Introduction

Testicular germ cell tumors (TGCTs) represent one of the few solid tumor types that, when metastasized, are curable by cisplatin-containing chemotherapy. The overall cure rate of metastasized TGCT is more than 80% (Einhorn, 2002). Most cell lines derived from TGCTs also display an unusually high sensitivity to DNA-damaging chemotherapeutic drugs (Masters et al., 1993; Huddart et al., 1995).

The tumor-suppressor protein p53 plays an important role in the response of cancer cells to DNA damage (Levine, 1997; Agarwal et al., 1998; Prives and Hall, 1999). Exposure to chemotherapeutic agents or -irradiation leads to stabilization and activation of p53, resulting in either cell cycle arrest or apoptotic cell death (Lundberg and Weinberg, 1999; Shen and White, 2001). p53-mediated apoptosis can occur via the death receptor pathway or the mitochondrial death pathway. p53 can induce both pathways by the transcriptional activation of Fas and DR5 or the proapoptotic Bcl-2 family members Bax, Bak, PUMA and Noxa (Shen and White, 2001). However, p53 also regulates the expression of proteins involved in the induction of cell cycle arrest such as p21^Waf1/Cip1 (p21), GADD45 or 14-3-3. Therefore, depending on which genes are activated by p53, tumor cells will go directly into apoptosis or go first into cell cycle arrest to repair the DNA damage.

Although the p53 gene is the most frequently mutated gene in human cancers (Greenblatt et al., 1994), almost no p53 mutations occur in human TGCTs (Lutzker, 1998). Lutzker et al. (2001) demonstrated in teratocarcinoma cells that a high-level wild-type p53 expression is required for apoptosis induction following low-level DNA damage. However, the exact mechanism(s) of p53-induced apoptosis by DNA damage in TGCTs is still unclear. Previously, we have shown that the sensitivity of TGCT cells to cisplatin is related to the presence of a functional p53- and Fas-dependent death pathway (de Jong et al., 1999; Spierings et al., 2003a). Furthermore, despite accumulation of p53 only minimal p21 induction occurs in TGCT cells upon chemotherapeutic drug exposure (Chresta et al., 1996; de Jong et al., 1999). In addition, most TGCTs lack p21 protein expression (Guillou et al., 1996; Bartkova et al., 2000; Datta et al., 2001). Several studies have shown that p21 is not only an important mediator of p53-induced cell cycle arrest (El-Deiry et al., 1993, 1994) but also of apoptosis suppression following DNA-damaging agents exposure (Gorospe et al., 1997; Gervais et al., 1998; Levkau et al., 1998; Asada et al., 1999; Zhang et al., 1999). The reduced p21 expression in TGCT cells may thus not only result in a less efficient inhibition of cell cycle progression but also fail to prevent rapid apoptosis induction upon DNA damage. Therefore, low p21 protein level is probably an additional explanation for the extreme sensitivity of TGCTs for apoptotic stimuli.

In the present study, potential mechanisms involved in the reduced p21 protein levels and the importance of the low p21 expression in the sensitivity of TGCT cells to apoptotic stimuli were studied. The results from this study clearly demonstrate that the low p21 protein expression is predominantly caused by reduced p21 gene transcription and sensitizes TGCT cells to the Fas death pathway.

Results

Reduced p21 protein level is related to the low p21 mRNA levels in TGCT cells

Previously, we found that p21 expression levels remained low in the TGCT cell lines Tera, 833KE and Scha in comparison to ovarian cancer cells A2780 despite the massive upregulation of p53 following cisplatin exposure (de Jong et al., 1999). As depicted in Figure 1a, Tera and A2780 cells revealed similar cisplatin-induced p53 protein accumulation, but only a marginal induction of p21 protein was observed in Tera cells in comparison to A2780 cells.

Figure 1.

Low p21 protein level is not due to increased degradation by the proteasome or cleavage by caspases. (a) Tera and A2780 cells were treated with different concentrations of cisplatin for 18 h and expression of p53 and p21 protein was examined by Western blot analysis. (b) Tera and A2780 cells were incubated for 6 h with 8 M cisplatin alone or with 10 M MG-132. Cells were analysed by immunoblotting for protein expression of p21 and p53. (c) Tera cells were incubated with cisplatin alone or with 50 M zVAD-fmk for 18 h. Cells were then analysed by immunoblotting for p21 expression and caspase-3 cleavage. Actin is shown as a loading control. A representative example of at least two independent experiments is shown

