The effects of triiodothyronine,hydrocortisone and insulin on lipid synthesis by cultured fibroblasts preincubated in a serum-free medium

Studies of lipid metabolism in cell cultures are usually carried out after preincubation of cells in media containing lipoprotein-deficient or delipidated serum. The artifacts produced during delipidation prevent the standardization of assays and the study of the role of hormones on lipid metabolism. We studied the effects of triiodothyronine, hydrocortisone, insulin and their combination on cholesterol and fatty acid synthesis in cultured human skin fibroblasts preincubated for 24 h in an artificial medium (medium A) consisting of equal volumes of Dulbecco's modified Eagle's and Ham's F-12 media enriched with transferrin, biotin and calcium pantothenate. In cells preincubated in medium A the incorporation of acetate to cholesterol and the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase were much lower than in cells preincubated in standard medium containing lipoprotein-deficient serum. Addition of the three hormones caused a marked stimulation of the incorporation of acetate to cholesterol (from 3.1 to 17.7 pmol/min per mg protein), an activity similar to that in cells preincubated in lipoprotein-deficient serum plus hormones. The stimulatory effect of the hormones on HMG-CoA reductase activity was smaller, from 11 to 26 pmol/min per mg protein compared to 83 pmol/min per mg protein in cells preincubated in lipoprotein-deficient serum plus hormones. Most of the stimulatory effect was due to insulin. The lack of coordinate response between these two parameters in cells preincubated in artificial medium could not be explained by (a) stimulation of a post-mevalonate step as measured by the incorporation of mevalonate to cholesterol; (b) the in vitro inactivation of HMG-CoA reductase by phosphorylation: incubation of fibroblast microsomes with Escherichia coli alkaline phosphatase resulted in a decrease in HMG-CoA reductase activity, in contrast to an increase in hepatic microsomes; (c) the presence of inhibitors of HMG-CoA reductase in the microsomal extract. In cells preincubated in medium A the incorporation of acetate to fatty acids and the activities of acetyl-CoA carboxylase and fatty acid synthetase were approximately equal to that of cells preincubated in standard medium containing lipoprotein-deficient serum. Hormones added to medium A caused a stimulation of incorporation of acetate to fatty acids (from 5.1 to 19.8 pmol/min per mg protein), the activity of acetyl-CoA carboxylase (from 494 to 820 pmol/min per mg protein) and of fatty acid synthetase (from 300 to 678 pmol/mg protein). These values were significantly higher than those obtained in cells preincubated with lipoprotein-deficient serum with or without hormones. Results indicate that (1) the artificial medium is useful in the study of lipid metabolism, especially of fatty acid synthesis; (2) the activity of HMG-CoA reductase cannot be taken as the sole index of cholesterol synthesis in cultured skin fibroblasts under all experimental conditions.     

Effect of testicular hyaluronidase on hyaluronate synthesis by human skin fibroblasts in culture

The effect of hyaluronidase treatment on the incorporation of [3H]glucosamine into hyaluronate in human skin fibroblast cultures was investigated. Fourth passage cells in confluent cultures were treated with hyaluronidase from bovine tests, Streptomyces and leech in Dulbecco's minimum essential medium in the presence of 3% fetal calf serum. The medium was removed from the control (non-treated) and the treated cultures and the washed cell layers were incubated with [3H]glucosamine and [35S]sulfate. [3H]Hyaluronate was separated by DEAE Trisacyl chromatography and identified by specific enzymic assays. Hyaluronidase treatment induced an increase in the amount of labelled hyaluronate secreted into the medium and into the pericellular compartment. This amount reached a plateau with increasing enzyme concentration and with the time of treatment. Oligosaccharides derived from hyaluronate did not produce this effect. The maximal increase was about 3-fold, and was not inhibited by exogenous hyaluronate (25-100 micrograms/ml) or by oligosaccharides from hyaluronate. Cycloheximide (0.03 mM) inhibited hyaluronate synthesis by 18% or less in the control cells and by 50% in the hyaluronidase-pretreated fibroblasts. No significant difference was found in the hyaluronate synthase activity between control and treated cells, at 60 min following treatment, indicating the reversibility of the effect. The persistence of the stimulation required the presence of hyaluronidase. The treatment of cells with specific hyaluronidases (from Streptomyces and leech) or with testicular hyaluronidase did not modify the labelling of the sulfated glycosaminoglycans. The incorporation kinetics of the [3H]glucosamine into labeled hyaluronate and the increased amount of non-labelled hyaluronate determined by radiometric assay indicated a specific stimulation of hyaluronate synthesis in the hyaluronidase-pretreated fibroblast cultures.     

