Biochimica et Biophysica Acta, 1013 (1989) 47-54 Elsevier 47 BBAMCR 12506 Differential responsiveness to phorbol esters correlates with differential expression of protein ldnase C in KG-1 and KG-la human mye|oid leukemia cells W. Craig Hooper 1, Robert T. Abraham i Curtis L. Ashendel 2 and Gayle E. Woloschak a s Department of Immunology, Mayo Clinic~ Foundation Rochester, MN and 2 Department of Medicinal Chemistry, Purdue UniversiO,, School of Pharmacy, West Lafayette, IN and 3 Argonne National Laboratory, Division of Biological and Medical Research, Argonne, IL (U.S.A.) (Received 30 December 1988) (Revised manuscript received 1 May 1989) Key words: Protein kinase C; Enzyme expression; Phorboi ester: Cell differentiation The KG-I myeioid leukemia cell line differentiates into macrophages in response to 12-O-tetradecanoylphorbol 13-acetate (TPA), while its spontaneous variant, the KG-|a cell line, is resistant to the differentiative effects of TPA. To determine the mechanism underlying these differential responses to TPA, experiments were performed to determine the relative numbers of TPA binding sites, protein kinase C (PKC) enzyme activity levels, PKC translocation responses and PKC gene expression in these cell lines. KG-Ia cells exhibited 40% fewer high affinity binding sites for TPA thau KG-I cells. Although PKC translocation from cytosol to the membrane fraction was observed in both cell types, total PKC activity, measured in vitro using type H|-S historic as substrate, was reduced by 70% in K~-|a cells. These biochemical differences between the parental line and the phorbol ester non-responsive variant were correlated with the depressed level of PKC-fl RNA abundance in KG-1a cells. Both lines expressed PKC-a RNA at comparable levels. Chronic exposure to TPA resulted in down-regulation of PKC enzyme activity in both cell lines, and a selective decrease in PKC-fl RNA transcripts in both cell types. In contrast, chronic TPA treatment had no effect on the levels of PKC-a RNA in KG-I and KG-Ia cells. Our results indicate a correlation between the level of PKC-fl expression and the responsiveness of myeloid lineage precursor cells to the differentiative effects of TPA. Many of the pleiotropic effects of the tumor promo- ter, TPA occur at the level of the cell membrane through the binding of specific receptors. Recent studies have provided evidence that this receptor is the Ca2+-sensi- tive phospholipid-dependent protein kinase (protein kinase C) (PKC) [1-3]. PKC is a multifunctional, serine/threonine-specific protein kinase whose activity is regulated physiologically by the intracellular free Ca 2+ concentration and 1,2-diacylglycerol (DAG) [4-6]. Abbreviations: 12-O-tetradecanoylphorbol 13-acetate; PKC, protein kinase C; DAG, diacylglyceroi; PlP 2, phosphatidylinositol 4,5-bis- phosphate; IP, inositol 1,4,5-triphosphate; PBS, phosphate-buffered saline, HBSS, Hank's balanced salt solution; BSA, bovine serum albumin; PMSF, phenylmethylsulfonylfluoride; PdBu, phorbol 12,13.dibutyrate. Correspondence (present address): W.C. Hooper, Centers for Disease Control, Division of Host Factors, DO2, 1600 Clifton Road, Atlanta, GA 30333, U.S.A. In certain receptor systems, occupancy by specific ligands initiates a signal transduction pathway in which phospholipase C hydrolyzes phosphatidylinositol 4,5- bisphosphate (PIP2) to form two intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) and DAG. IP a releases Ca 2+ from hormone,sensitive intraceilular stores and DAG increases the affinity of PKC for Ca 2 +. Thus, these second messengers synergize to activate PKC. TPA appears to act as a potent DAG analog and induces a direct activation of PKC in intact cells. How- ever, unlike DAG, the activating effect of TPA is pro- longed due to the relatively slow removal of TPA from cell membranes by metabolic degradation [4-7]. One of the myriad effects of TPA is the induction of terminal differentiation in a variety of cell lineages, including human myeloid leukemia ceils [8,9]. The hu- man promyelocytic leukemia cell line, HL-60, can be induced by TPA to differentiate into cells bearing a macrophage phenotype [10,11]. A similar effect has been observed with membrane-permeant DAG analogs, 076%4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)