The effect of differentiating doses of all-trans retinoic acid (ATRA, 10-6 M) and vitamin D3 (10-7 M) was investigated on the nuclear levels of endogenous ceramide and protein kinase C-ζ (PKC-ζ) catalytic activity in HL-60 myeloid cells. ATRA induced a parallel increase of ceramide and catalytically active PKC-ζ into the nuclear compartment of HL-60 cells (peak at 72 h). On the other hand, vitamin D3 increased the levels of nuclear ceramide and PKC-ζ activity to a lesser extent and with a delayed kinetics compared to ATRA (peak at 96 h). Pretreatment of HL-60 cells with high pharmacological concentrations of exogenously-added C2-ceramide (10-6 M) completely blocked the ATRA-mediated activation of nuclear PKC-ζ. Exogenous C2-ceramide (10-6 M) also inhibited the granulocytic differentiation induced by ATRA, whereas it did not affect monocytic differentiation mediated by vitamin D3. Transient transfection experiments performed with a plasmid construct containing a constitutively active mutated form of the PKC-ζ cDNA fused in 3' to a fluorescent tag protein (pEGFP-PKC-ζ) demonstrated that the overexpression of catalytically active PKC-ζ was not accompanied by the appearance of a differentiated morphology. These findings suggest that nuclear PKC-ζ is necessary but not sufficient to induce granulocytic differentiation of HL-60 myeloid malignant cells.
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