Acetate Utilization and Macromolecular Synthesis During Sporulation of Yeast

Acetate utilization and macromolecule synthesis during sporulation (meiosis) of Saccharomyces cerevisiae were studied. When diploid cells are transferred from glucose nutrient medium to acetate sporulation medium at early stationary phase, respiration of the exogenously supplied acetate proceeds without any apparent lag. At the completion of ascospore development, 62% of the acetate carbon consumed has been respired, 22% remains in the soluble pool, and 16% is incorporated into lipids, protein, nucleic acids, and other cell components. Measurements of the rate of protein synthesis during sporulation reveal two periods of maximal synthetic activity: an early phase coincidental with increases in deoxyribonucleic acid, ribonucleic acid, and protein cellular content and a later phase during ascospore formation. Experiments in which protein synthesis was inhibited at intervals during sporulation indicate that protein synthesis is required both for the initiation and completion of ascus development.     

Clonal selection in cultured human fibroblasts: role of protein synthetic errors

Protein synthetic error frequency, determined in cell-free extracts as delta leu/delta phe incorporation following poly(U) stimulation, has been found to decrease progressively in several strains of human diploid fibroblasts during their limited replicative lifespan. To explore the basis of this phenomenon, we followed a mass (uncloned) culture of one normal strain at 13 stages of its replicative lifespan. We found a progressive tenfold decline in error frequency that was inversely correlated with passage level (r = -.93, p less than .001). This could not be ascribed to the slow rates of replication associated with fibroblast senescence because slowing of growth by serum deprivation did not change error frequency. Additionally, terminal mass cultures maintained for 16 wk at saturation density to minimize cell selection did not change error frequency over this time. Error frequencies in 12 individual clones purified from the parental culture did not decline on repeated passage, either remaining constant or, in two clones, rising abruptly three- to five-fold after initial assays. Error frequencies of clones showed a weak inverse correlation with growth vigor but not with the maximum doubling number. We conclude that selective pressures favor more vigorously dividing clones with low protein synthetic error frequencies leading to their predominance in mass cultures.     

Protein SH-group concentration and RNA synthesis during the process of induction of proliferation in the stationary phase of cell culture growth]

The dynamics of intracellular protein SH-group (PSH) content was studied cytochemically in the course of stimulation of cell proliferation in stationary cultures of an established Chinese hamster cell line and of human diploid embryo fibroblasts. The results were compared with the pattern of RNA synthesis during the prereplicative period. In Chinese hamster cells immediately after medium changing in stationary cultures there is an augmentation of PSH content in parallel withe the increase in RNA synthesis rate. Later on, the rate of RNA synthesis and PSH content are seen decreasing followed by a new increase in the rate of RNA synthesis correlated with the second rise in PSH content. In stationary cultures of human diploid fibroblasts, there is also an increase in the rate of RNA synthesis and in the content of SH after medium changing, but the second wave of RNA synthesis and the second rise in PSH content are not pronounced. The variation in PSH content reflects the shift in the cell metabolism during the prereplicative period and is not attributed to changes in cell protein content.     

High-density lipoprotein that supports Ureaplasma urealyticum growth.

A high-density lipoprotein with growth-promoting activity for Ureaplasma urealyticum was purified in high yield from equine serum by ammonium sulfate fractionation and molecular filtration. Fractions enriched in growth-promoting activity represented 5% of the total serum protein, and 30 micrograms of the purified protein per ml gave an activity equivalent to that from 100 micrograms of whole serum per ml. The serum was totally replaced by purified lipoprotein when tested in a soy peptone-yeast dialysate or when added to a chemically defined synthetic medium. Polyacrylamide gel electrophoresis indicated that one major protein with growth-promoting activity is present. A total of 10 proteins were distinguished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 75% of the total contributed by two proteins with molecular weights of 160,000 and 170,000. A total of 90% of the lipoprotein was an alpha-protein with a mobility of 0.67 in two-dimensional immunoelectrophoresis (albumin = 1.0). The active component was further characterized as high-density lipoprotein by density ultracentrifugation. Two components with S = 6.4 and S = 15.8 were distinguished by velocity sedimentation. The lipid was removed from lipoprotein during its precipitation with acetone. The growth-promoting activity of delipidized protein was dependent upon the addition of exogenous cholesterol, and [14C]cholesterol was transferred to urea-plasmic cells in cultures containing the delipidized protein. A major portion of the [14C]cholesterol remained associated with the protein during filtration on Sepharose 4B columns.     

