TRAIL initiates activation of Caspase-8, which is blocked by the FLICE inhibitory protein (FLIP), resulting in resistance to apoptosis. Here we show that overexpression of FLIPL and FLIPS in ML1 cells, with low constitutive levels of FLIP, protects these cells against apoptosis induced by TRAIL, not only via Caspase-8 inhibition, but also via upregulation of anti-apoptotic molecules.

Methods: 1) Apoptosis was determined by Annexin V/ 7-AAD following treatment with TRAIL (100–500ng/ml), or TNFa (20–100ng/ml) in ML1 cells transduced with FLIPL.GFP (green fluorescent protein), FLIPS.GFP or Neo.GFP (control). 2) Caspase-8, Caspase-3, Bid, Bcl-xL, XIAP, phosphorylated (P)-IKBa and P-Akt were determined by western blots. 3) Active Caspase-3 was determined using EnzChek Caspase-3 assay kit.

Results: Both FLIPL and FLIPS transduction protected ML1 cells against apoptosis induced by TRAIL (300ng/ml), while no protection was observed in Neo.GFP cells. FLIPL had a more profound protective effect than FLIPS (Fig.1A). Both FLIPL and FLIPS, but not Neo.GFP, blocked Caspase-8 and Caspase-3 activation (Fig.1B); FLIPS cells showed two-fold higher levels of active Caspase-3 than FLIPL cells, consistent with higher apoptosis in FLIPS cells. Caspase-3 can be activated through Caspase-8 (extrinsic pathway) or via Caspase-8/Bid (intrinsic pathway). The latter was responsible for high active Caspase-3 levels in FLIPS cells as shown by the presence of cleaved Bid (t-Bid) (Fig.1B); cleavage of Bid was inhibited by combination of TRAIL and Z-IETD-FMK (Caspase-8 inhibitor). Anti-apoptotic molecules, including Bcl-xL, XIAP and FLIP are regulated by NF-kB and FLIP participates in an NF-kB auto-amplification loop. While Neo.GFP cells showed little Bcl-xL after 4h of TRAIL exposure and there was a twofold reduction in FLIPS cells, only a slight reduction of Bcl-xL was noted in FLIPL cells. FLIPL cells showed the lowest rates of apoptosis when exposed to TNFa and BMS543541, a specific inhibitor of IkB kinase (Fig. 1C). In the presence of BMS543541, phosphorylation of IkBa and levels of Bcl-xL and XIAP decreased in Neo.GFP and FLIPS but not in FLIPL cells. Additional data suggest that the PI3-kinase/Akt pathway is involved in constitutive NF-kB activation and differentially affected by FLIPL and FLIPS (Fig. 1D). Preliminary results in immunodeficient mice transplanted with transduced ML1 cells indicated the in vivo relevance of the differences between FLIPL and FLIPS with FLIPL cells engrafting earlier and showing earlier signs of sickness.

Conclusions: FLIPL and FLIPS conferred resistance to TRAIL induced apoptosis but showed differential effects: Caspase-8/Bid was involved in the apoptosis pathway in FLIPS, but not in FLIPL cells. FLIPL cells’ resistance was due not only to caspase inhibition but to the recruitment of downstream anti-apoptotic pathways such as NF-kB and PI3K/Akt. In vivo data further substantiated the antiapoptotic/pro-survival behavior of FLIPL cells.

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