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p21 protein level can be regulated by proteasomal degradation or caspase-mediated cleavage (Gervais et al., 1998; Levkau et al., 1998; Zhang et al., 1999; Naujokat and Hoffmann, 2002; Xiang et al., 2002). As shown in Figure 1b, p21 protein levels in A2780 cells increased considerably upon exposure to the proteasome inhibitor MG-132. However, although MG-132 exposure of Tera cells resulted in clear p21 accumulation, the p21 protein levels remained substantially lower than those observed in A2780 cells (a 10-fold increase in Tera and an 80-fold increase in A2780, respectively). Cisplatin combined with MG-132 did not increase p21 protein levels in Tera or A2780. Western blot analysis of p53, another target for proteasome degradation, revealed an increase of p53 upon MG-132 incubation, indicating that the proteasome-dependent degradation pathway was indeed inhibited in Tera cells. Similar results were observed with Scha cells (data not shown). Previously, we have shown that zVAD-fmk efficiently inhibits cisplatin-induced apoptosis of Tera cells (Spierings et al., 2003a). Although caspase-3 cleavage was completely inhibited by zVAD-fmk, it did not result in elevated p21 protein levels upon cisplatin exposure (Figure 1c).

To investigate whether a reduced transcription of the p21 gene might cause the low p21 protein expression in TGCTs, Tera and A2780 cells were incubated with or without 8 M cisplatin followed by analysis of the p21 mRNA expression. In Tera cells, p21 mRNA expression was relatively low in comparison to A2780 cells (Figure 2). Cisplatin treatment induced p21 mRNA in both cell lines. However, the p21 mRNA levels remained limited in Tera cells upon cisplatin exposure as compared to p21 mRNA levels in cisplatin-treated A2780 cells (14-fold difference). Comparable results were observed in the TGCT cell line Scha (data not shown). Altogether, these results suggest that the low p21 protein levels in Tera cells are predominantly caused by a low level of p21 gene transcription.

Figure 2.

Reduced p21 protein level is related to the low p21 mRNA levels in TGCT cells. Tera and A2780 cells were incubated for 24 h with or without 8 M cisplatin and p21 mRNA levels were determined by RT–PCR. Expression of GAPDH was used to control RNA integrity and quantity. Subsequent dilutions (0 (undiluted), 5- and 25-fold) of cDNA were used for RT–PCR. Relative optical density of p21 mRNA expression (25-fold dilution) in untreated (white bars) or cisplatin-treated (black bars) Tera and A2780 cells. p21 mRNA expression was corrected to GAPDH expression and is expressed in relation to untreated Tera cells (which was defined as 1). A representative example of at least three independent experiments is shown

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Irradiation but not cisplatin treatment induces p21 mRNA and protein levels

Several studies have demonstrated an increase in p21 mRNA and protein level in TGCT cells upon irradiation (Burger et al., 1998b, 1999; Malashicheva et al., 2000), while others reported a minimal induction of p21 following drug-induced DNA damage (Chresta et al., 1996; de Jong et al., 1999). Therefore, Tera cells were irradiated or exposed to cisplatin followed by analysis of p21 expression levels. Despite similar p53 accumulation, cisplatin marginally induced p21 protein levels whereas a massive induction of p21 protein was observed upon irradiation of Tera cells (Figure 3a). Comparable differences in p21 accumulation were obtained between cisplatin-treated and irradiated Scha cells (data not shown). In addition, irradiated Tera cells expressed 12-fold more p21 mRNA than cisplatin-treated Tera cells (Figure 3b). These results suggest that p53 is transcriptionally more active upon DNA damage caused by irradiation than by cisplatin, resulting in increased p21 levels.

Figure 3.

Irradiation but not cisplatin induces massive elevation in p21 mRNA and protein levels. Tera cells were irradiated or treated with cisplatin. After 24 h, the cells were analysed by immunoblotting for p53 and p21 protein expression (a) or by RT–PCR for p21 mRNA expression (b). Expression of GAPDH was used to control RNA integrity and quantity. Subsequent dilutions (0 (undiluted), 5- and 25-fold) of cDNA were used for RT–PCR. Relative optical density of p21 mRNA expression (25-fold dilution) in untreated (white bars), cisplatin-treated or irradiated (black bars) Tera cells. p21 mRNA expression was corrected to GAPDH expression and is expressed in relation to untreated Tera cells (which was defined as 1). A representative example of at least three independent experiments is shown

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Subcellular localization of p53 and p21 upon cisplatin treatment or irradiation

To exclude that cisplatin-induced p53 could not enter the nucleus to transcriptionally activate p21, the cellular localization of p53 upon cisplatin exposure or irradiation was studied. Therefore, nuclear- and cytosol-rich fractions were isolated from irradiated or cisplatin-treated Tera cells and evaluated for the presence of p53. Successful fractionation was verified by Western blot analysis for the nuclear retinoblastoma protein (Rb), which was only detectable in the nucleus. As shown in Figure 4, p53 was present in both the cytoplasm and the nucleus, without a clear difference in localization upon irradiation or cisplatin exposure. In contrast, while irradiation induced p21 expression predominantly in the cytoplasm, cisplatin only gave a minimal induction of p21 in the cytoplasm and the nucleus.

