INACTIVATION OF A C-MYB ESTROGEN RECEPTOR FUSION PROTEIN IN TRANSFORMED PRIMARY-CELLS LEADS TO GRANULOCYTE/MACROPHAGE DIFFERENTIATION AND DOWN-REGULATION OF C-KIT BUT NOT C-MYC OR CDC2/

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen recept or and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMY B-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-est radiol, the ERMYB cells display increased adherence, decreased clonoge nicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cell s grown in the presence or absence of beta-estradiol. Neither c-myc no r cdc2 expression was down-regulated after removal of beta-estradiol d emonstrating that differentiation is not a consequence of decreased tr ansactivation of these genes by ERMYB. While bcl-2 expression was redu ced by 50% in ERMYB cells grown in the absence of beta-estradiol, ther e was no increase in DNA laddering, suggesting that Myb was not protec ting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation o f ERMYB cells, and c-kit mRNA could be partially re-induced by the re- addition of beta-estradiol. Furthermore, a reporter construct containi ng the c-kit promoter was activated when cotransfected with a Myb expr ession vector, providing further evidence of a role for Myb in the reg ulation of c-kit.