Previous studies by this laboratory have linked signaling by the phosphatidyl inositol 3-kinase (PI3K), the """"""""classical"""""""" mitogen activated protein kinase (MAPK), the c-Jun NH2-terminal kinase (JNK) and the p38 MAPK pathways in cultures of primary hepatocytes to altered proliferative and survival behavior patterns. The studies in this competing renewal application propose to determine the mechanisms by which a novel anti-cancer chemotherapeutic agent acts on signal transduction pathway function and anti-apoptotic gene expression in primary hepatocytes, in concert with cytotoxic and cytoprotective bile acids. Hepatotoxicity can be a site of dose-limiting toxicity for several chemotherapeutic agents, notably the novel heat shock protein 90 (HSP90) modulating family of agents termed """"""""geldanamycins"""""""". Although geldanamycins are proposed to modulate tumor cell survival by decreasing the expression/stability of certain cytoprotective signaling proteins that bind to HSP90 e.g. Raf-1, Akt, Src, ERBB2, little is known about how or why non-transformed hepatocytes are, relatively speaking, so sensitive to these novel drugs. Cholestatic liver disease occurs when bile acids, synthesized in the liver, are restricted in their ability to leave the liver and enter the gall bladder. Cholestasis can often also occur during cancer chemotherapy. The accumulation of toxic bile acids in the liver, e.g. deoxycholic acid (DCA), leads to hepatocyte cell death. The mechanisms by which physiologic and cholestatic levels of bile acids interact with novel chemotherapeutic agents to alter the survival hepatocytes is unknown. Specific protection of liver function in the presence of toxic geldanamycins will improve the therapeutic window for these novel agents. Based on previous and continuing studies, this proposal has the following Specific Aims:
Specific Aim 1. Studies will determine the mechanisms by which the HSP90 inhibitors geldanamycin (NCS122750) and 17-allylamino-17demethoxygeldanamycin (17AAG; NSC330507) enhance the toxicity of bile acids in primary hepatocytes in vitro and the liver in vivo.
Specific Aim 2. Studies will determine the mechanisms by which bile acids and geldanamycins modulate plasma membrane receptor and non-receptor tyrosine kinase function and signaling into downstream pathways, which we predict is via the generation of free radicals by the mitochondria.
Specific Aim 3. As a direct outgrowth of work conducted during the initial funding period, new studies determine the mechanisms by which the cyclin dependent kinase (CDK) inhibitor p21 enhance p53 expressing, lowers Mdm2 levels, and reduces the apoptotic threshold to bile acids and 17AAG in primary hepatocytes.
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