed that GpG-intrastrand crosslinks were formed directly after cisplatin treatment, and at a higher level 6 h later (Figure 1B). This is in line with previous findings showing that following a 1 h cisplatin treatment the level of intrastrand crosslinks peaked after 4-6 h [10]. For repair experiments we therefore treated the cells with cisplatin for 1 h and determined the level of GpG crosslinks 6 h later (for the maximum level of GpG adducts) and 24 h later for determining their repair. The amount of GpG-intrastrand crosslinks measured 6 h post-treatment was set to 100%. The GpG-intrastrand crosslink levels measured 24 h post-treatment were corrected for dilution due to DNA synthesis during the recovery period to rule out a reduction of GpG damage because of DNA replication. Quantification revealed that the initial amount of GpG intrastrand crosslinks was about 50% lower in bladder cancer cells compared to TTC (Figure 1C). Post-treatment removal of GpG-intrastrand crosslinks in cells treated with cisplatin in shown in Figure 1D. No reduction in the level of GpG-intrastrand crosslinks was observed in XP12RO cells, which are deficient in the NER protein XPA and were therefore included as a control for repair deficiency. In MGH-U1 bladder cancer cells the amount of GpG-intrastrand crosslinks was significantly reduced by 45% after 24 h. This reduction was similar to that seen in TTC where the amount of GpG-intrastrand crosslinks was reduced by about 35% after 24 h. Since a repair capacity of cisplatin-induced intrastrand crosslinks in the range of 40-50% indicates repair proficiency [10], the data indicate that TTC are able to repair cisplatin-induced intrastrand adducts.

Induction and repair of cisplatin-induced ICLs was investigated using a modification of the comet assay, which permits detection of ICLs at the single cell level [10]. Cisplatin-induced ICLs peak between 7-9 h post-treatment (data not shown). Therefore, cells were treated with increasing concentrations of cisplatin for 1 h and the amount of ICLs was determined 7 h later. The level of ICLs increased in a concentration dependent manner (Figure 1E). To measure the repair of ICLs, cells were treated with cisplatin (15 μg/ml, 1 h) and the amount of ICLs was determined 7 and 24 h after treatment. As a control we used the ERCC1-deficient cell line 43-3B [11]. The level of ICLs did not change in 43-3B cells, which is in line with the observation that these cells are deficient in ICL repair. For MGH-U1 cells the ICL level was reduced by about 50% after 24 h suggesting proficiency of repair of cisplatin-induced ICLs in the bladder cancer cells (Figure 1F). No reduction in the ICL level after 24 h was observed in 833 K cells, and a small reduction in ICL level was observed in SuSa cells indicating impaired ICL repair. Statistical analysis confirmed the difference in ICL repair capacity between MGH-U1 and 833 K (p ≤ 0.001) and MGH-U1 and SuSa (p ≤ 0.001). Taken together, the data revealed that TTC are impaired in the repair of cisplatin-induced ICLs.

γH2AX formation can be used as a marker for DNA damage (notably DNA double-strand breaks) associated with ICLs [12]. We therefore treated the cells with cisplatin for 1 h and stained for γH2AX 24, 48 and 72 h post-treatment. Representative examples of γH2AX staining for MGH-U1 bladder and 833 K testis tumour cells are shown in Figure 2A. γH2AX immunofluorescence was measured in more than 400 cells per time point. The results were expressed as % of γH2AX positive cells, which were defined as having a fluorescence signal above 500 units (Figure 2B). Over 90% of MGH-U1 cells stained positive for γH2AX when analyzed 24 h after cisplatin treatment, and the percentage of positive cells was decreased 48 and 72 h post-treatment. In contrast, γH2AX formation after cisplatin treatment persisted in 833 K and SuSa cells (Figure 2B). It has been suggested that following the induction of interstrand crosslinks the persistence of γH2AX formation may result from defective processing of ICLs [12]. The data, therefore, support the conclusion of an impaired repair of ICLs in TTC.

