concanavalin-a and mezerein

The CDw50 antigen is a constitutively non-phosphorylated leukocyte surface molecule which becomes highly phosphorylated in all the normal and lymphoblastoid cells analyzed (peripheral blood mononuclear cells, Molt 4, CEM, 8402, Namalwa), after stimulation with tumor promoter agents (phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, mezerein). This phosphorylation is rapid (within 1-5 min), dose-dependent and results in the incorporation of PO(3-)4 groups on serine residues. Furthermore, the level of CDw50 phosphorylation induced by tumor promoter agents is decreased by the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. Activation of peripheral lymphocytes with concanavalin A, phytohemagglutinin and cross-linking of CD3 molecules also induces CDw50 phosphorylation, but the response is delayed and less intense than when tumor promoting agents are used. Treatment with any of the aforementioned agents is not accompanied by quantitative changes in the CDw50 surface expression. We therefore conclude that protein-kinase-C-mediated mechanisms are involved in phosphorylation, but not in regulation of the surface expression of the CDw50 leukocyte antigen.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Antigens, CD; Antigens, Differentiation; Cell Adhesion Molecules; Concanavalin A; Cross-Linking Reagents; Diterpenes; Down-Regulation; Electrophoresis, Gel, Two-Dimensional; Enzyme Activation; Flow Cytometry; Fluorescent Antibody Technique; Humans; Isoquinolines; Kinetics; Lymphocytes; Phorbol 12,13-Dibutyrate; Phosphorylation; Phytohemagglutinins; Piperazines; Precipitin Tests; Protein Kinase C; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate

Lymphocytes can be stimulated to proliferate in vitro by mitogens such as concanavalin A. The tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) can enhance this proliferation, partly because of an increase in interleukin 2 (IL-2) production. However, if lymphocytes are treated with TPA for 24 h before concanavalin A exposure, IL-2 production and proliferation are depressed. The target of the action of TPA is protein kinase C, which is activated after a short exposure but down-regulated after a longer one. This study was designed to determine if the modulation of IL-2 was separable from the modulation of protein kinase C. When phorbol esters phorbol 12-retinoate-13-acetate, phorbol 12,13-dibutyrate, 12-deoxyphorbol 13-phenylacetate, and 12-deoxyphorbol 13-phenylacetate-20-acetate, as well as nonphorbol tumor promoters mezerein, telocidin, and okadaic acid, were tested, all but okadaic acid reproduced the effects of TPA. However, 12-deoxyphorbol 13-phenylacetate and 12-deoxyphorbol 13-phenylacetate-20-acetate were required at nearly 100-fold higher concentrations than TPA to suppress IL-2 production, suppress mitogenesis, and cause down-regulation of protein kinase C. A comparison of structures indicated that an R group at the 12-position was less important for IL-2 production and mitogenesis than for down-regulation of protein kinase C and the suppression of mitogenesis. In no case was the modulation of protein kinase C separated from the effects on IL-2 production and proliferation.

Topics: Animals; Cattle; Cell Compartmentation; Concanavalin A; Diterpenes; Dose-Response Relationship, Drug; Down-Regulation; Ethers, Cyclic; In Vitro Techniques; Interleukin-2; Lymphocyte Activation; Lymphocytes; Lyngbya Toxins; Okadaic Acid; Phorbol Esters; Protein Kinase C; Terpenes