A rapid colorimetric microassay to detect agonists/antagonists of protein kinase C based on adherence of EL-4.IL-2 cells.
A rapid, colorimetric, microassay for detection of agents which are known agonists/antagonists of protein kinase C (PKC) was developed, utilizing their effects on adherence of EL-4.IL-2 cells. Cells that were incubated with agents which are known inducers of PKC activation, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), mezerein and indolactam V, readily adhered to wells of 96 well microtiter plates within 1-2 hours, whereas cells incubated with the negative PKC activator, 4 alpha-phorbol 12-myristate 13-acetate (4 alpha-PMA), which is structurally related to PMA (4 beta-PMA), did not adhere. The adherent cells withstood repeated vigorous washings with tissue culture medium. Adherence of EL-4.IL-2 cells in the presence of PMA could be blocked by the addition of two known inhibitors of PKC, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride and staurosporine. Detection of the presence of adherent cells was accomplished by the addition of a tetrazolium salt to culture wells and determination of the remaining viable cells by scanning using a multiwell spectrophotometer (ELISA reader). The EL-4.IL-2 adherence assay meets several important criteria for use as a primary screen in the detection of potential PKC agonists/antagonists, i.e. its selectivity, simplicity, rapid performance through automation and reproducibility.
Longley
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Division of Biomedical Marine Research, Harbor Branch Oceanographic Institution, Inc., Fort Pierce, FL 34946.
Harmody
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D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000032:analysis / Q000494:pharmacology; D000818:Animals; D002273:Carcinogens / Q000032:analysis / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D003124:Colorimetry; D004224:Diterpenes; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D007211:Indoles / Q000032:analysis / Q000494:pharmacology; D007546:Isoquinolines / Q000032:analysis / Q000494:pharmacology; D007769:Lactams / Q000032:analysis / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000032:analysis / Q000494:pharmacology; D010703:Phorbol Esters / Q000032:analysis* / Q000494:pharmacology; D010879:Piperazines / Q000032:analysis / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results; D019311:Staurosporine; D013729:Terpenes / Q000032:analysis / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000032:analysis / Q000494:pharmacology; D013945:Thymoma; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D002273:Carcinogens; D004224:Diterpenes; D007211:Indoles; D007546:Isoquinolines; D007769:Lactams; D010703:Phorbol Esters; D010879:Piperazines; D013729:Terpenes; C011309:mezerein; D015240:Phorbol 12,13-Dibutyrate; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C043969:indolactam V; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate