Okadaic acid (OA) is a tumor promoting polyether fatty acid, produced by dinoflagellates and concentrates in marine sponges and shellfish. The biological consequences of OA in MNNG-initiated T51B rat liver epithelial cells is tumor promotion. The molecular and biochemical consequences of OA action with respect to tumor promotion are not understood with the exception that OA binds to and inhibits the activity of three member of the protein ser/thr phosphatase family. Our progress during the previous four years demonstrates that OA increases EGFr mRNA levels, receptor number, phosphorylation state and sensitizes cells to the mitogenic actions of EGF. EGF-induced mitogenesis in T51B cells is extracellular Ca2+-dependent being mediated by an influx of Ca2+, PLD activation with diacyglycerol increase, c-jun and junB expression and transient interruption of gap junctional communication. In the next funding period the applicant will focus on OA effects on the EGFr and it phosphorylation state with respect to downstream signaling and tumor promotion. He will construct chimeric c-fms/EGFr, mutate specific ser/thr phosphorylation sites and express these chimeric receptors in T51B cells for purposes of answering the question: """"""""does OA function as a tumor promoter at the levels of the EGFr""""""""? The following SPECIFIC AIMS will be accomplished: 1) Construct a wild type chimeric growth factor receptor consisting of a human c-fms receptor extracellular domain linked to human EGFr transmembrane and intracellular domains. Express this chimeric receptor in T51B cells. Characterize the c-fms/EGFr chimera and determine the effects of OA on signal transduction by this receptor; 2) Map the OA-induced phosphorylation sites on the c-fms/EGFr; mutate these sites, construct chimeric mutant receptors and express these mutant receptors in T51B cells, 3) Determine the ability of the chimeric mutant receptor to signal down stream to immediate, intermediate and long-term markers of cell cycle progression in the presence and absence of OA; 4) Determine if OA can function as a tumor promoter in T51B cells expressing chimeric mutant receptors.
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