tLivin displays flexibility by promoting alternative cell death mechanisms

Abstract

Livin is a member of the Inhibitor of Apoptosis (IAP) protein family that inhibits apoptosis triggered by a variety of stimuli. We previously demonstrated that while Livin inhibits caspase activity, caspases can cleave Livin to produce a truncated protein, tLivin and that this newly formed tLivin paradoxically induces cell death. However to date, the mechanism of tLivin-induced cell death is not fully understood. In this study, we set out to characterize the form of cell death mediated by tLivin. Here we demonstrate that, unlike most death-promoting proteins, tLivin is a flexible inducer of cell death capable of promoting necrosis or apoptosis in different cell lines. The unusual flexibility of tLivin is displayed by its ability to activate an alternative form of cell death when apoptosis is inhibited. Thus, tLivin can promote more than one form of cell death in the same cell type. Interestingly, in cells where tLivin induces necrosis, deletion of the caspase binding BIR domain results in tLivin-induced apoptosis, suggesting the BIR domain can potentially hamper the ability of tLivin to induce apoptosis. We further elucidate that tLivin activates the JNK pathway and both tLivin-induced apoptosis and necrosis are partially mediated by JNK activity. Acquired resistance to apoptosis, common in many tumors, impinges on the efficiency of conventional anti-cancer agents that function primarily by inducing apoptosis. The ability of tLivin to induce death of apoptosis-compromised cells makes it an attractive candidate for targeted cancer therapy.

Figure 1. tLivin induces caspase-independent necrotic cell death of 293T cells.

293T cells were transfected with an empty vector (ev) or with vectors expressing either tBid, tLivin or tLivinΔBIR and harvested at the indicated time points post-transfection. Con, untransfected cells; (A) Cells were stained with propidium iodide (PI) and the percent of cell death was measured by flow cytometry. (B) Cells were analyzed by western blot for cleavage of caspases and PARP. (C) zVAD-fmk was added 1 h before transfection. Cells were harvested 24 h post-transfection and stained with PI. The percent of cell death was measured by flow cytometry. *p<0.05. (D) Cells were analyzed for DNA content by flow cytometry (top). Representative cell cycle histograms of tBID- and tLivin-transfected cells (bottom). (E) Cells were examined by transmission electron microscopy 18 h post-transfection with empty vector (I) or a tLivin-expressing vector (II, III). Cells were incubated with 10 mg/ml sodium azide for 4 h (IV) or 30 µg/ml etoposide for 24 h (V). Features of necrosis: cytoplasmic vacuolation (white arrow) (II, III, IV) and rounded mitochondria with disrupted internal structures (black arrows) (III, IV) are evident in cells transfected with tLivin and in cells treated with sodium azide. Apoptotic cells (treated with etoposide) exhibited blebbing of the plasma membrane (black arrow) (V). (F) Cells were analyzed for DNA content by flow cytometry. (G) zVAD-fmk at 20 µM was added 1 h before transfection and cells were harvested 24 h post-transfection. Each sample was divided into three aliquots and analyzed for cleavage of caspase 3 by western blot and for the percent of PI positive cells and DNA content by flow cytometry. ND, Not determined.

Figure 2. tLivin induces apoptosis of A549 cells.

Cells of A549 clones were treated with 2.5 µg/ml dox and harvested at the indicated time points. Con, untreated cells; etop, cells treated with 30 µg/ml etoposide for 48 h. (A) Cells were stained with PI and the percent of cell death was measured by flow cytometry (top). Cells were analyzed for tLivin and GAPDH protein levels by western blot (bottom). (B) Cleavage of caspase 3 and PARP was analyzed by western blot. (C) DNA content was analyzed by flow cytometry. (D) Cells were treated with 50 µM zVAD-fmk 1 h prior to addition of dox, harvested after 48 h, stained with PI and the percent of cell death was measured by flow cytometry. *p<0.05. (E) Cells were treated with 50 µM zVAD-fmk 1 h prior to addition of dox and harvested after 48 h. Each sample was divided into three aliquots and analyzed for cleavage of caspase 3 by western blot and for the percent of PI positive cells and DNA content by flow cytometry.

Figure 3. tLivin induces apoptosis of MelA1 cells.

Cells of MelA1 clones were treated with 2.5 µg/ml dox and harvested at the indicated time points. Con, untreated cells; sts, cells treated with 0.5 µM staurosporine for 10 h. (A) Cells were stained with PI and the percent of cell death was measured by flow cytometry (top). Cells were analyzed for tLivin and GAPDH protein levels by western blot (bottom). (B) Cleavage of caspase 3 and PARP was analyzed by western blot. (C) DNA content was analyzed by flow cytometry.

Figure 4. tLivin activates an alternative form of cell death when apoptosis is inhibited.

Cells of MelA1 clones were treated with 75 µM zVAD-fmk 1 h prior to addition of 2.5 µg/ml dox or 30 µM cisplatin and harvested after 36 h along with untreated cells (con). (A) Cells were stained with PI and the percent of cell death was measured by flow cytometry. *p<0.05. (B) Cells from each sample were divided into three aliquots and analyzed for cleavage of caspase 3 by western blot and for the percent of PI positive cells and DNA content by flow cytometry.

Figure 5. tLivin activates the JNK pathway.

(A) 293T cells were either transiently transfected with empty vector (ev), tBID- or tLivin-expressing vectors, treated with 30 µg/ml etoposide for 24 h (etop) or left untreated (con). Cells were harvested at the indicated time points post-transfection and the phosphorylation of JNK, ATF-2 and p38MAPK was analyzed by western blot. (B) Cells of MelA1 clones were harvested at the indicated time points following administration of 2.5 µg/ml dox or following 30 min treatment with 200 nM anisomycin (A), control cells (con) were left untreated. Phosphorylation of JNK and c-JUN was analyzed by western blot. (C) 293T cells were either transiently transfected with empty vector (ev), tLivin- or tLivinΔBIR-expressing vectors, treated with 200 nM anisomycin for 30 min (A) or left untreated (con). Cells were harvested at the indicated time points post-transfection and phosphorylation of JNK and c-JUN was analyzed by western blot.

Figure 6. tLivin-induced apoptosis and necrosis are partially mediated by activation of JNK.

(A) Cells of MelA1 clones were treated with 10 µM SP600125 1 h prior to administration of 2.5 µg/ml dox (where indicated) and harvested after 36 h along with untreated cells (con). Cells were stained with PI and the percent of cell death was measured by flow cytometry. *p<0.05. (B) 293T cells were transiently transfected with the indicated plasmids and harvested 24 h post-transfection along with untransfected cells (con). Cells were stained with PI and the percent of cell death was measured by flow cytometry. (C) 293T cells were transiently transfected with the indicated plasmids, harvested 24 h post-transfection and analyzed by western blot for the protein levels of p-c-JUN, c-JUN, JIP-1, tLivin and GAPDH.