Cell differentiation is regulated by a series of growth factors, many of which have now been defined. In the present proposal, we plan to study the cellular actions of hemopoietic growth factors using purified pertussis toxin and its A and B subunits as biologic probes to evaluate the role of guanine nucleotide binding components as transducing elements. Recent data linking the guanine nucleotide binding protein (Ni) with non-cyclic nucleotide mediators including the phosphoinositides and diacylglycerol and the recognition that oncogene products and growth factors may act through these pathways provide a new perspective for the inhibitory effects of pertussis toxin demonstrated previously. In initial studies, we have shown that purified pertussis toxin is capable of blocking the clonal growth of hematopoietic stem cells stimulated by the hematopoietic growth factors, colony stimulating factor-1 (CSF-1), interleukin-3 (IL-3) or granulocyte- macrophage colony stimulating factor (GM-CSF). In the present studies we will continue our evaluation of the effects of purified pertussis toxin, along with the A and B subunits, on the growth of purified hemopoietic stem cells or marrow populations depleted of specific subpopulations which have been stimulated by CSF-1. Of lesser priority, simila studies will be carried out with IL-3 and GM-CSF. We will utilize a homogeneous population of bone marrow derived macrophages to evaluate effects of pertussis toxin and its subunits on CSF-1-induced changes in morphology, protein degradation, protein synthesis, survival function and proliferation. We will characterize the effects of CSF-1 and pertussis toxin at the subcellular level using the same population of cells, evaluating 125I CSF-1 receptor binding and ADP- ribosylation of the guanine nucleotide regulatory protein. We will evaluate whether CSF-1 exerts its effects through linkage to the cyclic AMP or phosephoinositol metabolic pathways by measuring levels of cyclic AMP and phosphoinositide metabolites and will evaluate its effects on protein kinase C and calcium ion fluxes. Similar studies will be carried out studying the effects of IL-3 (or GM-CSF) on the factor dependent cell line (FDC-P1). Lastly, we will continue investigations of the effects of pertussis toxin and its subunits or growth factor production by the TC-1 adherent marrow cell line. These studies should enable us to elucidate the first step of hemopoietic growth factor activation on bone marrow cells and indicate which metabolic pathways will be appropriate for further investigation.
