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 levels of endogenous ceramide and protein kinase C-zeta (PKC-zeta) catalytic activity in HL-60 myeloid cells. ATRA induced a parallel increase of ceramide and catalytically active PKC-zeta 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-zeta 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-zeta. 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-zeta cDNA fused in 3' to a fluorescent tag protein (pEGFP-PKC-zeta) demonstrated that the overexpression of catalytically active PKC-zeta was not accompanied by the appearance of a differentiated morphology. These findings suggest that nuclear PKC-zeta is necessary but not sufficient to induce granulocytic differentiation of HL-60 myeloid malignant cells.
Bertolaso
L
L
Institute of Human Anatomy, University of Ferrara, Italy.
Gibellini
D
D
Secchiero
P
P
Previati
M
M
Falgione
D
D
Visani
G
G
Rizzoli
R
R
Capitani
S
S
Zauli
G
G
D015153:Blotting, Western; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D002762:Cholecalciferol / Q000494:pharmacology*; D006098:Granulocytes / Q000166:cytology*; D018922:HL-60 Cells; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology*; D000099221:Protein Kinase C zeta
D002518:Ceramides; D002762:Cholecalciferol; D011493:Protein Kinase C; D014212:Tretinoin; D000099221:Protein Kinase C zeta