The effects of phorbol ester on anabolism of lipids of cultured human skin fibroblasts

We have investigated the independent effects of phorbol ester (phorbol 12-myristate 13-acetate) on anabolism of the major lipid components in cultured diploid human skin fibroblasts. When we incubated these cells with [3H]acetate in serum-free medium for 18 h in the presence of 16 nM phorbol ester, [3H]acetate incorporation and the cellular content of cholesterol ester increased, and free cholesterol decreased. Enhancement of [3H]acetate incorporation into cholesterol ester was also observed when the cells were incubated with phorbol ester for 5 h in medium containing lipoprotein-deficient serum. Incorporation of [3H]galactose into glycosphingolipids increased many fold upon exposure of the cells either to fetal calf serum or separately to phorbol ester. Therefore, phorbol ester independently affects cholesterol and glycosphingolipid metabolism in a way that may be similar to that reported for serum low-density lipoproteins and unknown other factors in fetal calf serum. We have observed these effects of phorbol ester in the intact living cell. These findings should provide useful means for the study of metabolism of the plasma membrane lipid components in fibroblasts.     

Lipid requirement for cell cycling : The effect of selective inhibition of lipid synthesis

Tissue culture cells require lipid which must be provided exogenously or synthesized via endogenous pathways. The exogenous supplies can be largely removed by growing cells in medium containing delipidized serum. Pathways for synthesis of lipid can then be blocked at three steps: (1) fatty acids by removal of biotin, an essential coenzyme; (2) phosphatidylcholine and sphingomyelin by deleting choline from the growth medium; and (3) cholesterol by inhibiting HMG-CoA reductase with 25-hydroxycholesterol. Sustained proliferation is prevented when lipid synthesis is blocked at any one of these steps. Cell proliferation resumes upon restoring synthesis with biotin, choline, or mevalonate (the product of the HMG-CoA reductase reaction) or by providing the lipid end products oleic acid or cholesterol. Using a combined cytophotometric-autoradiographic analysis to determine cell cycle distributions we have demonstrated that prereplicative (G1) cell cycle arrests develop in parallel with the proliferative inhibition. Each of the G1 arrests can be reversed by restoring the synthetic pathways or their lipid products. These observations suggest a causal relationship between the supply of lipids and passage through G1.     

Retention of human skin fibroblast fatty acid modifications during maintenance culture

Summary The fatty acid composition of cultured human skin fibroblasts was modified by adding either oleic or linoleic acid to the growth medium. After the cultures became confluent, they were washed and transferred to different maintenance media in order to determine the stability of the various fatty acyl modifications. Some changes in fatty acid composition occurred under all conditions. When the maintenance medium was supplemented with fatty acid, the cellular neutral lipid and phospholipid fatty acyl composition were altered markedly within 16 to 24 hr. If no supplemental fatty acid was available during the maintenance period, however, the modified fatty acyl compositions were sufficiently retained so that appreciable differences between the cells enriched with oleate and linoleate persisted for at least 48 to 72 hr. This considerable degree of stability occurred when either 10% delipidized fetal bovine serum or 10% fetal bovine serum containing its inherent lipids were present in the maintenance medium. Although the triglyceride content of the fatty acid-modified cells was quite labile, neither the cholesterol nor phospholipid content changed appreciably during culture in any of the maintenance media. Since the fatty acid compositional differences persisted during several days of maintenance under certain conditions, these modified cultures appear to be a useful experimental system for assessing the effect of lipid structure on fairly long-term cellular functions. This work was supported by Arteriosclerosis Specialized Center of Research Grant HL14230 from the National Heart, Lung and Blood Institute, National Institutes of Health.     