Figure 4.

p21 is mainly localized in the cytoplasm upon irradiation. Tera cells were irradiated or treated with cisplatin. After 24 h, nuclear and cytoplasmic proteins were isolated as described under 'Materials and methods' and analysed by immunoblotting for expression of p53, p21 and Rb. Actin is shown as a loading control. A representative example of at least three independent experiments is shown

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Irradiated Tera cells are resistant to Fas-induced apoptosis

Recently, we have described that cisplatin treatment only results in a minimal p21 induction but sensitizes Tera cells to Fas-induced apoptosis (Spierings et al., 2003a). Several studies have shown that cytosolic p21 can inhibit the Fas death pathway by binding to and thereby inhibiting activation of caspase-3 (Suzuki et al., 1999, 2000a, 2000b; Glaser et al., 2001). To investigate whether the increased p21 protein level renders irradiated Tera cells resistant to Fas-induced apoptosis, Tera cells were exposed to 7C11, an agonistic anti-Fas antibody (Ab), 6 or 24 h after irradiation (Figure 5a and b). No additional apoptosis was induced in irradiated Tera cells. In contrast, Tera cells pretreated with low concentrations of cisplatin for 24 h were sensitive to Fas-induced apoptosis, increasing the apoptosis approximately twofold as compared to cisplatin only (Figure 5c). The difference in Fas sensitivity between irradiated and cisplatin-treated Tera cells was not caused by reduced Fas membrane expression. On the contrary, irradiated Tera cells revealed higher levels of Fas membrane expression in comparison to cisplatin treatment (Figure 6a and b, respectively). Evaluation of the expression of several apoptosis inhibitory proteins upon irradiation or cisplatin treatment revealed no clear changes in expression of FAP-1, cFlip_L, Bcl-2, Bcl-X_L, XIAP or the proapoptotic Bcl-2 family member Bax (Figure 6c). The nonspecific anti-XIAP immunoreactive molecule as indicated (^*) served as an internal loading control (Deveraux et al., 1999). These results suggest that high p21 protein levels induced by irradiation render Tera cells resistant to Fas-mediated apoptosis.

Figure 5.

Irradiated Tera cells are not sensitive to Fas-induced apoptosis. Tera cells were irradiation or treated with cisplatin. At 6 h (a) or 24 h (b) after irradiation and 24 h after cisplatin addition (c), medium (white bars) or 2 g/ml agonistic anti-Fas Ab, 7C11 (black bars) was added to the cells. Apoptosis was determined 20 h upon 7C11 exposure by acridine orange staining. Values shown are the means.d. of three independently performed experiments. ^* The percentage of apoptosis in cells treated with cisplatin and 7C11 differs from the percentage of apoptosis in cells treated with cisplatin only (P<0.05)

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Figure 6.

Resistance to Fas-induced apoptosis is related to increased p21 protein levels, but not to changes in apoptosis inhibitory proteins. Tera cells were irradiated (a) or treated with cisplatin (b). After 24 h, cells were analysed for Fas membrane expression plotted in arbitrary units (AU). Values shown are the means.d. of three independently performed experiments. (c) Irradiated or cisplatin-treated cells were analysed by immunoblotting for the expression of FAP-1, cFlip_L, Bcl-2, Bcl-X_L, Bax, XIAP and p21. A representative example of at least two independent experiments is shown

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p21 short interfering RNA sensitizes irradiated Tera cells to Fas-induced apoptosis

