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. 2004 Jan 8;23(1):39-48.
doi: 10.1038/sj.onc.1206978.

Rapid induction of mitochondrial events and caspase-independent apoptosis in Survivin-targeted melanoma cells

Tong Liu  1 , Brook Brouha, Douglas Grossman
Affiliations

Rapid induction of mitochondrial events and caspase-independent apoptosis in Survivin-targeted melanoma cells

Tong Liu et al. Oncogene. .

Abstract

The inhibitor of apoptosis (IAP) protein Survivin is expressed in most cancers and is a key factor in maintaining apoptosis resistance. Although several IAPs have been shown to act as direct inhibitors of caspases, the precise antiapoptotic function of Survivin remains controversial. To clarify the mechanism by which Survivin protects cells, we investigated the kinetics of apoptosis and apoptotic events following Survivin inhibition utilizing a melanoma cell line harboring a tetracycline-regulated Survivin dominant-negative mutant (Survivin-T34A). Blocking Survivin resulted in both caspase activation and apoptosis; however, the level of apoptosis was only partially reduced by caspase inhibition. Survivin blockade also resulted in mitochondrial events that preceded caspase activation, including depolarization and release of cytochrome c and Smac/DIABLO. Levels of other IAPs were not altered in Survivin-targeted cells, although modest cleavage of XIAP and Livin was observed. The earliest proapoptotic event observed in Survivin-targeted cells was nuclear translocation of mitochondrial apoptosis-inducing factor (AIF), known to trigger both apoptotic mitochondrial events and caspase-independent DNA fragmentation. These findings suggest that a key antiapoptotic function of Survivin relates to inhibition of mitochondrial and AIF-dependent apoptotic pathways, and its expression in melanoma and other cancers likely protects against both caspase-independent and -dependent apoptosis.

