The identity and combination of regulators which signal myeloid cells to either self-renew or mature, have not been clearly separated. The delineation and biochemical characterization of factor(s) which regulate these processes would help define the physiologic problem in leukemia, which is thought to result from an uncoupling of gene expression that governs cellular proliferation and differentiation. In this research project, it is proposed (1) to purify a human active granulocyte-macrophage/leukemia differentiation-inducing protein (GM-DF), (2) to define its relationship to species of colony stimulating factor (CSF), (3) to delineate the mechanisms of action by which GM-DF affects cell differentiation and (4) to determine its potential therapeutic application, by studying its in vitro target cell specificity and mechanism of action and its in vivo ability to inhibit leukemia. The following experimental techniques and design will be applied: (1) Biochemical purification with ammonium sulfate precipitation, Anion Exchange chromatography, Gel filtration, Affinity chromatography including Porcion Red Agarose, AH Sepharose, Controlled Glass Pore Bead, and Hydroxyapatite Isoelectric focusing and Waters Reverse phase High Pressure Liquid Chromatography. Fractions obtained at each point will be assayed for (a) GM-DF in agar cultures containing WEHI-3B(D+) or HL-60 cells, scored day 7 and 14 respectively for total and diffuse (differentiated) colonies; and (b) CSF, in the day 7 CFU-GM clonogenic assay in soft agar. (2) Purified GM-DF and partially purified preparations free of CSF, will be utilized in studies employing both fresh and cell line derived leukemic cells in suspension and agar cultures, in order to determine the target cell specificity for differentiation induction, kinetics and S phase specificity of GM-DF cell maturation promotion. Experiments employing GM-DF as anti-leukemic maturation therapy in syngeneic transplantable myelomonocytic leukemia, will be tested. Finally, while purification of GM-DF is awaited, it is planned to study the induction of GM-DF by Novo-Pyrexal (a highly purified preparation of endotoxin, derived from S. abortus equi.) in man, and in particular in patients with Acute Myelogenous Leukemia (ANLL) all FAB classifications, Chronic Myelogenous Leukemia (CML) in accelerated phase or in blast crisis, and Preleukemia or Myelodysplastic syndrome (MDS).
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