Differential responses of FLIP_Long and FLIP_Short-overexpressing human myeloid leukemia cells to TNF-α and TRAIL-initiated apoptotic signals

Objective

Clonal marrow cells from patients with early myelodysplastic syndrome (MDS) undergo apoptosis in response to tumor necrosis factor (TNF)−related apoptosis-inducing ligand (TRAIL). Cells from advanced MDS are resistant to TRAIL. Two isoforms of the Flice inhibitory protein (FLIP) short (FLIP_S) and FLIP long (FLIP_L), which modulate TRAIL signals, showed disease-stage−dependent differential regulation. Therefore, we aimed at characterizing potential differential effects of FLIP_L and FLIP_S, on TRAIL and TNF-α−induced apoptosis in model leukemic cell lines.

Materials and Methods

Using lentiviral constructs, FLIP_L and FLIP_S, as well as a green fluorescent protein control were overexpressed in ML-1 cells, which constitutively express very low levels of FLIP and are highly sensitive to apoptosis induction. Cells were then exposed to TRAIL or TNF-α, and effects on the extrinsic and intrinsic pathways of apoptosis induction were assessed.

Results

Overexpression of FLIP reduced TRAIL and TNF-α−induced apoptosis in ML-1 cells. However, while FLIP_L completely abrogated apoptosis, FLIP_S allowed for BID cleavage and caspase-3 activation. Concurrently, there was a decline of Bcl-xL and X-linked inhibitor of apoptosis protein (XIAP) in FLIP_S cells followed by apoptosis. Further, inhibition of nuclear factor-κB (NF-κB) activation in TNF-α−treated cells resulted in profound apoptosis in FLIP_S, but not in FLIP_L-overexpressing cells, consistent with the observations in patients with early stage MDS. Inhibition of NF-κB had only minimal effects on TRAIL signaling.

Conclusion

Thus, FLIP_L and FLIP_S exerted differential effects in myeloid leukemic cell lines in response to TRAIL and TNF-α. It might be possible to therapeutically exploit those differences with effector molecules specific for the FLIP isoforms.