Erythroid Differentiation Sensitizes K562 Leukemia Cells to TRAIL-Induced Apoptosis by Downregulation of c-FLIP

Abstract

Regulation of the apoptotic threshold is of great importance in the homeostasis of both differentiating and fully developed organ systems. Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells, in which the apoptotic threshold is often characteristically elevated. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied death receptor responses during erythroid differentiation of K562 erythroleukemia cells, which normally are highly resistant to tumor necrosis factor (TNF) alpha-, FasL-, and TRAIL-induced apoptosis. However, upon hemin-mediated erythroid differentiation, K562 cells specifically lost their resistance to TNF-related apoptosis-inducing ligand (TRAIL), which efficiently killed the differentiating cells independently of mitochondrial apoptotic signaling. Concomitantly with the increased sensitivity, the expression of both c-FLIP splicing variants, c-FLIP_L and c-FLIP_S, was downregulated, resulting in an altered caspase 8 recruitment and cleavage in the death-inducing signaling complex (DISC). Stable overexpression of both c-FLIP_L and c-FLIP_S rescued the cells from TRAIL-mediated apoptosis with isoform-specific effects on DISC-recruited caspase 8. Our results show that c-FLIP_L and c-FLIP_S potently control TRAIL responses, both by distinct regulatory features, and further imply that the differentiation state of malignant cells determines their sensitivity to death receptor signals.

ACKNOWLEDGMENTS

The first two authors contributed equally to this work.

We are grateful to Peter Krammer for caspase 8 and c-FLIP antibodies and Jürg Tschopp for c-FLIP constructs. We also thank the members of our laboratories for technical help and constructive criticism during the course of this study and Mika Korkeamäki for help with flow cytometry.

Financial support was obtained from the Academy of Finland, the Sigrid Juselius Foundation, and the Finnish Cancer Organizations. V.H. and M.P. were supported by the Turku Graduate School of Biomedical Sciences.