This application has as its fundamental premise the concept that the therapy of the acute myelocytic leukemias can be approached by the initiation of terminal differentiation and that this phenomenon can be induced by combinations of retinoic acid (RA) with granulocyte colony-stimulating factor (G-CSF) or vitamin D, (vit D3) to produce therapeutic benefit to patients with these diseases. To evaluate this possibility this proposal will characterize the molecular mechanisms by which G-CSF, RA and vit D3 through occupancy of their receptors produce synergistic induction of the differentiation of WEHI-3B murine myelomonocytic leukemia cells when G-CSF or vit D3 are used in admixture with RA. The application will examine the role of the G-CSF, vit D3 and RA receptors, as well as other portions of the signal transduction mechanism in the induction of the terminal differentiation of the WEHI-3B D+ leukemia and a differentiation resistant mutant (WEHI-3B D-) thereof to determine the importance of receptors for these inducers in the maturation process in this model of the acute myelocytic leukemias. Specifically, we will: (a) evaluate the synergistic interaction between RA and G-CSF on leukemia cell differentiation in selected differentiation competent WEHI-3B D+ and WEHI-3B D- clones transfected with expression plasmids for the G-CSF receptor (G-CSFR), for mutant receptor cDNAs having truncated cytoplasmic domains, and for RARalpha and RXRalpha, evaluating the effects of G-CSF and RA alone and in combination on the expression of RARalpha and RXRalpha mRNAs and proteins and the effects of these inducers on the expression of G-CSFR mRNA and protein; (b) evaluate the synergistic interaction between RA and vit D3 (and analogs thereof) on leukemia cell maturation in selected WEHI-3B D and WEHI-3B D+ clones transfected with an expression plasmid for the vit D3 receptor (VDR), determining the mechanism by which vit D3 and RA induce the expression of the VDR and the mechanism responsible for the lack of VDR expression in WEHI-3B D+ cells; (c) characterize the mechanism by which the scl gene functions as a repressor of the differentiation of WEHI-3B cells; and (d) ascertain whether properties deemed to be of importance to the attainment of a mature phenotype in the experimental WEHI-3B system employed are operative in other cell lines to ascertain the generality of the synergistic action of the combinations and in primary leukemia cells obtained from patients to determine whether these events correlate with the ability of primary leukemia cells to undergo terminal differentiation when exposed to combinations of G-CSF and RA, and vit D3 and RA.
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