The long-term objective of the proposed research are to define relationships between the synthesis of isoprenoid compounds and the cell replication cycle, and to determine mechnaisms that regulate the synthesis of isoprenoid compounds during the cell cycle. The results of the studies will provide basic information regarding the process of cell division that may be useful in the development of methods for controlling abnormal cell division. Chinese hamster ovary (CHO) cell cultures fractionated by centrifugal elutriation into cells in G1, S and G2+M phases of the cell cycle will be analyzed for concentrations of cholesterol, dolichol, dolichyl acyl esters, dolichyl phospate and coenzyme Q (CoQ). Cultures treated with mevinolin (a competitive inhibitor of HMG-CoA reductase) under conditions that result in G1-arrest of the cell cycle and, upon removal of the mevinolin sychronized progression through the cycle, will analyzed for the above compounds to determine changes in their concentrations. In related experiments the synthesis of isoprenoid compounds will be blocked as completely as possible, consistent with cell viability, by administration of mevinolin and 25-hydroxycholesterol and efforts will be made to restore growth to the G1-arrested cells by addition of dolichol, CoQ and cholesterol. The minimum level of (3 H) mevalonate required to permit the inhibited cells to transverse G1 will be determined and its incorporation into sterols, various forms of dolichol, CoQ, prenylated-polypeptides, tRNA, farnesyl-pyrophosphate and other intermediates will be assayed. The studies should reveal which isoprenoid compounds are required the cell cycle and the temporal order of their requirement. CHO mutants defective in the synthesis of an unidentified minor isoprenoid compound(s) that is required for G1 traverse will be selected and characterized as an aid to study of the role of isoprenoids in the cell cycle. Studies of the mechanisms by which HMG-CoA reductase is regulated will include measurements of inactive reductase, of its mRNA and of the concentrations of regulatory oxysterols in synchronized cells separated by centrifugal elutriation.
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| Kabakoff, B D; Doyle, J W; Kandutsch, A A (1990) Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth. Arch Biochem Biophys 276:382-9 |
| Rilling, H C; Bruenger, E; Epstein, W W et al. (1989) Prenylated proteins: demonstration of a thioether linkage to cysteine of proteins. Biochem Biophys Res Commun 163:143-8 |
