C-FMS DEPENDENT HL-60 CELL-DIFFERENTIATION AND REGULATION OF RB GENE-EXPRESSION

The dependence of induced myelomonocytic cell differentiation, and reg ulation of the RB tumor suppressor gene during this process, on the c- fms gene product, the CSF-1 lymphokine receptor, was determined in HL- 60 promyelocytic leukemia cells. Adding a monoclonal antibody with spe cificity for the c-fms gene product to cells treated with various indu cers of myelomonocytic or macrophage differentiation, including retino ic acid and 1,25-dihydroxy vitamin D3, inhibited the rate of different iation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greate r inhibition of differentiation. In a stable HL-60 transfectant overex pressing the CSF-1 receptor at the cell surface due to a constitutivel y driven c-fms trans gene, the rate of differentiation was enhanced co mpared to the wild type cell, consistent with a positive regulatory ro le for the CSF-1 receptor. The anti-fms antibody caused much less inhi bition of differentiation in the transfectants than in wild type cells , consistent with a larger number of receptors causing reduced sensiti vity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB express ion per cell, which offset the decrease normally caused by differentia tion inducers (1,25-dihydroxy vitamin D3 and retinoic acid). The chang es in RB expression preceded changes in the RB protein to the hypophos phorylated state. Most of the RB protein in proliferating cells was ph osphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of GO arrest. Thus the metabolic ca scade leading to myelomonocytic differentiation of HL-60 cells appears to be driven by a function of the c-fms protein. Inhibiting that proc ess by attacking this receptor impedes differentiation and also compro mises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide addit ional support for a potential role for down regulating RB expression i n promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF-1 actions. (C) 1993 Wiley-Liss, Inc.