Cisplatin induces the proapoptotic conformation of Bak in a deltaMEKK1-dependent manner

Abstract

In a panel of four human melanoma cell lines, equitoxic doses of cisplatin induced the proapoptotic conformation of the Bcl-2 family protein Bak prior to the execution phase of apoptosis. Because cisplatin-induced modulation of the related Bax protein was seen in only one cell line, a degree of specificity in the signal to Bak is indicated. Little is known about upstream regulation of Bak activity. In this study, we examined whether the apoptosis-specific pathway mediated by a kinase fragment of MEKK1 (DeltaMEKK1) is involved in the observed Bak modulation. We report that expression of a kinase-inactive fragment of MEKK1 (dominant negative MEKK [dnMEKK]) efficiently blocked cisplatin-induced modulation of Bak and cytochrome c release and consequently also reduced DEVDase activation and nuclear fragmentation. Accordingly, expression of a kinase-active MEKK1 fragment (dominant positive MEKK) was sufficient to induce modulation of Bak in three cell lines and to induce apoptosis in two of these. dnMEKK did not block cisplatin-induced c-Jun N-terminal kinase (JNK) activation, in agreement with a specifically proapoptotic role for the DeltaMEKK1 pathway. Finally, we show that reduction of Bak expression by antisense Bak reduced cisplatin-induced loss of mitochondrial integrity and caspase cleavage activity in breast cancer cell lines. In summary, we have identified Bak as a cisplatin-regulated component downstream in a proapoptotic, JNK-independent DeltaMEKK1 pathway.

Figures

FIG. 1

Cisplatin-induced modulation of Bak but not Bax. Human melanoma 224 cells were treated with cisplatin (20 μM, 16 h) or staurosporine (1 μM, 5 h), as indicated, before samples were prepared for FACS analysis of Bak or Bax-associated IFL. Control cells, gray peak; treated cells, black line. (A) Bak-associated IFL after cisplatin treatment; (B) Bax-associated IFL after cisplatin treatment; (C) Bax-associated IFL after staurosporine treatment; (D) Western blot showing Bak protein levels in control cells and after 16 h of cisplatin (CISPL) treatment. Tubulin (β-TUB) was used as an internal loading control. Ig2a, immunoglobulin 2a.

FIG. 2

Effect of dnMEKK on cisplatin-induced nuclear fragmentation. Cells were treated with 20 μM cisplatin in the presence or absence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. (A) Nuclear fragmentation in 224 cells after 16 and 24 h of cisplatin treatment; (B) Western blot showing expression of dnMEKK (37 kDa) in the same samples. A faint endogenous band is seen also in the extracts from cells which were not infected and is thus not due to leakage. The experiment was repeated with similar results.

FIG. 3

dnMEKK blocks upstream of cytochrome c release without blocking JNK activation. 224 cells were treated with 20 μM cisplatin or 15 μg of BA per ml in the presence or absence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before drug treatment. (A) Western blot for cytochrome c (CYT. C) in cytosolic extracts at 20 h after addition of cisplatin (CISPL). The membrane was also probed with an antibody against mitochondrial cytochrome oxidase subunit IV to ensure lack of mitochondrial contamination of the extracts (not shown). β-TUB., tubulin. (B) BA (BET. ACID)-induced DEVDase activity measured in extracts (50 μg) of cells at the indicated time points. Results are shown as fold activation relative to control cells. (C) Cisplatin-induced activation of JNK1 and -2, as seen by phosphorylation at the indicated time points in the presence or absence of dnMEKK, was assessed by Western blotting using an antibody specific for phosphorylated JNK1 and -2 (P-JNK1 and -2). Tubulin was used as an internal loading control.

FIG. 4

Delayed induction of dnMEKK expression blocks nuclear fragmentation. (A) Western blot showing 37-kDa dnMEKK expression at different time points after DOX addition. (B) 224 cells infected with adeno-dnMEKK were induced to express dnMEKK by addition of DOX at the indicated time points, which are relative to the time of cisplatin addition (20 μM) at 0 h. Nuclear fragmentation levels were assessed at 24 h and compared to fragmentation induced in the absence of adeno-dnMEKK.