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Figures

Figure 1
Figure 1
Tet-regulated induction of Survivin-T34A expression and apoptosis. Melanoma cells stably transfected with tet-Survivin-T34A (T34A-Sur) or tet-wild-type Survivin (WT-Sur) were incubated for the indicated times either in the presence or absence of tet. In this system, transcription is induced in the absence of tet. (a) Lysates (25 μg) were subjected to Western blotting with antibodies for Survivin (that recognize endogenous and both mutant and wild-type tet-regulated Survivin) or β-actin. Expression of the respective transgenes can be appreciated relative to the expression of endogenous Survivin seen in cells cultured with tet. (b) T34A-Sur (left panel) and WT-Sur (right panel) cells were cultured in the presence (filled circles) or absence (open circles) of tet as indicated. Cells were fixed and permeabilized, and apoptotic cell fractions were determined by propidium iodide staining and flow cytometry. The data shown are representative of four experiments performed. (c) Annexin V staining of T34A-Sur (open circles) and WT-Sur (filled circles) cells cultured in the absence of tet as indicated. Cells were stained with Annexin V-FITC, and positive cells quantitated by flow cytometry. Error bars reflect s.e.m. from two experiments
Figure 2
Figure 2
Caspase activation in Survivin-targeted cells. (a) Lysates (100 μg) from T34A-Sur cells cultured in the absence of tet for the indicated times were blotted for caspase-3 and -9. Markers indicate caspase-3 precursor (32 kDa) and cleavage fragments (20, 17 kDa), and caspase-9 precursor (45 kDa) and cleavage fragment (37 kDa). (b) Lysates (100 μg) from HeLa cells cultured in the absence or presence of TNF-α and cycloheximide, and from T34A-Sur cells cultured in the absence of tet for the indicated times were blotted for caspase-8 and Bid. Markers indicate caspase-8 precursor (55 kDa) and cleavage fragment (35 kDa), and Bid 23 kDa precursor. Additional caspase-8 cleavage fragment (20 kDa) is not shown. Cleaved fragment (tBid) is unstable and not visualized
Figure 3
Figure 3
Caspase-independent apoptosis in Survivin-targeted cells. (a) T34A-Sur cells were incubated in the absence (open circles) or presence of tet after exposure to 1200 J/cm2 UVB (filled circles). HeLa cells were treated with TNF-α and cycloheximide (CHX) (open squares). Cells were cultured in increasing concentrations of z-VAD-fmk as indicated, and after 48 h cells were collected and percent apoptotic cells was determined as in Figure 1. The data shown are representative of two experiments performed. (b) T34A-Sur cells were incubated in the presence or absence of tet and 20 μm z-VAD-fmk as indicated, and 48 h later, cell lysates were analysed for caspase activity. Error bars reflect s.e.m. from three measurements. (c) T34A-Sur cells were incubated in the presence or absence of tet, and with 100 μm pan-caspase inhibitor z-VAD-fmk or inhibitors of caspase-8 (z-IETD) or caspase-9 (z-LEHD) as indicated. After 48 h, cells were collected and percent apoptotic cells was determined as in Figure 1. Error bars reflect s.e.m. from two experiments
Figure 4
Figure 4
Survivin targeting does not significantly affect the levels of other apoptotic regulators. Lysates (25 μg) were prepared from T34A-Sur cells cultured in the absence of tet for the indicated times. Western blotting was performed for Bcl-2, Mcl-1, Bcl-XL, cIAP-1, cIAP-2, XIAP, Livin, p53, Apaf-1, Bax, and Actin as shown. Markers indicate size of proteins and cleavage fragments
Figure 5
Figure 5
Survivin targeting causes mitochondrial depolarization. T34A-Sur cells were cultured over a 24-h period in the presence or absence of tet, and stained with JC-1. Histograms of tet-deprived cells (dotted line) in each panel are overlaid onto the control histogram (cells cultured in the presence of tet, solid line). Shift to the left indicates decreased red fluorescence and loss in mitochondrial transmembrane potential. The geometric mean fluorescence values of tet-deprived cells are indicated in parentheses
Figure 6
Figure 6
Mitochondrial content release in Survivin-targeted cells. T34A-Sur cells were cultured in the absence of tet, and the presence or absence of 80 μm z-VAD-fmk, for the indicated times. Cells were then fractionated into mitochondrial (M) and cytosolic (C) components, electrophoresed and blotted for cytochrome c or Smac/DIABLO as indicated. Blots for VDAC and β-actin confirm equivalent loading among mitochondrial and cytosolic lysates, respectively. Amount loaded per lane of mitochondrial and cytosolic lysates was 20 and 50 μg, respectively
Figure 7
Figure 7
Mitochondrial release of AIF in Survivin-targeted cells. (a) T34A-Sur and WT-Sur cells were cultured in the absence of tet for the indicated times. Mitochondrial (M, 20 μg) and cytosolic (C, 50 μg) lysates were electrophoresed and blotted for AIF. Blots for VDAC and β-actin confirm equivalent loading among mitochondrial and cytosolic lysates, respectively. (b) T34A-Sur cells were cultured for 24 h in the presence of 2 mm thymidine, washed, then cultured in the absence of tet for the indicated times. Mitochondrial (M, 20 μg) and cytosolic (C, 50 μg) lysates were electrophoresed and blotted for AIF, caspase-3, and actin as indicated
Figure 8
Figure 8
Survivin targeting triggers rapid nuclear translocation of AIF. T34A-Sur cells were cultured in the absence of tet for 0, 4, or 24 h as indicated. Cells were stained with anti-AIF (red), Mitotracker (green), and DAPI (blue), and images were overlaid as shown. Yellow color indicates colocalization of AIF and mitochondria. Purple color indicates colocalization of AIF and nucleus
Figure 9
Figure 9
Diagram depicting antagonism of Survivin and AIF pathways. Survivin restrains AIF in mitochondria. Targeting Survivin causes release of AIF, which translocates to the nucleus and mediates caspase-independent apoptosis. AIF also triggers the conventional mitochondrial apoptotic pathway, characterized by mitochondrial release of cytochrome c and Smac/DIABLO that lead to caspase activation
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