The overall goal of this project has been to determine the sequence of events leading from the initial interaction of phorbol esters (PEs) with a cell to later effects of these tumor promoters on expression of specific cell products. We have used a pair of EL4 mouse thymona cell lines, one of which produces T Cell Growth Factor (TCGF = Interleukin 2) in response to PEs and the other which is unresponsive, to dissect specific steps in the sequence. Accumulating evidence, from our lab and others, supports the probable identity of the PE receptor (PE-R) with the calcium/phospholipid-dependent protein kinase C (PK-C). Induction of TCGF Production appears to require a transcriptional event. We are now addressing the steps between interaction of PE with PK-C and generation of a signal in the nucleus for stimulation of TCGF production. Two major projects will be pursued. I) The PK-C/PE-R molecule will be studied in detail to determine whether any differences in the enzyme structure, stability, activation, or subcellular localization can be detected between PE-responsive and -nonresponsive cells. This project will involve purification of the enzyme from both cell lines, analysis by peptide mapping, determination of phosphorylation state and activation, generation of monoclonal antibodies, and use of the antibodies for subcellular localization studies. Project II will focus on identifying PK-C substrates which may be involved in stimulating TCGF production. Potential substrates in cytosol have already been identified in in vitro studies and several prominent differences between sensitive and resistant cells have been noted. In vivo 32P labeling studies will be carried out next to determine which PK-C substrates are phosphorylated in response to PE treatment of cells. The identity of important substrates (eg, those which differ in the two cell lines) will then be determined by Western blotting and immunoprecipitation with antisera to suspected candidates. Included among the candidates to be considered will be several cell surface or intracellular receptors (eg, those for TCGF, insulin, transferrin, retinoic acid) and several oncogene products (eg, ras, abl, c-src, myb, myc). A third, smaller project will be directed at determining whether reactive oxygen species are involved in the PE effects and, if so, how that system might interface with the PK-C/phosphorylation effects.
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