Serum-free Waymouth's Medium Research Articles

Tyrosine phosphatase SHP2 regulates the expression of acyl-CoA synthetase ACSL4

Acyl-CoA synthetase 4 (ACSL4) is implicated in fatty acid metabolism with marked preference for arachidonic acid (AA). ACSL4 plays crucial roles in physiological functions such as steroid synthesis and in pathological processes such as tumorigenesis. However, factors regulating ACSL4 mRNA and/or protein levels are not fully described. Because ACSL4 protein expression requires tyrosine phosphatase activity, in this study we aimed to identify the tyrosine phosphatase involved in ACSL4 expression. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, reduced ACSL4 protein levels in ACSL4-rich breast cancer cells and steroidogenic cells. Indeed, overexpression of an active form of SHP2 increased ACSL4 protein levels in MA-10 Leydig steroidogenic cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on ACSL4. The effects could be specifically attributed to SHP2 because knockdown of the phosphatase reduced ACSL4 mRNA and protein levels. Through the action on ACSL4 protein levels, SHP2 affected AA-CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 led to increased (or decreased) steroid production. We describe for the first time the involvement of SHP2 activity in the regulation of the expression of the fatty acid-metabolizing enzyme ACSL4.

Gene expression analysis in human fetal retinal explants treated with docosahexaenoic acid.

To explore the effect of docosahexaenoic acid (DHA) on gene expression during human fetal retinal maturation. Human fetal retinal explants were cultured in serum-free Waymouth's medium supplemented with DHA or oleic acid (OA), using bovine serum albumin (BSA) as the vehicle. After 14 days in culture, fatty acid composition was assessed, and the abundance of 2400 cDNAs was examined with a human cDNA microarray system. Transcript abundance remained unchanged for 82% and 90% of genes in the explants with added DHA or OA, respectively. Decreased expression was detected in 4% and 9% of genes, in explants supplemented with DHA or OA, respectively, whereas, 14% of genes in explants exposed to DHA and only 0.4% of genes in explants treated with OA showed increased expression. Transcripts displaying changes in abundance in explants supplemented with DHA encode for proteins involved in diverse biological functions, including neurogenesis, neurotransmission, and refinement of connectivity. These gene expression changes were not observed in explants supplemented with OA. The effect of DHA deficiency on retinal function during human development can be partly explained by modifications in retinal gene expression by direct or indirect mechanisms.

Antisense inhibition of epidermal growth factor receptor decreases expression of human surfactant protein A.

Epidermal growth factor (EGF) stimulates surfactant protein A (SP-A) synthesis in fetal lung tissue through ligand binding to the EGF receptor. We hypothesized that inhibition of EGF receptor messenger RNA (mRNA) would block SP-A expression in human fetal lung tissue during alveolar type II cell differentiation in vitro. Midtrimester human fetal lung explants were maintained in serum-free Waymouth's medium for 3 to 5 d in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to the initiation codon region of EGF receptor mRNA. Sense and scrambled ONs similarly modified were used as additional controls. The concentration of EGF receptor mRNA was semiquantitatively determined by reverse transcriptase/polymerase chain reaction (RT-PCR). We found a significant 3-fold decrease in EGF receptor mRNA levels in the antisense-treated groups compared with the control group with no effect in the sense condition. Immunohistochemical staining revealed a decrease in the amount of staining for EGF receptor protein in distal pulmonary epithelial cells in the antisense-treated groups compared with either control or sense conditions. Treatment with antisense EGF receptor ON decreased both SP-A mRNA and protein compared with controls with no effect in the sense condition. The ONs did not affect tissue viability as measured by the release of lactate dehydrogenase. We conclude that selective degradation of EGF receptor mRNA with antisense ON treatment results in a decrease in SP-A expression in human fetal lung. These findings support the critical importance of the EGF receptor for the regulation of SP-A gene expression during human alveolar type II cell differentiation.

Novel Negative Regulatory Element in the Platelet-derived Growth Factor B Chain Promoter That Mediates ERK-dependent Transcriptional Repression

Platelet-derived growth factor (PDGF), which consists of an A and/or B chain, stimulates migration and proliferation in vascular smooth muscle cells as well as a large number of other cell types. Investigations over recent years have defined roles for several positive regulatory transcription factors in the PDGF-B promoter. However, little is known about the transcriptional mechanisms that negatively regulate this gene. Here, we used transient transfection and 5' deletion analysis to define a specific region in the PDGF-B promoter-mediating repression in vascular smooth muscle cells. Gel retardation assays revealed this region is bound by nuclear protein(s) in a specific manner. Supershift assays excluded the direct association of Sp1, Sp3, and Egr-1. Mutation of the negative regulatory element no longer supported nucleoprotein complex formation and, when introduced into the PDGF-B promoter, rescued the promoter from repression. Promoter activity was also restored by transfection of oligonucleotide decoys bearing the repressor binding site. The MEK1/2 inhibitor, PD98059, and a dominant negative construct generating inactive ERK1 increased reporter expression driven by the PDGF-B promoter. In contrast, the MEK inhibitor had no effect on the activity of the mutant PDGF-B promoter. These effects were cell type-specific, since neither suppression of the PDGF-B promoter nor nucleoprotein complex formation was observed in vascular endothelial cells. These findings define a distinct negative regulatory element in the PDGF-B promoter that interacts with nuclear protein(s) and inhibits PDGF-B promoter-dependent gene expression in an ERK-dependent manner.

