Tumor promoters are compounds that act synergistically with initiating carcinogens to induce tumor formation in mammalian cells. Phorbol esters are tumor promoters that activate protein kinase C (PKC), protein serine/threonine kinases that are dependent on calcium, diacylglycerol, and phospholipid for activity. Exposure of cells to phorbol esters also results in the activation of MAP kinase and S6 kinase, two messenger- independent serine/threonine kinases activated downstream of PKC whose activities are regulated by phosphorylation. S6 kinase is activated when phosphorylated by the MAP kinase. The mechanism of action of the MAP kinase is unknown; phosphorylation on both tyrosine and threonine residues is required. Cell lines that vary in their responsiveness to phorbol esters are useful tools in the study of the mechanism of action of tumor promoters. Wild-type EL4 murine thymoma cells, but not variant EL4 cells, respond to phorbol ester by initiating interleukin-2 transcription. Phorbol ester causes a rapid increase in MAP and S6 kinase activities in wild-type cells but not in variant cells. These kinases represent the ERK and RSK gene products, respectively. Wild-type cells, but not variant cells, produce an activating factor in response to phorbol ester. this factor enhances the phosphorylation of both the ERK and RSK kinases by an unknown mechanism when incubated in the presence of both kinases. This proposal investigates the hypothesis that the activation of downstream kinases is an essential pathway in mediating responses to tumor promoters. Portions of the proposal deal with the mechanism of activation of the ERK and RSK kinases, the roles of these kinases in mediating transcriptional activation, and pathways for PKC- dependent phospholipid hydrolysis.
The specific aims are as follows. 1) The mechanism by which activation of PKC is linked to activation of ERK and RSK will be investigated: a) The activating factor produced by wild-type EL4 cells in response to phorbol ester will be isolated, purified, and characterized, b) The effects of the cdc2 kinase complex on the phosphorylation of ERK and RSK will be further investigated, c) Differences in protein phosphorylation between intact wild-type and variant EL4 cells in response to phorbol ester will be examined. 2) The mechanisms by which EL4 cells develop resistance to phorbol ester will be studied: a) Additional phorbol ester-resistant variant cell lines will be developed, b) Expression of PKC isoforms will be determined in wild-type and variant cell lines, c) The effects of phorbol ester on ERK, RSK and activating factor will be assessed in both cell types, d) Phorbol ester-induced phosphorylation of FOS and jun will be compared in wild- type and variant cells, e) Hydrolysis of phosphatidylcholine in response to phorbol ester will be analyzed in these cell lines. In summary, the proposed work uses a combination of cellular and biochemical approaches to examine the mechanism of action of tumor promoters and to define the role of PKC in T-cell activation. The information gained will provide a basis for future approaches to cancer therapy and prevention.
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