Fatty acid synthesis and metabolism of phospholipid acyl groups in strain L mouse fibroblasts

Strain L mouse fibroblasts grown in medium supplemented with 2.5% delipidized horse serum were found capable of desaturating oleic and linoleic acid to dienoic and trienoic acid(s), respectively. Although 40-60% of de novo fatty acid synthesis from [2-3H]acetate was inhibited by the administration of exogenous oleic or linoleic acid, sterole synthesis was only slightly affected. Within 24-48 h after incorporation, phospholipid fatty acyl groups could undergo active exchange between phospholipids. After this dynamic transition period was over, not only were the phospholipid acyls retained but some vicinal fatty acyl pairs of phospholipid also appeared to be stable and remained together throughout the depletion period. At any time in the experiment, however, introduction of exogenous fatty acid perturbed this phospholipid acyl retention, delayed the time at which the phospholipid acyl groups no longer moved between phospholipids and also decreased the ultimate number of phospholipid acyl groups retained by strain L mouse fibroblasts.     

Improved maintenance of adult rat alveolar type II cell differentiation in vitro: effect of hydrocortisone and cyclic AMP

We have examined the effect of hydrocortisone and cyclic AMP on the maintenance of lipid synthesis in primary cultures of adult rat alveolar type II cells. These hormones were tested in the presence of either 1% or 5% charcoal-stripped rat serum (CS-rat serum). The effect of substratum on responsiveness to these hormones was evaluated by comparing cells cultured for 4 days on tissue culture plastic, on floating type I collagen gels, on rat lung fibroblast feeder layers on floating collagen gels (floating feeder layers), and on Engelbreth-Holm-Swarm (EHS) tumor basement membrane gels. Type II cells cultured on floating feeder layers in medium containing 1% CS-rat serum and 10(-5) M hydrocortisone plus 0.5 mM dibutyryl cyclic AMP exhibited significantly increased incorporation of [14C]acetate into total lipids (238% of control). The hormone combination also increased the relative percentage of acetate incorporated into phosphatidylglycerol (PG; 7.3% versus 1.9%) and saturated phosphatidylcholine (PC; 43.6% versus 37.6%). The percentage of acetate incorporated into neutral lipids was significantly decreased by the addition of hormones (28.6% versus 70.0%). The addition of hydrocortisone and cyclic AMP to medium containing 5% CS-rat serum resulted in an increase in the relative incorporation of acetate into saturated PC (51.2% versus 46.4%), but had no effect on the relative incorporation of acetate into PG or on the incorporation of acetate into total lipids. Type II cells cultured on EHS gels in medium containing 1% CS-rat serum plus hydrocortisone and cyclic AMP showed increased acetate incorporation into total lipids (204% of control) and a relative decrease in the percentage of acetate incorporated into neutral lipids (16.9% versus 47.0%). The hormone combination also increased the relative incorporation of acetate into PG (4.4% versus 2.5%) and saturated PC (49.9% versus 42.1%). Hydrocortisone and cyclic AMP added to medium containing 5% CS-rat serum concentration increased the relative incorporation of acetate into saturated PC by type II cells on EHS gels, but these additions had no effect on acetate incorporation into PG. No responses to these soluble factors were seen when type II cells were cultured on floating type I collagen gels without feeder layers or on tissue culture plastic. These data indicate that there are positive interactions between substratum, soluble factors and serum in the maintenance of differentiated function of adult rat alveolar type II cells in vitro.     

