CELL GROWTH DEPENDS ON MITOCHONDRIALLY-DERIVED CITRATE

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In a multi-stage experimental design, relationships will be investigated between selected aspects of the well-documented deregulated (continuously operating) cholesterogenesis pathway, and the induction of new DNA synthesis (as a prelude to cell proliferation) in tumors. Emphasis will be focused on: (1) the role of the mitochondrial tricarboxylate (citrate-malate) exchange carrier in the above phenomena; (2) the clarification of this laboratory's recent discovery of the ability of the mitochondrial citrate transport blocking agent 1,2,3-benzenetricarboxylate (BTC) to dramatically inhibit DNA synthesis and proliferation in cultured tumor cells; and (3) the detection, isolation and partial characterization of a mevalonate-derived, isoprenylated (and possibly phosphorylated) protein adduct from the cytosol of rapidly growing cells which may be required for DNA synthesis and cell cycling during tumorigenesis. (1) Morris hepatoma 3924A (with normal rat liver as control), as well as suspension cultures of murine B-cell lymphoma 70Z/3, (2) T-cell leukemia L5178Y and (3) ascites hepatoma MH129 (recently adapted to growth in suspension culture), which together comprise cancers of different germ layer origins, shall serve as material for propagation +/- BTC, and/or the source of post-mitochondrial supernatant (PMS) cell-free, lipid synthesizing systems. We shall study precursor carbon flux through sterol biosynthesis as a function of the action of BTC via 14C labelled intermediate isolation as well as by following 13C enriched precursor substrate flux via CMR, utilizing tumor PMS preparations. With newly available 3H-BTC, we will attempt to characterize the intracellular distribution of this inhibitor. The effects of BTC on DNA, RNA and protein synthesis during stages of the cell cycle in synchronized cell populations will be explored, in an effort to better understand how the mitochondrial citrate carrier inhibitor slows the growth of cells. Detection and partial characterization of the unique derivatized protein species will employ refinements of our already successful 2-D NEPHGE analysis and the autoradiograms generated from O'Farrell gels. The isolation of the apparently unique isoprenylated protein derivatives detected under cell proliferation conditions shall be a principal effort of these studies. Once isolated and purified, antibodies to these unique derivatives shall be raised to search for their production and cell fraction location during the cell cycle.