Neoplasia is a disorder characterized by abnormalities in proliferation and differentiation. Preliminary clinical trials using therapies designed to stimulate leukemic cell differentiation have yielded promising results. Phorbol esters are a group of agents which induce in vitro differentiation of human leukemic cells more potently than agents currently being evaluated for clinical use. However, mitogenic effects in other tissues preclude their use in a """"""""clinical setting. Phorbol esters (TPA) exert their effects by activating members of the protein kinase C (PKC) gene family. Because of differences among the seven PKC isoforms, it is hypothesized that individual isoforms may selectively mediate TPA-induced events. Thus, studies are warranted to directly assess the involvement of the individual isoforms in mediating the effects of TPA. Using the human monoblastoid U937 leukemic cell line which differentiates into a macrophage-like cell after exposure to TPA, we have: (1) demonstrated the existence of a previously uncharacterized PKC isoform; (2) observed that PKC zeta is contained in the U-937 cell and does not translocate or down regulate in response to TPA; (3) found that TPA treatment functionally induces a partial down regulation PKC a activity by altering its substrate specificity and (4) using PKC mutants prepared by recombinant polymerase chain reaction techniques that are constituitively activated in the absence of TPA determined that individual isoforms elicit qualitatively different effects on gene expression. This data form the basis for this revision of the previously submitted proposal. The goal of the current proposal is to determine the mechanism(s) by which TPA stimulates U937 differentiation. The main thrust of the proposal is to determine the role of individual isoforms in mediating TPA stimulated differentiation and other TPA-induced responses. Pursuant to this goal, we will: (1) directly examine the role of individual isoforms by assessing the ability of PKC mutants constituitively activated in the absence to TPA to elicit U937 differentiation and other TPA-induced responses; (2) analyze the role of individual isoforms on TPA-stimulated differentiation by selectively depleting isoforms with antisense DNA; (3) determine the factors activating PKC-zeta, the cellular responses elicited by activation of this isoform and examine the role of the second cysteine-rich repeat in the Cl domain in conferring TPA-responsiveness to PKC; (4) analyze the mechanisms responsible for TPA-induced alterations in PKC a substrate specificity; (5) identify and characterize a newly recognized PKC activity in the U937 cell; (6) determine the effects of altering endogenous PKC isoform expression on TPA-responsiveness in the U937 cell; and (7) to analyze the role of the DNA region 5' to the beta and gamma isoforms in regulating transcriptional activity of these genes in the U937 cell using CAT constructs containing these regions. By determining the role of individual isoforms in mediating TPA-induced differentiation and other phorbol ester stimulated responses, information obtained from these studies could have direct clinical implications in the treatment of leukemias. These results would focus the development of agonists selectively activating the involved isoform and could form the basis for designing PKC mutants which could be used as a selective form of gene therapy for certain leukemias.