Sterol metabolism and oral epithelial cell growth

Summary Previous studies have demonstrated that as the density of cultured oral epithelial cells increases, there is a concomitant increase in phospholipids and cholesterol ester synthesis and a decrease in that of cholesterol and sterol precursors. Other studies have suggested that the effects of exogenous cholesterol sulfate may be similar to growth responses and influence metabolic steps related to cell density. To further examine this possibility, in the present study lipid synthesis was monitored in hamster cheek pouch epithelial cells in cultures established at different cells densities and in the presence of varying amounts of exogenous cholesterol sulfate. Cell [¹⁴C]acetate incorporation into lipids was measured in cultures established at four densities ranging from very subconfluent to very dense (postconfluent) in two media, Dulbecco’s modified Eagle’s medium (DMEM) with 5% fetal bovine serum and KSFM, a non-serum containing keratinocyte medium. Results indicated that the relative proportion of radiolabel incorporated into different lipid classes changed with cell density. In DMEM, the percentage of radiolabel incorporated into total phospholipids and fatty acids increased significantly with increasing cell density whereas percent incorporation into cholesterol, sterol precursors, and cholesterol esters significantly decreased. In KSFM cultures, proportionate phospholipids labeling was significantly increased in more dense cultures whereas cholesterol and cholesterol esters labeling was significantly decreased. In subconfluent and confluent cultures exposed to 10 or 25μM cholesterol sulfate, the relative proportions of phospholipid labeling also increased significantly compared to dimethyl sulfoxide (solvent) controls, whereas sterol precursors, fatty acids, and cholesterol esters labeling was signifcantly decreased. These results indicate that cholesterol sulfate can affect cellular lipid synthesis in a manner similar to that which occurs with increasing cell density, and strengthen the hypothesis that cholesterol sulfate may regulate lipid metabolic pathways related to growth and differentiation.     

Promotion of tick cell growth by proline and fractions from tick eggs

Tick cell lines representing three genera (Rhipicephalus appendiculatus, Dermacentor variabilis and Boophilus microplus) were grown in basal medium containing different supplements. Their effect on cell growth was measured by determining the rate at which cultures were transferred. The optimal concentration of the foetal bovine serum was 5% for the Boophilus line and 10% for the other two. Addition.of tick egg extract (TEE) increased the splitting rate 1.5–2.4 times during a period of 30 days, and changed the morphology of some cells. The crude TEE was delipidized and the protein and lipid fractions were analyzed for biological activity. The delipidized protein fraction of the TEE improved the splitting rate to about the same degree as the crude TEE. Adding l-proline to the basal medium increased the splitting rate 1.2–1.9-fold, or nearly to the same degree as did the crude TEE. The addition of the lipid and aqueous polar fractions of TEE did not improve cell growth.     

The effect of serum components on sterol biosynthesis in L cells

L cells were cultivated in test medium which contained 14C-sodium acetate, and the amount of labeled digitonin-precipitable sterol was assayed in medium and cells. Increasing concentrations of whole serum in the medium had two effects: depressed cellular synthesis and enhanced release of synthesized sterol from the cells. In experiments with delipidized serum containing unesterified cholesterol, cellular sterol synthesis decreased as free cholesterol concentration in the medium increased. In other experiments using medium containing increasing lecithin concentration and no exogenous sterol, the concentration of lecithin markedly influenced the distribution of synthesized sterol between the cells and the medium which then directly influenced the amount of sterol synthesized. These experiments indicate that cell sterol synthesis is regulated by internal levels of free sterol. This, in turn, is a function of cellular sterol flux which is regulated by the concentration and composition of serum lipoprotein in the medium.     

Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: Modulating effect of retinoic acid

Summary Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 μM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.     

Inhibition of dolichyl phosphate biosynthesis by compactin in cultured rat hepatocytes

Isolated rat hepatocytes were cultured in monolayer for about 24 h. During this period the cells exhibited constant protein and lipid synthesis. When the culture medium contained compactin, a competitive inhibitor of the 3-hydroxyl-3-methylglutary-coenzyme-A reductase, dolichyl-P synthesis was inhibited by 91% at the end of the incubation, as estimated by the incorporation of [3H]acetate and by 77% as estimated by the incorporation of 32Pi. These results indicate that in primary cultures of rat hepatocytes dolichyl monophosphate is mainly synthesized through a de novo process, while phosphorylation through the CTP-mediated kinase is of limited functional importance.     

