dolichols and Cell-Transformation--Viral

Serum depletion of exponentially growing normal human fibroblasts resulted in a moderate depression of the activity of HMG-CoA reductase which occurred simultaneously to the onset of growth arrest of the cells. Specific inhibition of HMG-CoA reductase using mevinolin also resulted in growth arrest. PDGF counteracted the suppressive effect of serum depletion on HMG-CoA reductase activity and cell growth. The growth inhibitory effect of serum depletion and mevinolin was correlated to a decreased biosynthesis of dolichols, in particular of dolichol-20. If PDGF was present in the serum-free medium a high rate of dolichol synthesis was maintained. This effect was mediated not only through an increased HMG-CoA reductase activity. PDGF also increased the incorporation of mevalonate into dolichols, once again into dolichol-20 in particular. In contrast to HDF, the growth of virus-transformed human fibroblasts was not decreased following serum depletion. This was correlated to a sustained activity of HMG-CoA reductase and a sustained dolichol-20 synthesis. In order to block growth and dolichol synthesis of the transformed fibroblasts a stronger inhibition of HMG-CoA reductase activity was required than in the normal cells. Conditioned medium isolated from the transformed cells was found to maintain a high growth rate and a high HMG-CoA reductase activity in serum-depleted HDF. In addition, the incorporation of mevalonate into dolichols was increased. The present data raise the possibility that PDGF or related factors, through autocrine loops, may contribute to the maintenance of a high dolichol synthesis in tumor cells.

Topics: Butadienes; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Culture Media; Dolichols; Fibroblasts; Hemiterpenes; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mevalonic Acid; Pentanes; Platelet-Derived Growth Factor; Simian virus 40

The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase was higher in SV40-transformed human fibroblasts (90-VA VI) than in normal ones (HDF). In both these cell types mevinolin (25 microM) caused an 85-90% depression of HMG CoA reductase activity and of the incorporation of [3H]acetate into sterols. In HDF this was coupled to an efficient block of cell growth, whereas the growth of 90-VA VI was only slightly reduced by mevinolin. In HDF, mevinolin (25 microM) also abolished essentially all dilichol synthesis, as measured by incorporation of [3H]acetate. In contrast, dolichol synthesis remained unaltered, or was increased, in mevinolin-treated 90-VA VI. We suggest that these different responses of dolichol synthesis may depend on different substrate affinities of the rate-limiting enzyme in the dolichol pathway. However, if 90-VA VI was treated with 25-hydroxycholesterol (25-OH), an alternative inhibitor of HMG CoA reductase, the cellular growth as well as dolichol synthesis was significantly decreased. Since the inhibitory effect of 25OH on HMG CoA reductase activity did not exceed that of mevinolin, it seems that 25-OH, besides HMG CoA reductase, inhibits steps distal to HMG CoA reductase. This notion was further supported by the finding that addition of mevalonate did not prevent the 25-OH-induced growth inhibition. However, if dolichol was added along with 25-OH, the block was partially prevented, indicating that a critical level of de novo synthesis of dolichol for cellular growth.

Topics: Cell Division; Cell Transformation, Viral; Culture Media; Dolichols; Gene Expression Regulation, Enzymologic; Humans; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; In Vitro Techniques; Lovastatin; Mevalonic Acid; Simian virus 40; Sterols

Effects of treatment with serum-free medium and 25-hydroxycholesterol (25-OH) on the cell cycle of simian virus 40-transformed 3T3 fibroblasts, designated SV-3T3 cells, were studied and compared with simultaneous effects on the activity of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase and incorporation of [3H]mevalonic acid into cholesterol, Coenzyme Q, and dolichol. The data confirm our previous finding (O. Larsson and A. Zetterberg, Cancer Res., 46: 1233-1239, 1986) that 25-OH inhibits the cell cycle traverse of SV-3T3 cells specifically in early G1. In contrast, treatment with serum-free medium had no effect on cell cycle progression. The effect of 25-OH on the cell cycle traverse was correlated to a substantial decrease in the activity of HMG CoA reductase, whereas there was no change in the rate of [3H]mevalonic acid incorporated into cholesterol, Coenzyme Q, and dolichol. When the cells were exposed to serum-free medium, there was no depression of activity of HMG CoA reductase, and the rate of [3H]mevalonic acid incorporated into dolichol and cholesterol was not affected in any appreciable degree. In contrast the rate of Coenzyme Q synthesis was substantially decreased as a result of serum depletion. A similar decrease in Coenzyme Q synthesis was also achieved by treating the cells with cholesterol-poor serum. This indicates that the rate of Coenzyme Q synthesis is dependent on the concentration of cholesterol in the culture medium. In order to analyze whether some of the products in the mevalonic acid biosynthetic pathway may be of importance in the control of G1 traverse and cell proliferation of SV-3T3 cells, cholesterol, Coenzyme Q, and dolichol were added as supplements to cells treated with 25-OH. It was shown that dolichol was capable of overcoming the 25-OH-induced inhibition of G1 traverse efficiently, whereas cholesterol and Coenzyme Q were considerably less effective. Considered together with the fact that the activity of HMG CoA reductase and incorporation of mevalonic acid into dolichol were unaffected following serum-free treatment, the results suggest that maintenance of a certain level of de novo synthesis of dolichol may contribute to the capability of SV-3T3 cells to proliferate in serum-free medium.

Topics: Cell Cycle; Cell Division; Cell Line; Cell Transformation, Viral; Dolichols; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Mevalonic Acid; Simian virus 40; Ubiquinone