To determine if p21 is indeed responsible for the observed differences in sensitivity to Fas-induced apoptosis between irradiated and cisplatin-treated cells, short interfering RNA (siRNA) were used to suppress endogenous p21 mRNA. SiRNAs are highly efficient for the selective silencing of gene expression (Elbashir et al., 2001a, 2001b). After transfection of p21 specific (p21 siRNA I and II) or control luciferase (Luc siRNA) siRNA molecules, Tera cells were irradiated, followed by the addition of 7C11. After 20 h exposure to 7C11, apoptosis was determined by acridine orange staining (Figure 7a–d) and PARP cleavage (Figure 7e). Control cells only exposed to the transfection agent Oligofectamine (control) or transfected with Luc siRNA molecules were still resistant to Fas-induced apoptosis (Figure 7a and b, respectively). In contrast, Tera cells transfected with p21 siRNA became sensitive to stimulation of the Fas receptor following irradiation. Using p21 siRNA I molecules, apoptosis was approximately 1.7-fold increased compared to irradiation only (Figure 7c). p21 siRNA II molecules were slightly more effective, increasing apoptosis approximately 2.1-fold compared to irradiation only (Figure 7d). Both p21 siRNA sets inhibited the irradiation-induced increase in p21 protein (Figure 7f). These results indicate an important role of p21 in the sensitivity to Fas-induced apoptosis of Tera cells.

Figure 7.

Decreasing the expression of p21 by RNA interference sensitizes irradiated Tera cells to Fas-induced apoptosis. Tera cells were exposed to Oligofectamine alone (control, (a)) or transfected with siRNA duplexes directed against the luciferase gene (Luc, (b)) as control siRNA or the p21 gene (p21 I and II, (c) and (d), respectively). Cells were irradiated 48 h after transfection. At 6 h upon irradiation, medium (white bars) or 2 g/ml anti-Fas Ab, 7C11 (black bars) was added to the cells. Apoptosis was determined by acridine orange staining ((a)–(d)) and by PARP cleavage (e). Values shown are the means.d. of three independently performed experiments. ^* The percentage of apoptosis in irradiated cells treated with 7C11 differs from the percentage of apoptosis in irradiated cells (P<0.05). (f) Expression of p21 was analysed by immunoblotting in Tera cells exposed to Oligofectamine alone (control) or transfected with Luc or p21 siRNA molecules. A representative example of at least two independent experiments is shown

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p21 is not involved in irradiation-induced G₂/M arrest or caspase-3 inhibition

The cellular p21 localization determines its function as an inhibitor of cell cycle progression (nuclear) or apoptosis (cytoplasmic) (Asada et al., 1999; Suzuki et al., 1999, 2000a, 2000b; Glaser et al., 2001). The cytosolic p21 localization in Tera cells (Figure 4) suggests that the main function of p21 is not inhibition of cell cycle progression. Therefore, analysis of cell cycle distribution by propidium iodide (PI) staining was performed to investigate the role of p21 in the regulation of cell cycle progression upon irradiation. Figure 8 shows that despite the reduced p21 protein levels, p21 siRNA-transfected Tera cells still underwent an irradiation-induced G₂ cell cycle arrest. In addition, complex formation between p21 and caspase-3 was examined to investigate a possible apoptosis inhibitory role of cytoplasmic p21. However, although p21 or caspase-3 was clearly immunoprecipitated using antibodies specific for p21 or caspase-3, respectively (data not shown), no caspase-3 or p21 was coprecipitated. The lack of complex formation with caspase-3 and the lack of involvement in cell cycle arrest indicates that p21 executes its role as an apoptosis inhibitor by a different mechanism.

Figure 8.

p21 is not involved in the irradiation-induced G₂/M cell cycle arrest. Tera cells were exposed to Oligofectamine alone (control) or transfected with siRNA duplexes directed against the luciferase gene (Luc siRNA) as control siRNA or the p21 gene (p21 siRNA I and II). Cells were irradiated 48 h after transfection. At 24 h upon irradiation, cell cycle distribution was analysed by flow cytometry. Cells were analysed for DNA content by PI staining. The percentages of cells in the G₀/G₁, S and G₂/M phase are indicated. A representative example of at least two independent experiments is shown

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Discussion

In the present study, potential mechanisms involved in the reduced p21 protein levels and the involvement of p21 in the sensitivity of TGCT cells to apoptotic stimuli have been studied. We demonstrate that the low p21 protein expression in TGCT cells upon cisplatin exposure is related to a reduced p21 gene transcription and not to an increased proteasome- or caspase-mediated breakdown. In contrast to cisplatin treatment, irradiation of TGCT cells resulted in substantial induction of p21 protein levels caused by increased p21 gene transcription. Cisplatin-treated TGCT cells expressing low p21 protein levels are Fas-sensitive, whereas the elevated p21 protein levels in irradiated TGCT cells render them resistant to Fas-induced apoptosis.

