Apparent coordination of the biosynthesis of lipids in cultured cells: its relationship to the regulation of the membrane sterol:phospholipid ratio and cell cycling.

Abstract

The coordination of the syntheses of the several cellular lipid classes with one another and with cell cycle control were investigated in proliferating L6 myoblasts and fibroblasts . Cells cultured in lipid-depleted medium containing one of two inhibitors of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholesterol or compactin, display a rapid, dose-dependent inhibition of cholesterol synthesis. Inhibition of the syntheses of each of the other lipid classes is first apparent after the rate of sterol synthesis is depressed severalfold . 24 h after the addition of the inhibitor, the syntheses of DNA, RNA, and protein also decline . The inhibition of sterol synthesis leads to a threefold reduction in the sterol : phospholipid ratio that parallels the development of proliferative and G, cell cycle arrests and alterations in cellular morphology . All of these responses are reversed upon reinitiation of cholesterol synthesis or addition of exogenous cholesterol .A comparison of the timing of these responses with respect to the development of the G, arrest indicates that the primary factor limiting cell cycling is the availability of cholesterol provided either from an exogenous source or by de novo synthesis. The G, arrest appears to be responsible for the general inhibition of macromolecular synthesis in proliferating cells treated with 25-hydroxycholesterol . In contrast, the apparent coordinated inhibition of lipid synthesis is not a consequence of the G, arrest but may in fact give rise to it .Sequential inhibition of lipid syntheses is also observed in cycling cells when the synthesis of choline-containing lipids is blocked by choline deprivation and is observed in association with G, arrests caused by confluence or differentiation . In the nonproliferating cells, the syntheses of lipid and protein do not appear coupled .The study of the mechanism and control of membrane biogenesis is still in its infancy. One of the many unexplored areas is the coordination between the synthesis of membranes and the synthesis of DNA and cell cycling. We have begun to study the coupling between membrane lipid and macromolecular syntheses by culturing cells in lipid-depleted medium (LDM)' 'Abbreviations used in this paper: LDM, lipid-depleted medium ; 25-OH, 25-hydroxycholesterol; CMF-PBS, calcium, magnesium-free, 810 containing inhibitors of the synthesis of selective lipid pathways . In this way the cells are deprived of both the exogenous and endogenous source of a particular lipid.By this approach evidence for coordinated control of membrane and DNA syntheses has been found (7). The lipid phosphate-buffered saline ; Versene, 0.02% disodium EDTA in CMF-PBS; DMEM, Dulbecco's minimal essential medium; GLC, gas-liquid chromatography .

Phosphatidyl choline and the growth in serum‐free medium of vascular endothelial and smooth muscle cells, and corneal endothelial cells

“…Consistent with this hypothesis are the recent reports linking membrane phospholipid synthesis, in particular that of PC, to DNA synthesis (Hirata and Axelrod, 1980) and passage through the cell cycle (Cornell and Horwitz, 1980). These studies (Cornell and Honvitz, 1980;Hirata and Axelrod, 1980) emphasize that changes in phospholipid synthesis can lead to alterations in membrane composition and fluidity which may be important in triggering cell growth.…”

Growth stimulation of Rous sarcoma virus‐transformed BHK cells by biotin and serum lipids

“…6, Fig. 1) (Holley et al, 1974;Cornell et al, 1977; Cornell and Horwitz, 1980). Bettger et al showed that a lipid supplement improved the clonal growth of human diploid fibroblasts in a defined medium (Bettger et al, 1981).…”

“…L6 myoblasts cultured in medipm containing delipidized serum and biotin were arrested in G1 when an inhibitor of hydroxymethylglutaryl-CoA reductase, 25-hydroxycholestero1, was added to the medium (Cornell and Honvitz, 1980). Lipid synthesis occurred after the removal of 25-hydroxycholesterol and the cells then synthesized DNA and multiplied (Cornell and Horwitz, 1980). Phosphatidylinositol synthesis was increased in serum starved cells after serum or growth factor stimulation (Ristow et al, 1980).…”

Cholesterol availability modulates myoblast fusion.

“…To measure the protein synthesis rate by myoblasts in the presence of cycloheximide, [''H]leucine (2 pCi/ml) was added simultaneously with 10 kg/ml cycloheximide. At 10, 30, 45, 70, and 100 min thereafter, the radioactivity incorporated into TCA-precipitable material was determined by adding 5% TCA directly to the culture plates as described in the accompanying paper (5) . The rate of glycoprotein synthesis of 52-h myoblast cultures was estimated by adding ['"C]fucose (0 .5 yCi/ml) after 50 h in culture and measuring the radioactivity incorporated into cold 5% TCA-precipitable material at 52 h .…”