The exact mechanism of how initial cisplatin lesions are recognized and the signal becomes transmitted to the apoptotic machinery is still not entirely clear [13]. However, both Chk1 and Chk2 have been implicated in cisplatin damage signaling [14, 15]. We therefore investigated the phosphorylation of Chk1 and Chk2 at different times following cisplatin treatment. PARP-1 cleavage was also investigated as a hallmark of apoptosis. Representative examples of immunoblots are shown in Figure 3A, demonstrating that cisplatin induces activation of Chk1_Ser317 and Chk2_Thr68 as well as PARP-1 cleavage. PARP-1 cleavage was clearly more pronounced in TTC lines than in MGH-U1 (Figure 3A), which is in line with the higher apoptotic response in these cells following cisplatin. Chk1 was transiently induced with a peak level in all lines at 24 h after the onset of cisplatin treatment. For Chk2 a marked difference was observed 3 d after treatment where the activation occurred at higher level in TTC lines than in MGH-U1 cells (Figure 3A and 3B). This was taken to indicate that checkpoint activation occurs as a sustained response in TTC, presumably because of the presence of non-repaired DNA lesions.

The endonuclease ERCC1-XPF is implicated in the repair of cisplatin-induced ICLs [16]. As the level of ERCC1-XPF is low in TTC [7] we hypothesized that ERCC1-XPF is the rate-limiting factor responsible for the observed impaired ICL repair in TTC. 833 K cells were transiently transfected with the bi-cistronic mammalian expression vector pEF6(XPF-IRES-ERCC1) and the levels of ERCC1 and XPF proteins were investigated. Immunoblot analysis showed an increase in both ERCC1 and XPF proteins over time (Figure 4A). Over-expression of ERCC1-XPF did not increase the level of ICL repair in MGH-U1 (data not shown). However, over-expression of ERCC1-XPF in 833 K cells resulted in significant repair of cisplatin-induced ICLs (Figure 4B). When 833 K cells were transfected without DNA (mock) or with a vector control (vector) we observed no repair of ICLs (Figure 4B). Statistical analysis confirmed the difference in ICL repair between 833 K untransfected cells and ERCC1-XPF over-expressing cells (p ≤ 0.001). Altogether these data suggest that over-expression of ERCC1-XPF abrogated the ICL repair deficiency in TTC.

To investigate whether the increased ICL repair has an impact on cellular sensitivity towards cisplatin, ERCC1-XPF was over-expressed in 833 K cells before treatment with cisplatin. The cells were treated with cisplatin (12 and 15 μg/ml, 1 h) and apoptosis was determined 96 h post-treatment. Over-expression of ERCC1-XPF clearly reduced the frequency of cells in subG1 indicating that ERCC1-XPF had a protective effect on apoptosis induction by cisplatin (Figure 4C). These findings suggest that low levels of ERCC1-XPF contribute to cisplatin sensitivity in TTC. We should note that the decrease in sensitivity was not dramatic, most likely because of a low transfection efficiency usually observed with TTC which are difficult to transfect because of their extreme sensitivity towards most experimental manipulations.

As a proof of principle for demonstrating a contribution of ERCC1-XPF for cisplatin sensitivity, we down-regulated ERCC1-XPF in MGH-U1 cells using siRNA against ERCC1. ERCC1 and XPF form a tight complex [17]. The proteins are unstable and are rapidly degraded without its partner [18]. It is therefore possible to reduce both ERCC1 and XPF protein using siRNA against ERCC1. We transiently transfected MGH-U1 cells with ERCC1 siRNA and investigated ERCC1 and XPF levels by immunoblotting. A strong decrease of both ERCC1 and XPF protein levels was observed (Figure 5A). To investigate the effect of down-regulation of ERCC1-XPF on cisplatin-induced ICL damage, cells were transfected with ERCC1 siRNA, treated with cisplatin for 1 h and investigated for gH2AX formation as a marker for damage related to cisplatin ICLs 24 and 48 h later (Figure 5B). Between 80 and 90% of cells stained positive for γH2AX when analyzed 24 h after cisplatin treatment. In MGH-U1 parental cells and cells transfected with control siRNA the percentage of positive cells was decreased 48 h post-treatment. In contrast, γH2AX formation after cisplatin treatment persisted in MGH-U1 cells transfected with ERCC1 siRNA (Figure 5B). These data indicate that down-regulation of ERCC1-XPF lead to impaired processing of ICLs in bladder cancer cells. To further investigate the effect of down-regulation of ERCC1-XPF on cisplatin sensitivity, cells were transfected with ERCC1 siRNA, treated with cisplatin, and apoptosis was determined 48 and 72 h later. Down-regulation of ERCC1-XPF increased the sensitivity to cisplatin, as shown by the higher level of apoptosis (Figure 5C). The increase in sensitivity was statistically significant but small, perhaps due to the relatively long cultivation period following after siRNA transfection, and variations in transfection efficiency. The data support the view that ERCC1-XPF is a key factor in determining cisplatin sensitivity of TTC.