Although several reports have investigated the importance of p53 in chemosensitivity of TGCTs, the results are rather contradictory. In murine F19 teratocarcinoma cells, it has been demonstrated that the p53 expression levels are important for a rapid apoptosis induction in response to chemotherapy (Lutzker et al., 2001). A correlation between wild-type p53 expression and chemosensitivity was also observed in human TGCT cell lines (Chresta et al., 1996; Arriola et al., 1999; de Jong et al., 1999). In addition, a cell line expressing mutant p53 was derived from a cisplatin-resistant human TGCT and exhibited relative resistance to cisplatin and reduced apoptotic cell death compared to a cell line expressing wild-type p53 that was derived from a sensitive TGCT (Houldsworth et al., 1998). In contrast, another panel of TGCT cell lines expressing either wild-type or mutant p53 did not confirm these results, since no relation between cisplatin sensitivity, apoptosis induction and p53 status was observed (Burger et al., 1997, 1998a). An immunohistochemical study investigating p53 expression in drug-responsive and nonresponsive TGCT tumors indicated that p53 levels do not determine the sensitivity of TGCTs to chemotherapy (Kersemaekers et al., 2002a). Owing to the high p53 expression often observed in TGCTs, it has been hypothesized that p53 is a functionally inactive protein in TGCT cells but becomes activated by DNA damage to facilitate apoptosis induction. However, the concomitant overexpression of p53 and mdm2 observed in TGCT cell lines and TGCT tumors suggests the presence of transcriptionally active p53 in vivo (Riou et al., 1995; Kersemaekers et al., 2002a; de Jong et al., 1999). Moreover, the Fas death receptor, whose expression is also regulated by p53, is often widely expressed in human TGCTs. For example, Hara et al. (2001) showed that 73 and 56% of the tested TGCTs with or without seminomatous elements, respectively, expressed Fas. A report of Kersemaekers et al. (2002b) revealed Fas expression in teratomas and spermatocytic seminoma but not in seminomas or nonseminomas. In contrast, Sugihara et al. (1997) found expression of Fas in all seminomas, embryonal carcinomas and yolk sac tumors examined. Interestingly, although p53 regulates the expression of mdm2, Fas and p21, several studies have shown that human TGCTs lack p21 protein expression (Guillou et al., 1996; Bartkova et al., 2000; Datta et al., 2001). In addition, despite accumulation of p53 upon exposure to chemotherapeutic drugs, TGCT cell lines show a minimal induction of p21 in comparison to other wild-type p53 expressing cancer cell lines (Chresta et al., 1996; de Jong et al., 1999). In contrast, a clear increase in transcription of the Fas gene was observed in cisplatin-treated TGCT cells (Spierings et al., 2003a). These observations indicate that in TGCTs, cisplatin-induced p53 shifts the balance between proapoptotic signaling and cell cycle arrest (e.g., stimulating DNA repair) towards proapoptosis as reflected in a decreased p21 gene transcription and increased Fas gene transcription (Spierings et al., 2003b). Results obtained in the present study support this hypothesis, showing that reduced p21 protein levels in TGCT cells are related to low p21 transcription, even in the presence of accumulated p53 upon cisplatin treatment. In contrast to other studies (Gervais et al., 1998; Levkau et al., 1998; Zhang et al., 1999; Xiang et al., 2002), p21 was not cleaved by caspases in cisplatin-treated TGCT cells. Although proteasome inhibition resulted in elevated p21 protein expression in human TGCT cells, the fold of induction in p21 protein level was much less in comparison to A2780 ovarian cancer cells. Malashicheva et al. (2000) showed in mouse TGCT cells that p21 protein is degraded by a proteasome-dependent mechanism resulting in the lack of p21 protein expression despite the presence of p21 mRNA. They also detected a strong induction of p21 mRNA 20 h following irradiation. Our results are in agreement with those of Malashicheva et al. (2000). We detected a 10-fold increase in p21 protein level after a short treatment (6 h) with a proteasome inhibitor, while a massive p21 mRNA induction was found 24 h after irradiation. In contrast to Malashicheva et al. (2000), we also measured p21 protein levels 24 h after irradiation and demonstrated a massive increase in p21 protein levels. Therefore, the reduced p21 protein levels in TGCT cells upon cisplatin treatment are predominantly due to the reduced p21 gene transcription in TGCTs. We can, however, not exclude the involvement of p21 proteasomal degradation, because p21 protein can also stimulate its own transcription by stimulating p300 transactivation of p53 (Snowden et al., 2000). Whether a reduced p53 transcriptional activity, as suggested above, is indeed responsible for the low p21 mRNA levels and not a decreased stability of the p21 mRNA has not been proven yet.

