Pro- and anti-apoptotic effects of p53 in cisplatin-treated human testicular cancer are cell context-dependent

Abstract

In murine testicular cancer (TC) cells wild-type p53 contributes to sensitivity to DNA-damaging drugs in a dose-dependent way. In human TC, however, the role of wild-type p53 functionality in chemotherapeutic response remains elusive. We analyzed functionality of wild-type p53 in cisplatin sensitivity in the human TC setting using a p53 short interfering (si)RNA approach. The cisplatin-sensitive TC cell line (Tera), the subline with acquired cisplatin resistance (Tera-CP) and a panel of intrinsically resistant TC cell lines (Scha and 2102EP), all expressing wild-type p53, were used. p53 and p53 transcriptional targets MDM2 and p21 (Waf1/Cip1) (p21) were expressed in a p53 transactivation-dependent way in all TC cell lines. Following cisplatin exposure, expression levels of p53 increased, with a subsequent increase in MDM2 and p21 mRNA and protein levels and Fas cell membrane levels. Downregulation of p53 with siRNA lowered cisplatin-induced apoptosis in Tera and Tera-CP, which was associated with a diminished Fas membrane expression. In contrast, p53 suppression augmented cisplatin-induced apoptosis in Scha and 2102EP and concomitantly strongly suppressed MDM2 and p21 mRNA and protein expression. Our results indicate that p53 is involved in transactivation of pro- and anti-apoptotic genes in untreated and cisplatin-treated TC cells, but subtle differences are present between TC cell lines. The opposite role of p53 in cisplatin-induced apoptosis among TC cell lines demonstrates the importance of the cellular context for the p53 transactivation phenotype in TC cells.

Figure 1. Large differences in p53, p21 and MDM2 expression levels between TC cell lines. (A) Expression levels in TC cells were determined using western blot of proteins in whole-cell lysates, treatment as indicated. Immunoblotting was performed as described in “Materials and Methods.” Each lane was loaded with 20 μg protein. Actin was used as a loading control. A representative example of at least three independent experiments is shown. (B) Differences in relative p53, MDM2 and p21 expression levels of the TC cell lines. (C) Relative expression level of miR-17–5p.

Figure 2. Cisplatin treatment for 24 h resulted in apoptosis in TC cells. Apoptosis was determined with several different assays using cell morphology, caspase-3 activity and PARP cleavage as read-outs. Cisplatin concentration-dependent apoptosis (0, 4 and 8 µM) is related to the cell survival (see Table 1). (A) The percentage of apoptotic cells was determined with fluorescence microscopy on acridine orange stained cells, (B) caspase-3 activity was determined in whole-cell lysates using a fluorescence assay and (C) PARP cleavage was determined with immunoblotting on whole-cell lysates after exposure of the cells to cisplatin. Counting of apoptotic cells, caspase-3 activity assay and immunoblotting were performed as described in “Materials and Methods.” Values are the mean ± SD of three experiments.

Figure 3. Suppression of p53 has opposite effects on cisplatin-induced apoptosis in different TC cell lines. TC cells were either untransfected (con), or transfected with p53 siRNA (p53) or negative control siRNA (neg) and after 24 h treated with cisplatin (0, 4 or 8 μM) for an additional 24 h. Note that Tera-CP cells are treated with cisplatin for 48 h. (A) Transfection with p53 siRNA suppressed p53, p21 and MDM2 expression in Tera and Scha cells also after cisplatin treatment (4 μM) for 24 h. PARP cleavage, however, was reduced in p53-suppressed Tera cells but enhanced in p53-suppressed Scha cells following cisplatin treatment. Protein expression was determined in whole-cell lysates using immunoblotting as described in “Materials and Methods.” Each lane was loaded with 20 μg protein. Actin was used as a loading control. To detect p21 in lysates of Tera, membranes had to be exposed to X-ray films longer than compared with Scha. A representative example of three independent experiments is shown. (B) Apoptotic cells were visualized with fluorescence microscopy on acridine orange-stained cells with acridine orange using a fluorescence microscopy counted with on stained cells. Values are the mean ± SD of three experiments. *p < 0.05; **p < 0.01; ***p < 0.005; compared with matching negative control siRNA (neg)-transfected cells.

Figure 4. Suppression of p53 has opposite effects on cisplatin-induced caspase-3 activity in different TC cell lines. Cells were either transfected with negative control siRNA (neg), and with p53 siRNA set I (p53I) or p53 siRNA set II (p53II). Following siRNA treatment for 24 h, cells were treated with cisplatin (4 μM) for an additional 24 h. Note that Tera-CP cells are treated with cisplatin for 48 h. (A) Both p53 siRNA sets suppressed p53 expression in Tera-CP and 2102EP cells, also after cisplatin treatment (4 μM) of the p53-suppressed cells. PARP cleavage, however, was reduced in p53-suppressed Tera-CP cells and enhanced in p53-suppressed 2102EP cells following cisplatin treatment. Protein expression was determined in whole-cell lysates using immunoblotting as described in “Materials and Methods.” Each lane was loaded with 20 μg protein. Actin was used as a loading control. A representative example of three independent experiments is shown. (B) Caspase-3 activity was determined in whole-cell lysates using a fluorescence assay as described in “Materials and Methods.” Values are the mean ± SD of three experiments. *p < 0.05; **p < 0.01; ***p < 0.005; compared with matching negative control siRNA (neg)-transfected cells.

Figure 5. Cisplatin-induced p21 and MDM2 mRNA and Fas surface expression in TC cells is strongly reduced in p53-suppressed TC cells. Cells were either transfected with p53 siRNA (p53 I) or negative control siRNA (neg). Following siRNA treatment for 24 h, cells were treated with cisplatin (4 μM) for an additional 24 h. (A) Quantitative real-time PCR was used to determine p21 and MDM2 mRNA levels that were normalized to the level of GAPDH in the same sample. (B) Fas membrane expression was determined with flow cytometry and indicated as mean fluorescence intensity (MFI). Values are the mean ± SD of three experiments. *p < 0.05; **p < 0.01; ***p < 0.005; compared with matching negative control siRNA (neg)-transfected cells.

Figure 6. Proposed simplified model showing the mechanisms through which p53 can either be involved in a pro- or anti-apoptotic response to cisplatin treatment in TC cells. Cisplatin-induced DNA damage activates p53, which, in turn, transcribes p21 and MDM2, and induces Fas membrane expression. Recently, we have identified the Fas pathway to be involved in cisplatin-induced apoptosis in cisplatin-sensitive TC cell lines. Moreover, we demonstrated that high cytoplasmic p21 expression inhibits both Fas death receptor-mediated apoptosis and cisplatin-induced apoptosis. Here, we propose the following model. In TC cells hardly expressing p21, cisplatin-induced p53 activation results in enhanced Fas death receptor expression and Fas death receptor-mediated apoptosis. In addition, cisplatin-induced activation of p53 may also lead to enhanced intrinsic (mitochondrial) apoptosis. Thus, p53 has a pro-apoptotic function. TC cells, resistant to cisplatin, express relatively high cytoplasmic p21 levels that are strongly dependent on p53 transcriptional activity and lower Oct4 and miR-106b family members. In these cells, cisplatin-induced p53 activation also results in enhanced Fas death receptor expression, although to a lesser extent. However, the relatively high cytoplasmic p21 expression levels are blocking both cisplatin-induced extrinsic and intrinsic apoptosis. In these cisplatin-resistant TC cells, p53 has an anti-apoptotic function as transcriptional activator of p21 and MDM2.