Regulation of Myelopoiesis In Vitro: Partial Replacement of Colony-Stimulating Factors by Tumor-Promoting Phorbol Esters

Abstract

AbstractTumor-promoting phorbol esters such as 12–0-tetradecanoyl-phorbol-13-acetate (TPA) or phorbol-12,13-didecanoate (PDD) stimulate freshly isolated murine bone marrow cells in vitro to form colonies of myeloid cells in semisolid medium without added colony-stimulating factors (CSF), while phorbol and its nonpromoting derivatives do not induce marrow colony formation. TPA-stimulated colonies are composed primarily of monocyte-macrophage cells with admixed granulocytes in some colonies, and cell dose-response curves for colony formation suggest clonal origin of the colonies. The cells forming colonies in the presence of TPA alone appear to be a subset of marrow granulocyte-macrophage colony-forming cells (GM-CFC) characterized by larger mean cell size (determined by velocity sedimentation), and TPA-stimulated colony formation is inhibited by dexamethasone and prostaglandin E1, agents that also inhibit GM-CFC colony formation stimulated by CSF. The active phorbol esters can apparently replace exogenous CSF in agar cultures of murine bone marrow and may do so by either (1) directly stimulating GM-CFC. (2) inducing endogenous CSF secretion, or (3) increasing GM-CFC’s responsiveness to CSF. We were unable to demonstrate induction of CSF secretion by marrow cells in liquid culture in the presence of TPA. We did find that TPA synergistically enhanced colony formation stimulated by suboptimal concentrations of CSF from two sources, and this effect of TPA was concentration dependent. PDD also enhanced colony formation stimulated by suboptimal CSF concentrations, while phorbol had no effect. In long-term flask cultures of normal murine marrow, TPA caused rapid, concentration-dependent, terminal differentiation (to macrophages), and decline in the progenitor cell compartment. Our data indicate that tumor-promoting phorbol esters may alter the function of cell surface receptors for CSF on GM-CFC, causing stimulation of proliferation and differentiation in the absence of CSF or at very low concentrations of CSF. These compounds should be important in the study of hematopoietic regulation.