833 K and SuSa human TGCT cell lines, MGH-U1 bladder carcinoma cells and XPA-deficient XP12RO were described previously [7, 23]. 43-3B is an ERCC1-deficient CHO hamster cell line [11]. All cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine (PAA) and 5% antibiotics (penicilline/streptavidine). For drug treatment cells were incubated with cisplatin for 1 h at 37°C in a humidified atmosphere.

Apoptosis was measured by flow cytometry (sub-G₁ content). After treatment with cisplatin and postincubation in fresh medium, cells were harvested, fixed with ethanol (70%) and stained with propidium iodide (17 μg/ml) after RNase (30 μg/ml) digestion. Samples were analyzed on a FACSCalibur (Becton Dickinson, Germany). Accumulated data were analyzed using WinMDI Software.

Colony formation assays were performed according to [8].

ERCC1 and XPF cDNAs were cut from plasmid pET30B(+)ERCC41 [37]. XPF cDNA between restriction sites XbaI and Not I was ligated into the mammalian expression vector pEF6 (Invitrogen) digested with SpeI and NotI. ERCC1 cDNA between XbaI restriction sites was ligated into pEF6(XPF) digested with XbaI. IRES sequence was amplified by PCR using primers with 5'ends complementary for the NotI restriction site. The PCR product was digested with NotI and ligated into pEF6(XPF-ERCC1). The resulting vector pEF6(XPF-IRES-ERCC1) was used for transfection studies.

To over-express ERCC1-XPF transient transfections were performed. Cells were incubated for 24 h with medium containing Effectene transfection reagent (Qiagen) and 2 μg of vector pEF6(XPF-IRES-ERCC1) or 2 μg of vector pEF6 containing no insert. After transfection the cells were washed and treated with cisplatin for 1 h. Transient knock-down of ERCC1 was achieved by transient transfection of 10 nM ERCC1 siRNA (Dharmacon RNA technologies: D-006311-02). siRNA was delivered using Dharmacon siRNA transfection reagent according to the manufacturer's instructions. A non-targeting siRNA was used in control experiments and was purchased from Qiagen (AllStars Negative Control siRNA).

Cells were treated with cisplatin for 1 h and harvested immediately or incubated in fresh medium for another 6 or 24 h. DNA was isolated using the Master-Pure™ Complete DNA and RNA Purification kit (Epicentre^® Biotechnologies, USA). For each time point 4 μg DNA was used in duplicate for detection of cisplatin-induced GpG adducts. DNA was denatured (95°C for 10 min) and placed on ice immediately. Ice-cold ammonium acetate was added (final concentration 1 M), DNA was applied to a nylon Hybond-N+ membrane (Amersham) using a slot blot apparatus (Hybridot Manifold, Bethesda Research Laboratories). The membrane was washed with ammonium acetate (1 M), incubated in 5 × SSC for 5 min and washed in water. The DNA was fixed onto the membrane by baking at 80°C for 2 h. The membrane was blotted in PBS/0,2% Tween-100, 5% non-fat dry milk for 2 h, incubated with an antibody specific for GpG-intrastrand crosslinks [9] at a dilution of 1/500 at 4°C overnight. Peroxidase-conjugated secondary anti-rat antibody (1/2,000) was used for 2 h in blocking buffer. GpG-intrastrand crosslinks were visualized by chemiluminescence. SynGene software was used for quantification. The percentage of lesions remaining at 24 h was calculated in comparison to the lesions present 6 h post-treatment. To assess DNA synthesis during the recovery period, cells were labeled with 50 nCi/ml [¹⁴C]thymidine for 24 h prior to cisplatin treatment. Dilution factors (specific activity of DNA at time point/specific activity of DNA at 0 h) were determined for the 24 h repair period.

