ACCUMULATION OF CATALYTICALLY ACTIVE PKC-ZETA INTO THE NUCLEUS OF HL-60 CELL-LINE PLAYS A KEY ROLE IN THE INDUCTION OF GRANULOCYTIC DIFFERENTIATION MEDIATED BY ALL-TRANS-RETINOIC ACID

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 le vels of endogenous ceramide and protein kinase C-zeta (PKC-zeta) catal ytic activity in HL-60 myeloid cells. ATRA induced a parallel increase of ceramide and catalytically active PKC-zeta into the nuclear compar tment of HL-60 cells (peak at 72 h). On the other hand, vitamin D3 inc reased the levels of nuclear ceramide and PKC-zeta activity to a lesse r extent and with a delayed kinetics compared to ATRA (peak at 96 h). Pretreatment of HL-60 cells with high pharmacological concentrations o f exogenously-added C-2-ceramide (10(-6) M) completely blocked the ATR A-mediated activation of nuclear PKC-zeta. Exogenous C-2-ceramide (10( -6) M) also inhibited the granulocytic differentiation induced by ATRA , whereas it did not affect monocytic differentiation mediated by vita min D3. Transient transfection experiments performed with a plasmid co nstruct containing a constitutively active mutated form of the PKC-zet a cDNA fused in 3' to a fluorescent tag protein (pEGFP-PKC-zeta) demon strated that the overexpression of catalytically active PKC-zeta was n ot accompanied by the appearance of a differentiated morphology. These findings suggest that nuclear PKC-zeta is necessary but not sufficien t to induce granulocytic differentiation of HL-60 myeloid malignant ce lls.