A major mediator of hepatocyte regeneration following partial hepatectomy is Hepatocyte Growth Factor (HGF), whose receptor is a membrane tyrosine kinase known to activate the lipid kinase phosphatidylinositol 3'-kinase (PI 3'-kinase) in HGF-stimulated cells. This Pilot and Feasibility study will examine the role of PI 3'-kinase during hepatocyte proliferation, using, the minimal deviation rat hepatoma line Fao as a model system. Fao cells retain many liver- specific functions. Moreover, Fao cells can be moved from a quiescent state into a proliferative state by the addition of insulin in the absence of serum or other growth factors. As insulin shares with HGF the ability to activate PI 3'-kinase, these cell provide a useful model system in which to study the regulation of hepatocyte proliferation. The approach use in this proposal is to modulate the activity of PI 3'-kinase in Fao cells through the introduction of activated or dominant negative mutants; alterations in insulin- stimulated PI 3'-kinase activity will be correlated with changes in the sensitivity of insulin-induced mitogenic responses. Activated PI 3'-kinase mutants will be created by introducing sequences that direct the addition of lipid moieties, thereby causing the constitutive association of PI 3'-kinase with intracellular membranes. Dominant negative mutants will be created by mutating residues in the regulatory and catalytic subunits of PI 3'-kinase that are homologous to critical residues in related proteins. The effect of these perturbations on insulin-stimulated proliferation, as well as insulin stimulation of liver-specific metabolic enzymes, will be determined. These studies should be a valuable initial inquiry into the role of PI 3'-kinase in hepatocyte physiology.
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