This proposal is a continuation of studies examining soluble factors controlling or modulating human, myeloid leukemia cell growth. In the past, the actions of colony-stimulating factor (CSF) on normal and acute non-lymphocytic leukemia cell (ANNL) growth were compared. Monoclonal antibodies to transferrin (Tf) receptors were employed to examine roles of Tf, Fe, and Tf receptors in myeloid leukemia cell growth. In this proposal, specialized serum-free, Tf-free, and protein-free cultures of myeloid leukemia cell lines will be used to assess cytokinetic effects of Tf, Fe, insulin, and somatomedins on ANNL cell growth. These effects will be correlated with effects on cellular oncogene(c-onc) gene and Tf receptor expression. The c-onc gene responses to these nutritionally-related molecules wil be contrasted to cell line responses to CSF. For the latter studies, HL60 and KG-1 cell lines induced to differentiate and to respond to CSF will be used. Cytokinetic responses to CSF will also be assessed, and coupling of CSF response to differentiation-related, cell surface antigens assssed. CSF effects on self-renewal will be examined using colony and continuous cultures of fresh ANLL cells and leukemia cell lines treated with differentiation-inducers and CSF. Anti-polypeptide, CSF antibodies will be prepared and tested for effects on normal cells and differentiation-induced cell lines and ANLL cells. To continue studies of Tf, Fe and Tf receptors in ANLL cell growth, regulation of Tf receptor gene expression will be studied in Fe-loaded and serum-free cultured cells, ANLL cells induced to differentiate, cells cultured with anti-Tf receptor antibodies, and cells in transition from resting to proliferating states. A TfR cDNA will be used for this purpose and Tf receptor transcript levels correlated with rates of specific protein and mRNA synthesis. Studies of Tf's role in ANLL growth will also examine mechanisms by which Tf enhances CSF release from normal cells and increases ANLL cell growth. For these studies, effects of internalized ligands such as Tf, alpha2-macroglobulin, LDL, and Fc receptor bound immunoglobulin on CSF generation and Tf receptor turnover will be determined. The Tf receptor positive adherent blood and marrow cells will be characterized using monoclonal antibodies, and effects of Tf compared to Ca ionophore. Finally, Tf effects on long-term normal and ANLL marrow growth will be assessed.
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