The detection of interstrand crosslinking was investigated using a modification of single cell gel electrophoresis (comet assay) as described previously [10]. Exponentially growing cells were treated with cisplatin for 1 h, harvested after 7 h and 24 h and diluted to a density of 2.5 × 10⁴ cells/ml. All cisplatin-treated samples and one control were subjected to 8 Gy X-irradiation to induce random strand breakage, one unirradiated control was also included. The cells were lysed and subjected to electrophoresis. The presence of ICLs retards migration of the irradiated DNA during electrophoresis, resulting in reduced tail moment compared to control cells. To prevent repair of DNA breaks after irradiation, cells were kept on ice. Immediately after irradiation the cells were embedded in 0.5% low melting point agarose on microscope slides which were pre-coated with with 0,5% low melting point agarose. After the agarose solidified, the slides were placed in cold lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Na-Laurylsarcosinate, pH 10) and incubated for 1 h at 4°C, followed by incubation in alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) for 30 min to denature DNA. Electrophoresis was carried out for 15 min at 25 V in the dark. Following electrophoresis the slides were incubated in neutralization solution (0.4 M Tris pH7.5) 3 × for 5 min, rinsed with H₂O and fixed with absolute ethanol for 5 min. Slides were allowed to dry overnight, stained with propidium iodide (50 μg/ml), and comets were analyzed using a Nikon MIKROPHOT FXA fluorescence microscope. Fifty cells per slide were analyzed using Komet 4.0.2 Assay Software (Kinetic Imaging Ltd, Liverpool). The value of the tail moment was used to describe the rate of migration of DNA out of the nucleus during electrophoresis. The tail moment is calculated as product of percentage of DNA in the comet tail and distance between the head and tail. The presence of ICLs retards migration of the irradiated DNA during electrophoresis, resulting in a reduced tail moment compared to the untreated control. The amount of ICLs was therefore determined by comparing the tail moment of the irradiated cisplatin-treated samples with irradiated untreated samples and unirradiated untreated controls. The level of interstrand crosslinking is proportional to the decrease in tail moment (DTM) in the irradiated drug treated sample compared to the irradiated untreated control. The % DTM was calculated using the following formula described by [10]: % DTM = [1 - (TM_{D IR} - TM_{C U})/(TM_{C IR} - T_{C U})] × 100, where TM_{D IR} is the mean tail moment of the cisplatin treated irradiated sample, TM_{C IR} is the mean tail moment of the irradiated control sample and TM_{C U} is the mean tail moment of the unirradiated control sample. Statistical analysis using the software program SPSS (SPSS Inc., USA) was performed using % DTM of 150-200 cells. Kruskal-Wallis one-way analysis of variance by ranks was used to compare whether ICL repair capacity differed within the group of four cell lines. To compare two independent groups of sampled data Mann-Whitney U test was used.

Cells were plated at a density of 3 × 10⁵ per dish on cover slips in 60 mm dishes and incubated for 24 h at 37°C. The cells were treated with cisplatin (6 μg/ml) for 1 h and incubated in fresh medium for 24, 48 and 72 h, respectively. Cells were quickly rinsed with PBS and then fixed with 4% formaldehyde for 20 min at RT, followed by ice-cold methanol absolute at -20°C for up to 72 h. The fixed cells were blocked with 5% BSA in PBS/0.3% Triton-X100 for 1 h at RT and then incubated with a 1:1,000 dilution of anti-phospho H2AX_Ser139 antibody (Millipore) over night at 4°C. After washing 3 times in PBS the samples were incubated with a 1:500 dilution of a Alexa-fluor 488 conjugated goat anti-mouse antibody (Invitrogen) for 1-2 h at RT in the dark, followed by staining with DAPI (100 ng/ml) for 30 min. 10 μl mounting medium was added, and the cover slips containing the cells were mounted onto microscope slides. Fluorescence images were captured using a Zeiss microscope (ImageM1 AX10) and analysed using Metafer software (MetaSystems).

Protein extraction and SDS gel electrophoresis were perfomed according to [7]. The antibodies used were: XPF 1/5,000 dilution of polyclonal antibody RA1 [21]; ERCC1 1/1,500 dilution of polyclonal antibody RWO18 [17]; RPA2 1/5,000 dilution of monoclonal antibody 9H8 (NeoMarkers); phospho-Chk1 1/1,000 dilution of polyclonal antibody (Bethyl Laboratories); phospho-Chk2 1/1,000 dilution of polyclonal antibody (Epitomics); PARP-1 1/1,000 dilution of monoclonal antibody raised against amino acids 22-219 (BD); anti-rabbit IgG 1/2,000 dilution (DAKO) or anti-mouse IgG 1/5,000 dilution (DAKO). RPA2, which is a housekeeping protein, was used as loading control since our previous data showed relatively little variation in RPA2 levels for different cancer cell lines [7].