Although p21 gene transcription can be initiated by several other transcription factors such as E2F, C/EBP, AP2 or BRCA1 (Zeng et al., 1997; Somasundaram et al., 1997; Hiyama et al., 1998; Cram et al., 1998), p53 has often been shown to be important for the induction of p21 upon exposure to DNA-damaging agents (El-Deiry et al., 1993, 1994; Michieli et al., 1994; Butz et al., 1998). In addition, the p53 family protein member p73 also activates transcription of the p21 gene upon DNA damage (Vikhanskaya et al., 2000; Fillippovich et al., 2001; Stiewe et al., 2002). However, the N-terminally truncated, transactivation-deficient p73 isoform, TA-p73 (also called N-p73), is a dominant-negative inhibitor of both p53 and p73 blocking their transcriptional activity (Grob et al., 2001; Stiewe et al., 2002; Zaika et al., 2002). Interestingly, while cisplatin treatment of TGCT cells gave a selective induction of TA-p73, no change in expression of this protein was observed upon irradiation (data not shown). Based on these findings, it is conceivable that the absence of p21 protein upregulation is related to the selective accumulation of TA-p73 upon cisplatin exposure. In addition, the activation of p73 is regulated by the tyrosine kinase c-Abl (Agami et al., 1999; Gong et al., 1999; Levrero et al., 2000; Tsai and Yuan, 2003). In contrast to irradiation, cisplatin causes a sustained activation of c-Abl and subsequent stabilization of p73 (Agami et al., 1999; Gong et al., 1999; Levrero et al., 2000). Since TA-p73 can also be phosphorylated and stabilized by c-Abl (Tsai and Yuan, 2003), it is tempting to speculate that the continued cisplatin-induced stabilization of TA-p73 might further contribute to the reduced p21 protein levels.

Irradiation can sensitize tumor cells to Fas-induced apoptosis (Fulda et al., 1998; Ogawa et al., 1998; Sheard et al., 1999; Kimura and Gelmann, 2000). In the present study, however, irradiated TGCT cells were Fas-resistant despite the high Fas membrane expression. This cell cycle-independent effect was due to increased accumulation of p21 in the cytoplasm, which blocked Fas-induced apoptosis. These findings are supported by the study with siRNA demonstrating restoration of Fas sensitivity in response to p21 downregulation. Several studies have shown that p21 inhibits death receptor Fas- and DR4/DR5-mediated apoptosis (Suzuki et al., 1999, 2000a, 2000b; Xu and El-Deiry, 2000; Glaser et al., 2001; Xiang et al., 2002). Different pathways have been recognized that mediate this p21 effect. A direct inhibitory binding between p21 and caspase-3 might occur, which could not be detected in the present study (data not shown). Furthermore, p21 can inhibit apoptosis induced by apoptosis signal-regulating kinase 1 (ASK1) (Asada et al., 1999; Schepers et al., 2003). Since Fas ligation induces recruitment of Daxx, which interacts with and thereby activates ASK1 (Chang et al., 1998; Charette et al., 2000; Ko et al., 2001), inhibition of Daxx–ASK1-induced apoptosis might be another mechanism of p21 to cause Fas resistance. Alternatively, the inhibitory effect of p21 on Fas-induced apoptosis of irradiated TGCT cells could be at the level of Bcl-X_L. Increased p21 protein levels indirectly suppress deamidation, i.e. inactivation, of Bcl-X_L via activated Rb (Deverman et al., 2002; Johnstone, 2002). These various p21-inhibitory pathways are currently the subjects of further research.

In conclusion, the present study shows that the low p21 protein expression observed in TGCT cells upon cisplatin treatment is caused by reduced p21 gene transcription and sensitizes these cells to the Fas death pathway.

Materials and methods

Cell lines

The TGCT cell lines Tera and Scha were described previously (Sark et al., 1995). Both Tera and Scha revealed no Fas mutations (Spierings et al., 2003a). No p53 mutations were detected in Tera (Burger et al., 1997). The human ovarian cancer cell line A2780 was used as a control for functional wild-type p53 (Skilling et al., 1996) expressing cells with high p21 protein levels. The wild-type p53 gene status of the TGCT cell lines and A2780 was verified using SSCP. Primer sets for exons 2–11 of the p53 genome were as described by Dijkhuizen et al. (1996). All cell lines grow as monolayers in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum (both from Life Technologies, Breda, the Netherlands) in a humidified atmosphere at 37°C and 5% CO₂.

