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Low p21Waf1/Cip1 protein level sensitizes testicular germ cell tumor cells to Fas-mediated apoptosis
Diana C J Spierings, Elisabeth G E de Vries, Alja J Stel, Nelina te Rietstap, Edo Vellenga and Steven de Jong
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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
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
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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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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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 mean
s.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)
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, cFlipL, Bcl-2, Bcl-XL, Bax, XIAP and p21. A representative example of at least two independent experiments is shown
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
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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