Sphinganine potentiation of cellular differentiation induced by various anti-leukemia drugs in human leukemia cell line HL-60

A slight induction of cellular differentiation (myelocytes and granulocytes) of HL-60 cells occurred after treatment with anti-tumor agents etoposide (VP-16), mitoxantrone (MXT), mitomycin C (MMC), actinomycin D (Act-D) or novobiocin (NOVO). Addition of sphinganine (SP), an inhibitor of protein kinase C (PKC) enhanced (2-3fold) the VP-16, MXT, MMC or Act-D-induced differentiation but not the NOVO-induced differentiation. No induction of differentiation was observed with 5-fluorouracil (5-FU) in the absence or presence of SP. The addition of SP in the fresh medium after the removal of VP-16, MXT, or MMC (0.5 h treatment) enhanced the induction of differentiation. In contrast, SP post-treatment did not have any effect on enhancing the differentiation which was induced by Act-D short exposure (0.5 h). In an attempt to characterize the biochemical requirements for potentiation of VP-16-induced differentiation, we examined the effects of calcium depletion using calcium chelator ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) or calcium channel blocker verapamil. Potentiation of VP-16-induced differentiation by SP was not observed in EGTA- or verapamiltreated cells. Calcium supplementation to the cells during the treatment with EGTA restored the SP-potentiation of VP-16-induced differentiation. Our results also showed that the induction of differentiation was accompanied by a decrease in PKC activity (70% of the control). PKC activity decreased to a greater extent (50% of control) in SP potentiation of differentiation induction. Our results suggested that calcium-dependent biological action of antitumor agents and the inhibition of PKC activity are required for SP-potentiation of differentiation induction.