H cess in the adult, where about 3 x lo9 erythroid cells/kg and lo9 myeloid celis/kg are generated each day. Twenty-five years ago, in vitro assays were developed to detect factors that could stimulate the growth of colonies from immature bone marrow (BM)-derived progenitors.'.' These systems have made it possible to identify and characterize the factors regulating myelopoiesis. The application of molecular biologic approaches to study hematopoiesis facilitated the cloning of genes encoding the hematopoietic growth factors and their production in mammalian cells, yeast, and bacteria in quantities sufficient for in vitro and in vivo studies. Granulocyte-macrophage colonystimulating factor (GM-CSF) is one of a family of glycoprotein cytokines that have potent effects in stimulating proliferation, maturation, and function of hematopoietic cells. Although GM-CSF was the first, several hematopoietic growth factors are now used in clinical trials. Most of the effects observed using GM-CSF in vitro have been shown to occur in vivo either in animal models or in human subjects. In this review, I shall summarize the basic science studies on GM-CSF, because others have recently reviewed the literature on the clinical applications of GM-CSF and other hematopoietins.'-' GENOMIC ORGANIZATION AND CHROMOSOMAL LOCALIZATION

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor-and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF-or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of hematopoietic progenitor cells and enhances the differentiated functions of mature granulocytes and monocytes/macrophages (12, 18). The pleiotropic biological activities of GM-CSF are mediated by a high-affinity receptor which consists of an alpha subunit that binds ligand with low affinity and a beta subunit that confers high-affinity binding to the receptor-ligand complex and is required for signal transduction. Both subunits are members of the hematopoietin receptor superfamily, which includes the receptors for interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, and IL-7, granulocyte colony-stimulating factor, erythropoietin, growth hormone, and prolactin (1, 7). The 120-kDa beta subunit of the human GM-CSF receptor (13-common) is also shared with the high-affinity human IL-3 and IL-5 receptors (23). Phosphorylation of similar sets of proteins has been demonstrated in factor-responsive cells stimulated by either GM-CSF or 20). In addition, pathways involving p2lras, c-Raf, and p42,4...

By in situ chromosomal hybridization, the GM-CSF and FMS genes were localized to human chromosome 5 at bands q23 to q31, and at band 5q33, respectively. These genes encode proteins involved in the regulation of hematopoiesis, and are located within a chromosome region frequently deleted in patients with neoplastic myeloid disorders. Both genes were deleted in the 5q-chromosome from bone marrow cells of two patients with refractory anemia and a del(5)(q15q33.3). The GM-CSF gene alone was deleted in a third patient with acute nonlymphocytic leukemia (ANLL) who has a smaller deletion, del(5)(q22q33.1). Leukemia cells from a fourth patient who has ANLL and does not have a del(5q), but who has a rearranged chromosome 5 that is missing bands q31.3 to q33.1 [ins(21;5)(q22;q31.3q33.1)] were used to sublocalize these genes; both genes were present on the rearranged chromosome 5. Thus, the deletion of one or both of these genes may be important in the pathogenesis of myelodysplastic syndromes or of ANLL.

Human granulocyte colony-stimulating factor (G-CSF) is a regulatory glycoprotein that stimulates the production of neutrophilic granulocytes from committed hematopoietic progenitor cells both in vitro and in vivo. In this report, we show that biosynthetic (recombinant) human G-CSF enhances colony formation by normal human bone marrow and the human myeloid leukemic cell lines, HL-60 and KG-1, as well as nonhematopoietic small cell lung cancer lines, H128 and H69. G-CSF also modulates multiple differentiated functions of human neutrophils, including enhanced oxidative metabolism in response to f- Met-Leu-Phe (f-MLP), increased antibody-dependent cell-mediated cytotoxicity (ADCC), and augmented arachidonic acid release in response to ionophore and chemotactic agents. These effects are all maximal at a concentration of 100 to 500 pmol/L. Using 125I-labeled recombinant human G-CSF, high affinity binding sites were identified on human neutrophils, the myeloid leukemia cell lines KG-1 and HL-60, and the small cell carcinoma cell lines, H128 and H69. G-CSF receptor numbers ranged between 138 and 285 sites per cell with a kd of 77 to 140 pmol/L, consistent with the concentrations of G-CSF that elicit biologic responses in vitro. Decreased specific binding of 125l-G-CSF by human neutrophils was consistently observed in the presence of excess unlabeled human granulocyte-macrophage colony-stimulating factor (GM-CSF), suggesting competition or down modulation by GM-CSF of the G- CSF receptor.

Human erythroid-potentiating activity (EPA) is a 28,000 mol wt glycoprotein that stimulates the growth of erythroid progenitors in vitro and enhances colony formation by the K562 human erythroleukemia cell line. EPA has potent protease inhibitory activity, and is also referred to as tissue inhibitor of metalloproteinases (TIMP). We observed that colony formation by K562 cells in semi-solid medium containing reduced fetal calf serum (FCS) is not directly proportional to the number of cells plated, suggesting production of autostimulatory factors by K562 cells. Using radioimmunoprecipitation and a bioassay for EPA, medium conditioned by K562 cells was found to contain high levels of biologically active EPA; Northern hybridization analysis confirmed the expression of EPA mRNA. Radiolabeled EPA was used to identify cell surface receptors on K562 cells. Together, these results suggest that EPA may act as an autocrine growth factor for K562 cells.