Western blot analysis

After irradiation or treatment with cisplatin (Pharmachemie BV, Haarlem, the Netherlands), alone or in combination with 7C11 (Immunotech, Marseille, France), MG-132 or zVAD-fmk (both from Calbiochem, Breda, the Netherlands), cells were harvested and lysates were examined by Western blot analysis as described previously (Spierings et al., 2003a). The following Abs were applied: mouse anti-p53-DO-1, mouse anti-Rb, mouse anti-Bcl-2, rabbit anti-Bcl-X_L, rabbit anti-Bax, goat anti-FAP-1 Abs and goat anti-caspase-3 Abs were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-caspase-3 Ab was obtained from Becton Dickinson. Mouse anti-Rb and mouse anti-p21 were purchased from Oncogene Research (Cambridge, MA, USA). Rabbit anti-PARP Ab was obtained from Roche Diagnostics (Almere, the Netherlands) and mouse anti-XIAP Ab from Transduction Laboratories (Lexington, KY, USA). Mouse anti-actin was obtained from ICN Biomedicals (Zoetermeer, the Netherlands). Mouse anti-cFlip Ab NF6 (Scaffidi et al., 1999) was a gift from Dr M Peter (University of Chicago, IL, USA). Binding of these antibodies was determined using horseradish peroxidase (HRP)-conjugated secondary Abs (all from DAKO, Glostrup, Denmark) and visualized with the ECL-chemiluminescence kit of Roche Diagnostics.

Reverse transcription–polymerase chain reaction (RT–PCR)

RT–PCR was performed as previously described (Spierings et al., 2003a). A 331 bp p21 fragment was amplified in 30 cycles with the primers 5'-CGA CTG TGA TGC GCT AAT GG-3' (sense) and 5'-CCG TTT TCG ACC CTG AGA G-3' (antisense). Human glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) was used as a control to normalize the amount of template RNA. Primer sequences for GAPDH were 5'-CAC CAC CAT GGA GAA GGC TGG-3' and 5'-CCA AAG TTG TCA TGG ATG ACC-3' (sense and antisense, respectively), which resulted in a 200 bp fragment after 24 cycles. PCR products were electrophorized in a 2% agarose gel in 1 Tris-borate EDTA buffer. Densitometry analysis was performed with Diversity One PDI software (Amersham, Pharmacia Biotech, Roosendaal, the Netherlands).

Irradiation

Cells from exponentially growing cultures were harvested and resuspended into a density of 1.2 10⁶/300 l and -irradiated in round-bottom tubes using a ¹³⁷Cs -ray machine (IBL 637; CIS Biointernational, Gif-sur-Yvette, France) at a dose rate of 0.783 Gy/min. Irradiated cells were plated directly in fresh tissue culture medium.

Acridine orange apoptosis assay

Irradiated cells were plated in 96-well tissue-culture plates. After 6 or 24 h, medium (control) or 7C11 was added to the irradiated cells. For cisplatin treatment, cells were plated in 96-well tissue-culture plates and incubated with cisplatin alone or in combination with 7C11. After 20 h incubation with this anti-Fas Ab, acridine orange (10 g/ml) was added to each well to distinguish apoptotic cells from viable cells. Staining intensity was determined by fluorescence microscopy and apoptosis was defined by the appearance of apoptotic bodies and/or chromatin condensation. Apoptosis was expressed as percentage apoptotic cells in a culture.

Preparation of fractionated proteins

Irradiated, cisplatin-treated or untreated cells were harvested and washed with ice-cold phosphate-buffered saline (PBS: 6.4 mM Na₂HPO₄; 1.5 mM KH₂PO₄; 0.14 mM NaCl; 2.7 mM KCl; pH=7.2) and suspended in 400 l buffer A (10 mM Hepes/KOH, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). After incubation on ice for 10 min, 25 l 10% Nonidet P-40 (NP-40) was added and directly vortexed for 10 s. The nuclear-rich fraction was pelleted by centrifugation at 23 000 g for 1 min and the supernatant was collected as the cytosol-rich fraction. The pellet was washed with buffer A, dissolved in buffer B (20 mM Hepes/NaOH, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) and incubated on ice for 20 min and vortexed every 5 min. After centrifugation at 23 000 g for 5 min, the supernatant was collected as the nuclear-rich fraction. Each fraction was examined by Western blot analysis as described above.

Detection of Fas membrane expression

Irradiated, cisplatin-treated or untreated cells were stained with a phycoerythrin (PE)-conjugated Ab against Fas (DX2 from Becton Dickinson, Erembodegem-Aalst, Belgium) for 30 min on ice. Subsequently, cells were washed and analysed by flow cytometry (Epics Elite, Coulter-Electronics, Hialeah, FL, USA). The mean fluorescence intensity was determined by comparison of the fluorescence intensity of unlabeled cells.

