Abstract
Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)
Similar content being viewed by others
References
Nishizuka Y: Signal transduction: crosstalk. Trends Biochem Sci 17: 367, 1992
Asaoka Y, Nakamura S, Yoshida K and Nishizuka Y: Protein kinase C, calcium and phospholipid degradation. Trends Bochem Sci 17: 414–417, 1992
Bell RM and Burns DJ: Lipid activation of protein kinase C. J Biol Chem 266: 4661–4664, 1991
Dekker LV and Parker PJ: Protein kinase C — a question of specificity. Trends Biochem Sc 19: 73–75, 1994
Nishizuka Y: The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334: pp 661–665, 1988
Houslay MD: Crosstalk: a pivotal role for protein kinase C modulating relationships between signal transduction pathways. J Biochem 195: 9–27, 1991
Bradshaw D, Hill CH, Nixon JS and Wilkinson SE: Therapeutic potential of Protein Kinase C inhibitors. Agents Actions 38: 135–147, 1993
Meyer T, Regenass U, Fabro D, Alter, E, Rossel J, Muller M, Caravatt G and Matter A: A derivative of staurosporine (CGP41251) shows selectivity for PKC inhibitionin vitro and anti-proliferative as well asin vivo anti-tumor activity. Int J Cancer 43: 851–856, 1989
Hallam TJ: Functional significance of protein kinase C in human T cell activity: a new therapeutic class? Clin Expt Rheumatology 11: 131–135, 1993
Prendiville J et al.: A phase I study of intravenous bryostatin I in patients with advanced cancer. Brit J Cancer 68: 418–424, 1993
Kumar CC: Setting up reporter-gene based assay systems for screening antineoplastic drugs. Pharmaceutical Technology 15: 26–34, 1991
Jones RE, Defeo-Jones D, McAvoy EM, Vuocolo GA, Wegrzyn RJ, Haskell KM and Oliff A: Mammalian cell lines engineered to identify inhibitors of specific signal transduction pathways. Oncogene 6: 745–751, 1991
Selden RF, Howie KB, Rowe ME, Goodman HM and Moore DD: Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol Cell Biol 6: 3173–3179, 1986
Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P and Karin M: Phorbol ester-inducible genes contain a commoncis element recognized by a TPA-modulatedtrans-acting factor. Cell 49: 729–739, 1987
Toullec D, Pianetti P, Coste H, Belle vergue P, Grand-Perret T, Ajakane, Baudet V, Boissin P, Boursier E, Loriolle F, Duhanel L, Charon D and Kirilovsky J: The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266: 15771–15781, 1991
Tamaoki T, Nemoto H, Takahashi I, Kato Y, Murimoto M and Tomita F: Staurosporine, a potent inhibitor of phospholipid/Ca2++ dependent protein kinase. Biochem Biophys Res Comm 135: 397–402, 1986
Hannun Y, Loomis C, Merril A and Bell RM: Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate bindingin vitro and in human platelets. J Biol Chem 261: 12604–12609, 1986
Akiayma T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M and Fukami Y: Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262: 5592–5595, 1987
Hashimoto Y, Nakayama T, Teramoto T, Kato H, Watanabe T, Kinoshita M, Tsukamoto K, Tokunaga K, Kurokawa K, Nakanishi S, Matsuda Y and Nonomura Y: Potent and preferential inhibitor of Ca++/calmodulin-dependent protein kinase II by by K252a and its derivative KT5926. Biochem Biophys Res Comm 181: 423–429, 1991
Kashiwada Y, Huang L, Ballas LM, Jiang JB, Janzen W and Lee KH: Hexahydroxybiphenyl derivatives as inhibitors of protein kinase C. J Med Chem 37: 195–200, 1994
Herbert JM, Augereau JM, Gleya J and Maffrand JP: Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Comm 172: 993–999, 1990
Evans FJ, Parker PJ, Oliver AR, Thomas S, Ryves WJ, Evans AT, Gordge P and Sharma P: Phorbol ester activation of the isotypes of protein kinase C from bovine and rat brain. Biochem Soc Transact 19: 397–402, 1991
Ryves WJ, Evans AT, Oliver AR, Parker PJ and Evans FJ: Activation of the PKC-isotypes a, b, g, s, e, by phorbol esters of different biological activities. FEBS Letters 288: 5–9, 1991
Tamaoki T and Nakano H: Potent and specific inhibitors of protein kinase C of microbial origin. Bio Technology 8: 732–735, 1990
Wilkinson SE, Parker PJ and Nixon JS: Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J 294: 335–337, 1993
Basu A: Potential of Protein kinase C as a target for anti cancer treatment. Pharmac Ther 59: 257–262, 1993
Schachtele C, Seifert R and Osswald H: Stimulus-dependent inhibition of platelet aggregation by the protein kinase C inhibitors polymyxin B, H-7 and staurosporine. Biochem Biophys Res Comm 151: 542–547, 1988
Kase H, Iwahashi K, Nakanishi S, Matsuda Y, Yamoda K, Takahashi M, Murakata C, Sato A and Kaneko M: K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Comm 142: 436–440, 1987
Hidaka H, Inagaki M, Kawamoto S and Sasaki Y: Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23: 5036–41, 1984
Loomis C and Bell RM: Sangivamycin, a nucleoside analogue, is a potent inhibitor of protein kinase C. J Biol Chem 263: 1682–1692, 1988
Rotenberg SA, Smiley S, Ueffing M, Krauss RS, Chen LB and Weinstein IB: Inhibition of rodent protein kinase C by the anti carcinoma agent dequalinium. Cancer Res 50: 677–685, 1990
Muid RE, Dale MM, Davis PD, Elliot LM, Hill CH, Kumar H, Lawton G, Tworney BM, Wadsworth J, Wilkinson SE and Nixon JS: A novel conformationally restricted PKC inhibitor Ro 31-8425, inhibits human neutrophil superoxide generation by soluble particulate and post-receptor stimuli. FEBS Lett 293: 169–172, 1991
Dale I and Gesher A: Effects of activators of protein kinase C including bryostatins 1 and 2 on the growth of A549 human lung carcinoma cells. Int J Cancer 43: 158–163, 1989
Housey GM, Johnson MD, Hsiao WLW, O'Brian CA, Murphy JP, Kirschmeier P and Weinstein IB: Overproduction of protein kinase C causes disordered growth control in rat fibroblasts. Cell 52: 343–354, 1988
Hsiao WL, Housey GM, Johnson MD and Weinstein IB: Cells that overproduce PKC are more susceptible to transformation by an activated H-ras oncogene. Mol Cell Biol 9: 2641–2647, 1989
Cacace A, Guadagno SN, Krauss RS, Fabbro D and Weinstein IB: The epsilon isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts. Oncogene 8: pp 2095–2104, 1993
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sista, P., Edmiston, S., Darges, J.W. et al. A cell-based reporter assay for the identification of protein kinase C activators and inhibitors. Mol Cell Biochem 141, 129–134 (1994). https://doi.org/10.1007/BF00926176
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00926176