Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation

Abstract

Testicular germ cell tumours (TGCT) represent the most common malignancies in young males. Whereas in 1970s, the survival rate in patients with metastatic testicular tumours was only 5%, these days, 80% of the patients treated by modern chemotherapy will survive their disease. The drug that revolutionised the cure rate for patients with metastatic testicular tumours was cisdiamminedichloroplatinum (cisplatin, CDDP). In vitro experiments on neoplastic germ cell lines showed that their exquisite sensitivity to CDDP could be attributed to p53-dependent and -independent pathways. Applying cDNA macroarray, semiquantitative RT-PCR and Western blot analyses, blocking experiments, caspase activity assays, and morphological methods, we sought here to define the p53-independent pathway(s) involved in the CDDP-induced apoptosis. For this purpose, we used the human TGCT cell line NCCIT, the mutated p53 of which is known to remain inactive during the course of CDDP-induced apoptosis. Our experiments showed that within hours of CDDP application, two prototype members of the 'mitogen-activated protein kinase' (MAPK) family, designated 'MAPK ERK kinase' (MEK) and 'extracellular signal-regulated kinase' (ERK), were dually phosphorylated and caspase-3 became active. Functional assays using MEK inhibitors demonstrated that the phosphorylation of MEK and ERK was required for the activation of caspase-3 as the executing caspase. Interestingly, experiments with the human malignant germ cell line NTERA, which is known to possess wild-type p53, revealed the same results. Thus, our data suggest that CDDP mediates its p53-independent apoptosis-inducing effect on the malignant human testicular germ cells--at least partially--through activation of the MEK-ERK signalling pathway. July 2004

Figure 1

Dose- and time-dependent effects of CDDP on NCCIT cells. (A) Tumour cells were treated with different CDDP concentrations. After 24 h, the apoptotic rate of tumour cells was assessed by MGG staining, immunocytochemistry for the active form of caspase-3 and ISEL for DNA fragmentation. Representative photomicrographs of active caspase-3 (black cytoplasmatic signals) and of DNA fragmentation (black nuclear signals) in untreated (control) and drug-treated NCCIT cells illustrate increased apoptosis of tumour cells treated with 50 μM CDDP when compared to the control. The diagram shows quantification of tumour cell apoptosis with MGG staining following application of different CDDP concentrations. (B) Tumour cells were treated with 50 μM CDDP. After 6, 9, 12 and 24 h, the apoptotic rate of tumour cells was assessed. Representative photomicrographs of active caspase-3 and of DNA fragmentation in untreated and CDDP-treated NCCIT cells illustrate increased apoptosis of tumour cells 24 h after drug treatment. The diagram shows quantification of tumour cell apoptosis with MGG staining in time dependency. Data presented in (A) and (B) are the mean of triplicates; similar results were obtained in three separate experiments.

Figure 2

MEK1/2 mRNA expression in untreated and CDDP-treated NCCIT cells. (A) Two RNA preparations from untreated and CDDP-treated NCCIT cells were reverse transcribed and hybridised to cDNA macroarray membrane filters. Sections from two representative autoradiographies show sustained expression of MEK1/2 in untreated (control) and CDDP-treated (50 μM) NCCIT cells 6 h after drug treatment (arrows). (B) semiquantitative real-time RT–PCR of MEK1 and MEK2 mRNA confirms that the expression level of both transcripts remains constant in CDDP-treated NCCIT cells (open arrows, left) compared to untreated ones (closed arrows, right). (C) as control, the expression level of MEK1 and MEK2 mRNA after heat-induced necrosis of the NCCIT cell line was analysed. Results demonstrate that in the course of heat-induced necrosis, the expression level of MEK mRNAs is not changed (-•-) compared to untreated ones (-▪-).

Figure 3

CDDP-induced activation of the MEK/ERK pathway in NCCIT cells. NCCIT cells were treated with 50 μM CDDP for 2 h, after which cell lysates were investigated for the expression and activation (phosphorylation) of MEK1/2 and ERK1/2 by Western blot analysis in the time course. Upper panel illustrates the expression of MEK1/2 and its activation (phosphorylation) following CDDP treatment. Lower panel demonstrates results from Western blots of ERK1/2 and its activated (phosphorylated) form pERK1/2 in the time course after CDDP treatment. Note that MEK1 and ERK2 are more strongly phosphorylated than MEK2 and ERK1. Expression of actin was used to control equal protein loading.

Figure 4

Blocking of CDDP-induced apoptosis by MEK inhibitors. To investigate whether phosphorylation of ERK1/2 is required for the CDDP-induced apoptosis of NCCIT cells, two different MEK inhibitors, U0126 and PD98059, were applied in addition to 50 μM CDDP. (A) Western blot analysis with an anti-phospho-ERK1/2 antibody shows the inhibition of ERK1/2 activation by MEK1/2 inhibitors (U0126, 10 and 30 μM; PD98059, 30 and 50 μM). (B) The diagram shows quantification of tumour cell apoptosis with MGG staining dependent on ERK blockage by different concentration of MEK inhibitors after 24 h. Data are the mean of triplicates; similar results were obtained in three separate experiments. (C) Flow cytometric DNA analysis of NCCIT cells treated with the MEK inhibitor U0126 (50 μM) demonstrates that following drug treatment, the cell cycle of NCCIT cells is not changed.

Figure 5

MEK–ERK activation results in caspase activity. (A) NCCIT cells were treated with CDDP (50 μM) in the presence or absence of the MEK inhibitor U0126 (30 μM) as described in Materials and methods. After cell lysis, the activity of caspase-3, -8 and -9 was determined using specific fluorogenic substrates: Ac-DEVD-MCA (for caspase-3), Ac-IETD-MCA (for caspase-8) and Ac-LEHD-MCA (for caspase-9). The data are expressed as the increase in fluorescence as a function of time. Note that the Y-axis ranges from 0 to 20 for caspase-3, from 0 to 2 for caspase-8 and from 0 to 1.5 for caspase-9. (B) NCCIT cells were treated with CDDP (50 μM) in the presence or absence of Ac-DEVD-CHO as caspase-3 inhibitor, or Ac-IETD-CHO as caspase-8 inhibitor or Z-LEHD-FMK as caspase-9 inhibitor. Diagrams show quantification of tumour cell apoptosis with MGG staining in time dependency. (C) NCCIT cells were treated with CDDP (50 μM) in the presence or absence of Ac-IETD-CHO as caspase-8 inhibitor or Z-LEHD-FMK as caspase-9 inhibitor. After cell lysis, the activity of caspase-3 was determined. Results show the increase in fluorescence as a function of time. Data presented in (A–C) are the mean of triplicates; similar results were obtained in three separate experiments.

Figure 6

Selectivity of CDDP-induced apoptosis. NTERA cells were treated with 50 μM CDDP in the presence or absence of the MEK inhibitors U0126 (30 μM) or PD98059 (50 μM), as described in Material and methods. (A) After 24 h, tumour cells were lysed and investigated for the expression and activation (phosphorylation) of ERK1/2 by Western blot analysis. (B) the diagram shows quantification of tumour cell apoptosis with MGG staining dependent on ERK blockage by MEK inhibitors after 24 h. Data are the mean of triplicates; similar results were obtained in three separate experiments.

Figure 7

Specificity of CDDP-induced apoptosis. (A) NCCIT cells were treated with CDDP (50 μM), vinblastin (30 μM) or H₂O₂ (1200 μM). After 24 h, the apoptotic rate of tumour cells was assessed by immunocytochemistry for the active form of caspase-3 and MGG staining. Representative photomicrographs of caspase-3 (black cytoplasmatic signals) in NCCIT cells illustrate increased caspase activity in tumour cells following treatment. The diagram shows quantification of tumour cell apoptosis with MGG staining. Data are the mean of triplicates; similar results were obtained in three separate experiments. A fraction of tumour cells was lysed and investigated for the expression and activation (phosphorylation) of ERK1/2 by Western blot analysis. (B) To examine the role of the MEK–ERK signalling pathway in the course of necrosis, NCCIT cells underwent heat (45°C up to 60 min) or mechanical stress (multiple pipetting). Results demonstrate that stress stimuli leading to cell death without primary DNA damage (heat- or mechanically induced necrosis) also elicit ERK phosphorylation.