Expression of p53, Bcl-2 and Bax in cisplatin-induced apoptosis in testicular germ cell tumour cell lines.

H Burger,G Stoter,Cj Kortland,Awm Boersma,K Nooter

doi:10.1038/bjc.1998.257

Abstract

We examined the sensitivity for cisplatin-induced apoptosis in a panel of four testicular germ cell tumour (TGCT) cell lines and monitored the cellular expression of the apoptosis-related proteins p53, Bcl-2 and Bax. Three of four TGCT cell lines (NT2, NCCIT and S2) were hypersensitive for cisplatin-induced apoptosis, while the TGCT cell line 2102 EP appeared to be resistant for cisplatin-induced apoptosis, even at relatively high drug concentrations (12.5 microM). For all four cell lines, the induction of apoptosis by cisplatin correlated with drug sensitivity in the MTT assay. The differences in chemosensitivity and induction of apoptosis could not be attributed to differences in cellular platinum accumulation, DNA platination or platinum-DNA adduct removal. We next analysed the relationship between p53 status and cisplatin-induced up-regulation of p53, and the susceptibility to cisplatin-induced apoptosis. Wild-type p53 containing NT2 and 2102 EP cells showed p53 up-regulation upon drug treatment, and NCCIT (mutant p53) and S2 (no p53 protein) cells did not. Consistently, the increase in wild-type p53 protein in NT2 and 2102 EP cells led to an increase in mRNA level of the p53 downstream gene p21/WAF/CIP, whereas mutant p53-containing NCCIT cells and p53-non-expressing S2 cells could not transactivate this p53-responsive gene. As NT2, NCCIT and S2 were readily triggered into apoptosis, while 2102 EP cells failed to undergo cisplatin-induced apoptosis, our data suggest that the presence of wild-type and/or transactivation-competent p53 might not be an absolute prerequisite for efficient induction of apoptosis in TGCT cell lines. Also endogenous levels of Bcl-2 and Bax expression did not correlate with cisplatin-induced apoptosis. In addition, the endogenous Bcl-2 and Bax expression was not affected by cisplatin treatment. The present study suggests that, at least in our panel of TGCT cell lines, hypersensitivity for cisplatin-induced apoptosis might not be necessarily correlated with the presence of wild-type p53 and is probably not associated with Bcl-2 and Bax expression.

Highlights

Was determined at t = 48 h after cisplatin treatment (12.5 gM, 2 h) by Western blot analysis with C-2-1 0, a Poly(ADPribose) polymerase (PARP)-specific MAb. dThe p53 gene status of the testicular germ cell tumour (TGCT) cell lines was determined by p53 cDNA sequencing and revealed that NCCIT cells are hemizygous for p53 containing one mutated allele carrying 1 -bp deletion. aExpression of the p53 protein was monitored by Western blot analysis with DO1, a p53-specific MAb
We investigated the susceptibility of a panel of four TGCT cell lines to cisplatin-induced apoptosis and evaluated the role of p53, Bcl-2 and Bax
The cell lines were incubated for 2 h with cisplatin, and apoptosis was assayed by morphology and by proteolytic cleavage of PARP, a nuclear enzyme involved in DNA repair, which is generally regarded as an early marker of chemotherapy-induced apoptosis (Kaufmann et al, 1993)

Summary

Introduction

We investigated the expression of p53, Bcl-2 and Bax in drug-induced apoptosis in a panel of well-defined TGCT cell lines. Was determined at t = 48 h after cisplatin treatment (12.5 gM, 2 h) by Western blot analysis with C-2-1 0, a PARP-specific MAb. dThe p53 gene status of the TGCT cell lines was determined by p53 cDNA sequencing and revealed that NCCIT cells are hemizygous for p53 containing one mutated allele carrying 1 -bp deletion.

Results

Conclusion