Phorbol ester (PE) tumor promoters inhibit adipose conversion of BALB/c 3T3 preadipocytes whereas they induce differentiation of the human promyelocytic leukemia cell line, HL-60. The objective of this proposal is to elucidate the biochemical and molecular mechanisms by which PEs can affect differentiation. The biology of the differentiation program of the preadipocytes, and of the inhibition of differentiation by PEs, has been very well-characterized; and cDNA probes that encode some of the proteins that alter in amount during differentiation are now available. These probes will be used to compare the accumulation of the mRNAs for 5 of these proteins in differentiating BALB/c 3T3 preadipocytes and in 12-0-tetradecanoylphorbol-13-acetate (TPA)-treated, non-differentiating cells. TPA-induced alterations in the accumulation of a specific mRNA or its protein product will be analyzed to determine if the tumor promoter affects transcription or turnover of that message or its translatability. Since the promoter is known to stimulate lactic acid production by these cells which markedly decreases the medium pH, the possibility that in some cases TPA may create culture conditions that inhibit proper transcription or translation of the message will be investigated. In HL-60 cells, the initial interaction of PEs with their receptor, protein kinase-C (PK-C), is followed by rapid translocation of cytosolic PK-C to membrane components. To study the biological significance of this translocation in differentiation, the appropriate immunological and molecular probes will be prepared in this laboratory or obtained from others. Antibodies to purified PK-C will be used to characterize PK-C and its subcellular localization in control and TPA-treated HL-60 cells and in variants that are reversibly resistant to the induction of differentiation by PEs. The antibodies will also be used to immunoscreen an existing human cDNA expression library for PK-C cDNA clones; these probes for PK-C mRNA will be used to analyze the transcriptional control of PK-C gene expression in the parental and variant HL-60 cells. The proposed studies with these two cell systems can increase our understanding of the initial interaction between cells and PEs and the role of subsequent events that may occur, in the control of cell differentiation by these compounds.