FIG. 5

dnMEKK blocks cisplatin-induced DEVDase activity. 224 cells were treated with 20 μM cisplatin for the indicated time periods, in the presence or absence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. After harvest, cells were aliquoted for apoptosis and DEVDase assays and for western blotting. Apoptosis levels, seen as nuclear fragmentation, are shown above the relevant bars. Filled bars, DEVDase activity against a synthetic substrate (Ac-DEVD-AMC), measured in cell extracts at the indicated time points. Results are shown as induction of activity relative to that in control samples. Empty bars, cleavage of DNA-PK, as assessed by densitometric scanning of the 160-kDa proteolytic fragment on Western blot films. Results are shown as fragment levels relative to that in control samples.

FIG. 6

Kinetics of cisplatin-induced DEVDase and caspase 9/ LEHDase activities. 224 cells were treated with 20 μM cisplatin for the indicated time periods in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Empty bars, cisplatin only; filled bars, cisplatin treatment in the presence of dnMEKK. (A) DEVDase activity on the synthetic substrate Ac-DEVD-AMC was assessed twice for each duplicate sample. The inhibitor DEVD-CHO reduced all activity to background in all parallel samples. Results are shown as fold increase in activity in untreated (control [C]) cells. (B) Caspase 9/LEHDase activity on the synthetic substrate Ac-LEHD-AMC. The inhibitor LEHD-CHO reduced all activity to background in all parallel samples. (C) Cell extracts (30 μg/lane) were analyzed by Western blotting of caspase 9. The 35-kDa cleavage fragment represents the active form. β-TUB., tubulin; CISPL, cisplatin.

FIG. 7

dnMEKK blocks cisplatin-induced modulation of Bak. (A) 224 cells were treated with 20 μM cisplatin for 16 h in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Shown are overlaid FACS graphs indicating Bak-associated IFL. Gray peak, control cells; dark lines, cisplatin treatment in the presence or absence of dnMEKK, as indicated. (B) Quantitation of cisplatin-induced Bak modulation in the presence or absence of dnMEKK in 224 and AA cell lines. Cells were treated with LD50s of cisplatin (CISPL.). Results are shown as fold increase in median IFL signal relative to controls.

FIG. 8

Induction of Bak modulation and apoptosis by dpMEKK. (A) Cells were infected with adeno-dpMEKK, and Bak-associated IFL was assessed at 24 h after addition of DOX. For comparison, Bak modulation at 16 h induced by LD50s of cisplatin (CISPL) is also shown. Results are shown as fold increase in median IFL signal compared to controls. (B) Cells were infected with adeno-dpMEKK, and nuclear fragmentation was assessed at 24 h after addition of DOX. Background levels in control cells varied but did not exceed 6%.

FIG. 9

Induction of Bax modulation. Bax-associated IFL was assessed at 16 h after cisplatin (CISPL) addition or at 24 h after addition of DOX to induce dpMEKK expression in adeno-dpMEKK-infected cells. The effect of staurosporine (STSN; 1 μM, 5 h) on Bax modulation in 224, AA, and FM55 cells is also shown.

FIG. 10

Reduced apoptotic responses in cells expressing antisense Bak. Parental MCF-7 and two subclones with stable expression of antisense Bak were treated with 20 μM cisplatin for 21 h. (A) Caspase activity was assessed by ELISA-based quantitation of the caspase-cleaved fragment of cytokeratin 18 in cell lysates as instructed by the manufacturer (Apoptosense ELISA kit; Peviva AB). Results are shown as fold increase in the levels of fragment specifically recognized by the antibody M30. (B) The mitochondrial transmembrane potential was determined in duplicate samples as retention of tetramethylrhodamine ethyl ester (TMRE; Molecular Probes Inc.), a cationic, lipophilic fluorochrome dye that accumulates in the negatively charged mitochondrial matrix. Depolarization of mitochondria, as seen during apoptosis, is quantitated as loss of TMRE fluorescent signal assessed by flow cytometry. Results are shown as fold increase in the fraction of cells with depolarized mitochondria, i.e., cells showing a median TMRE signal less than 5% of the median signal in controls. AS #8 and AS #9 are clones stably expressing antisense Bak.