Characterization of caspase proteases in cytokine-dependent myeloid progenitor cells using enzyme affinity labeling

Bone marrow-derived myeloid progenitor cells are dependent on the presence of cytokines such as interleukin-3 (IL-3) for their survival. The withdrawa l of IL-3 from IL-3-dependent myeloid progenitors results in death via an a poptotic program. Previous studies have shown that IL-3 withdrawal induces the activities of caspase proteases. However, the molecular identities of m yeloid progenitor caspases have not been determined. In this study, we used an affinity labeling reagent (biotin-YVAD-acyloxymethyl ketone) that binds to processed active caspase subunits, to study caspase activation in 32D a nd FDCP-1 myeloid progenitor cells. After IL-3 withdrawal, we detected affi nity labeling of caspase subunits of 20, 17, and 16 kDa in both cell lines. Surprisingly, affinity labeling of the 20- and 17-kDa proteins, but not th e 16-kDa protein, was also detected in healthy cells maintained in the pres ence of IL-3. By contrast, in cytokine-independent cell lines, affinity lab eling of caspase subunits was detected only after treatment with an apoptot ic stimulus. Immunoblotting experiments showed that caspase-3 constitutes a t least a portion of the 20- and 17-kDa affinity-labeled proteins detected in the myeloid progenitor cell lines. Taken together, these data provide di rect evidence of caspase activation in cytokine-dependent myeloid progenito rs, and suggest that unique apoptotic pathways may exist in these cells. J. Cell. Biochem. 73:79-89, 1999. (C) 1999 Wiley-Liss, Inc.