1,25-dihydroxyvitamin D3 primes acute promyelocytic cells for TPA-induced monocytic differentiation through both PKC and tyrosine phosphorylation cascades.

NB4 cells are the only in vitro model of differentiation in acute promyelocytic leukemia (APL). Although these cells respond to all-trans-retinoic acid to form neutrophils, our group has recently shown that these cells are capable of terminal monocytic differentiation in response to combined treatment with 1,25-dihydroxyvitamin D3 (1,25 D3) and 12-O-tetradecanoylphorbol-13-acetate (TPA). We show here that the agents need not be present simultaneously, but may be added sequentially. TPA treatment prior to 1,25 D3 led to the appearance of adherent cells; however, when 1,25 D3 treatment preceded TPA treatment cells expressed all differentiation markers reflective of terminal differentiation. This priming effect of 1,25 D3 was both dose and time dependent. Increasing the interval between 1,25 D3 and TPA treatment caused a decrease in this priming potential indicative of limited commitment inducing capacity of 1,25 D3. In order to characterize the mechanism of action of 1,25 D3 and TPA, chemical inhibitors of phosphorylation were used. Staurosporine and bisindolymaleimide GF 109203X treatment prior to and during 1,25 D3 treatment or TPA treatment caused attenuation of the differentiation response. Experiments utilizing tyrosine kinase and phosphatase inhibitors supported the hypothesis that 1,25 D3 signaling was mediated by both serine/threonine and tyrosine phosphorylation cascades. Results from this study provide evidence to support the hypothesis that 1,25 D3 signaling occurs via nongenomic mechanisms which when combined with the signaling effects of TPA, allow for the terminal differentiation of APL cells. This model should be used to develop new differentiation therapies for APL and other leukemias.