Effects of cholesterol sulfate on lipid metabolism in cultured human keratinocytes and fibroblasts

Effects of cholesterol sulfate on acetate incorporation into lipid fractions were examined in normal human fibroblast and keratinocyte cultures. Inhibition of sterologenesis in normal fibroblast cultures by cholesterol sulfate was less profound than that produced by either lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate also inhibited sterologenesis in low density lipoprotein receptor-deficient fibroblasts and inhibited both sterologenesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in keratinocytes. Cholesterol sulfate increased incorporation of acetate into fatty acid-containing lipids in preconfluent cultures of both cell types in lipoprotein-depleted media. Similar effects were not observed either in response to lipoprotein-containing serum or 25-hydroxycholesterol. Cholesterol sulfate had no effect on oleic acid incorporation into diglycerides, triglycerides, or phospholipid fractions; neither did it inhibit acid lipase activity; nor did it inhibit fatty acid oxidation, indicating that cholesterol sulfate does not inhibit catabolism of acyl lipids. Because cholesterol sulfate had similar effects on fatty acid metabolism in steroid sulfatase-deficient fibroblasts lines, desulfation to cholesterol is not a prerequisite. Cholesterol sulfate did not significantly affect incorporation of oleic acid into sterol esters in fibroblast cultures, but in contrast, inhibited sterol esterification in keratinocyte cultures. These data suggest a novel role for cholesterol sulfate as a modulator of cellular lipid biosynthesis.     

Studies on the stimulation of dolichol-mediated glycosylation by dexamethasone in HeLa cells.

The effect of the addition of 1 microM-dexamethasone on the temporal sequence of the glycosylation process has been studied in HeLa S3G cells. In the presence of delipidized serum, dexamethasone caused an increase in 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and an accelerated synthesis of dolichols. These events were followed by an increase in the transfer of mannose from GDP-mannose to mannolipid. An increase in the rate of synthesis of lipid-linked oligosaccharides and a stimulation of glycosylation were observed in cells grown in the presence of delipidized serum in the culture medium. The data are consistent with the view that cellular syntheses of lipids and glycoproteins are co-ordinately controlled.     

Incorporation of radioactive precursors into glycosaminoglycans by rat muscle fibroblasts exposed to a solubilized rat bone matrix fraction

Confluent cultures of rat muscle fibroblastic cells respond by increased glycosaminoglycan (GAG) synthesis when cultured in medium containing a solubilized bone matrix fraction (SBM) at a concentration of 100 micrograms/ml. The metabolism of the GAG associated with the cell pellet, the cell surface and the tissue culture medium fractions was studied, in the presence and absence of SBM, by measuring the incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the isolated GAG. Net synthesis of hyaluronic acid and of chondroitin sulfate in the medium fraction increased more rapidly in cultures containing SBM compared to controls, and the accumulation of labelled GAG in the medium of the treated cultures was approximately linear with respect to the length of incubation. The addition of SBM also resulted in increased incorporation of 3H and of 35S into the GAG of the cell surface and cell pellet fractions. In these fractions, stimulation of incorporation of radioactivity occurred in two waves: an early, relatively minor increase and a later relatively major increase. The relatively major stimulation of radioactivity into the GAG of the cell surface fraction occurred between 24 and 48 h and was independent of any apparent effect of serum.     

Growth stimulation of rous sarcoma virus-transformed BHK cells by biotin and serum lipids

Biotin or a serum lipid extract stimulated proliferation of G1 arrested Rous sarcoma virus-transformed BHK cells in modified Eagle's MEM (BM). The cells could be maintained continuously in BM plus biotin (BMB), but not in BM plus serum lipid extract (BM X L). Avidin inhibited growth stimulation when added to BMB, but did not inhibit growth when added to BM X L. 14C-acetate incorporation into total cellular lipids was stimulated in BMB, but not in BM. Thin-layer chromatography of the labeled cellular lipid extract indicated that relatively large amounts of 14C-acetate were incorporated into phosphatidylserine and little into the other major phospholipids. In the neutral lipids, the largest amount of incorporation was in cholesterol. G1 arrested cells multiplied rapidly in BM supplemented with dialyzed serum (BM X DS), but they did not multiply in BM with delipidized serum (BM X DLS). The addition of biotin or serum lipid extract to BM X DLS stimulated growth. Growth stimulation in BM X DLS by biotin was inhibited by avidin, but avidin had no effect on growth stimulation by serum lipid extract. Biotin stimulated additional multiplication in BM X DS and avidin inhibited this additional growth stimulation. These results suggest that growth stimulation requires lipids supplied by serum lipids or by de novo synthesis stimulated by biotin. In the absence of serum, the stimulation of the synthesis of growth factor(s) by biotin are also required for continuous multiplication.