RNA interference

Small interfering RNAs (siRNAs) specific for human p21 were designed conforming to the sequence AA(N19)TT, where AA and TT are present in the p21 open reading frame at a spacing of 19 nucleotides. Two sets of two single-stranded RNA molecules directed against p21 were synthesized by Eurogentec (Seraing, Belgium). Sequences for p21 siRNA I molecules were 5'-GAC CAU GUG GAC CUG UCA CTdT-3' (sense) and 5'-GUG ACA GGU CCA CAU GGU CdTdT-3' (antisense). Sequences for p21 siRNA II molecules were 5'-CUU CGA CUU UGU CAC CGA GTdT-3' (sense) and 5'-CUC GGU GAC AAA GUC GAA GTdT-3' (antisense). Single-stranded RNA molecules specific for the luciferase (Luc) gene served as control (Elbashir et al., 2001a). The sequences for Luc RNA molecules were 5'-CUU ACG CUG AGU ACU UCG AdTdT-3' (sense) and 5'-UCG AAG UAC UCA GCG UAA GdTdT-3' (antisense). For annealing to form RNA duplexes, 20 M of both single-stranded RNAs were incubated in annealing buffer supplied by Eurogentec (50 mM Tris, pH 7.5–8.0, 100 mM NaCl in RNase-free distilled water) for 1–5 min at 90°C and cooled down to room temperature in approximately 1 h. Tera cells (0.4 10⁶/well) were transfected in six-well plates with 10 l of 20 M siRNA duplexes using Oligofectamine reagent according to the manufacturer's instructions (Invitrogen BV, Breda, the Netherlands). After 48 h, cells were harvested, irradiated and plated in 96 wells or six wells for an apoptosis assay or for protein isolation, respectively. At 6 h after irradiation, medium or 2 g/ml 7C11 was added. After 20 h, percentage apoptosis was determined by acridine orange apoptosis assay or the cells were lysed for protein analysis. FACS analysis of Tera cells transfected with fluorescein-5-isothiocyanate (FITC)-labeled oligonucleotides revealed a transfection efficiency of 86%.

Cell cycle analysis

Cell cycle distribution was determined as described previously (de Jong et al., 1993). Briefly, cells were harvested, washed with ice-cold PBS, fixed in 70% ethanol and stored at 4°C. The cells were washed once with PBS and resuspended in PBS containing 40 g/ml RNase (Sigma-Aldrich Chemie BV, Zwijndrecht, the Netherlands) followed by incubation at 37°C for 15 min. The cells were stained with 40 g/ml PI (Sigma-Aldrich Chemie BV) for 30 min at 37°C and analysed on a FACScalibur flow cytometer (Beckton Dickinson Medical Systems, Sharon, MA, USA). Percentages of cells within each cell cycle compartment (G₀/G₁, S or G₂/M phase) were calculated using the DNA cell cycle analysis software Modfit (version 5.2, Verity Software House Inc. Topsham, ME, USA).

Immunoprecipitation

Irradiated or untreated Tera cells were harvested and washed once in cold PBS. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% NP-40, 0.5 mM sodium orthovanadate (Na₃OV₄), 1 mM PMSF, and complete protease inhibitors (Roche Diagnostics)) for 15 min on ice. After centrifugation at 2500 g at 4°C for 10 min, protein concentration of the supernatant was determined and equal amounts of proteins were used for the immunoprecipitation. Anti-p21 Ab (C-19 from Santa Cruz; 2 g/ml) or anti-caspase-3 (Transduction Laboratories; 2 g/ml) was added to the lysates and reacted at 4°C for 1 h. Immune complexes were precipitated using 30 l protein A-sepharose (Amersham, Pharmacia Biotech) and washed three times in 1.5 ml lysis buffer. The precipitate was examined for the presence of p21 and caspase-3 by Western blot analysis as described above. Goat HRP-conjugated secondary Ab specific for mouse IgG1 (Southern Biotechnologies, Birmingham, AL, USA) was used for the detection of p21. HRP-conjugated Protein G (Sigma-Aldrich Chemie BV) was used for detection of the goat caspase-3 Ab.

Statistics

Statistical analysis was performed using the Student's t-test. P-values 0.05 were considered to be significant.

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Acknowledgements

This study was supported by a grant from the Therapeutic Proteins for Chronic Diseases program of the University of Groningen, and by Grant RUG 99-1880 from the Dutch Cancer Society.