Glucocorticoid regulation of epithelial sodium channel genes in human fetal lung.

Pulmonary epithelial Na+ channels (ENaC), composed of three distinct subunits (alpha, beta, and gamma), play a critical role in the regulation of fluid reabsorption from airspaces of late-gestation fetal lung. We studied the expression of ENaC subunit genes in cultured human fetal lung. All three mRNAs were expressed at low levels in second trimester lung (13-32% of adult values at 24 wk gestation). There was a spontaneous increase of approximately threefold over preculture values of all three subunits within 24 h of explant culture in serum-free Waymouth's medium. Dexamethasone (Dex) induced all three mRNAs by two- to threefold. Maximal induction was noted by 8 h with 30-100 nM Dex and half-maximal stimulation with 3-10 nM Dex. Cycloheximide decreased basal expression of all three subunits by 8 h but did not alter the response to Dex. Actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), inhibitors of RNA polymerase II, decreased the basal and the Dex-induced expression of all three subunits with a more marked effect on human hENaC-gamma than on hENaC-alpha or hENaC-beta. Under conditions where transcription was blocked by actinomycin D or DRB, Dex did not alter the stability of the three mRNAs. Triiodothyronine (T3) at low (2 nM) or high (100 nM) concentrations had no effect on the expression of the three subunits in the presence or absence of low (10 nM) or high (100 nM) concentrations of Dex for 8 or 24 h. Similarly, 8-bromoadenosine 3',5'-cyclic monophosphate (2 microM) had no effect on basal or Dex-induced increase in the three subunits. We conclude that the three Na+ channel subunit genes are expressed in second trimester human fetal lung and are coordinately upregulated by glucocorticoid hormones but not by T3 or adenosine 3',5'-cyclic monophosphate. Glucocorticoid induction is receptor mediated, is primarily transcriptional, and does not require the induction of an intermediate protein for transcriptional enhancement. We speculate that induction of lung ENaC may contribute to the beneficial effects of antenatal glucocorticoids in premature babies.

Adenovirus-mediated gene transfer to human fetal lung ex vivo

Gene therapy of the fetus or newborn infant is a potentially useful approach for prevention or treatment of specific lung diseases. To begin to address issues such as efficiency, duration, and cellular distribution of transgene expression, we studied transduction of human lung cells by recombinant, replication-deficient adenovirus containing the lacZ gene driven by the beta-actin promoter and cytomegalovirus enhancer (H5.010CBlacZ). Human fetal lungs of 20- to 24-wk gestation received approximately 10(11) viral particles by instillation into a major bronchus, and the tissue was cultured as explants in serum-free Waymouth's medium. beta-galactosidase staining (X-gal) was detected by 24 h in defined regions of treated tissue and localized to epithelial cells of airways and terminal saccules. beta-galactosidase activity in homogenate of treated tissue was maximal 3-5 days after exposure to virus, ranging from 0.2 to 1.5 A420.min-1.mg protein-1 in four experiments (control values were approximately 0.001). When virus was added directly to lung explants in culture, beta-galactosidase was expressed in most of the peripheral cells and rarely in interior cells, the level of activity was dose dependent between 10(8) and 10(11) viral particles/ml, and transgene expression was sustained for at least 28 days. Treatment of isolated cultured cells with virus resulted in equivalent staining of both epithelial cells and fibroblasts. We conclude that fetal lung cells are efficiently transduced by recombinant adenovirus, indicating the feasibility of gene therapy in the infant or fetus. Cultured fetal lung may be useful for testing gene constructs being considered for therapy.

Restoration in vitro of normal rates of very-low-density lipoprotein triacylglycerol and apoprotein B secretion in hepatocyte cultures from diabetic rats.

Hepatocytes derived from diabetic rats were cultured in serum-free Waymouth's medium containing various supplements, after an initial 4 h period during which the cells were allowed to attach to the culture dish in the presence of foetal-bovine serum (10%). After removal of serum, these cells secreted much less very-low-density lipoprotein (VLDL) apoprotein B (apoB) and triacylglycerol than those derived from normal rats when cultured for 24 h in the basal medium. Inclusion of oleate (0.75 mM) in the medium initially increased the output of apoB and triacylglycerol, but the rates remained lower than those observed in normal hepatocytes and declined to zero after 72 h. This time-dependent decline in VLDL output was prevented by addition of dexamethasone to the oleate-containing medium. Levels of apoB and triacylglycerol output characteristic of normal hepatocytes could only be completely restored, however, by further addition of a mixture of lipogenic substrates (lactate plus pyruvate) to the medium. Restoration of normal levels of VLDL secretion in diabetic hepatocytes in vitro by this means was accompanied by a normal inhibitory response of apoB and triacylglycerol output to short-term (24 h) treatment with insulin or glucagon. Exposure of the cells to insulin for 72 h enhanced the secretion of VLDL, whereas treatment with glucagon for the same period potentiated the original inhibitory effect. The defective secretion of VLDL apoB observed when diabetic hepatocytes were cultured in the basal medium for 24 h could also be rectified by inclusion of a mixture of oleate (0.75 mM), lactate (10 mM), pyruvate (1 mM), dexamethasone (1 microM) and insulin (78 nM) in the medium during the 4 h attachment period in the presence of serum. Under these conditions, the increase in the secretory response of triacylglycerol was not so pronounced.

Long-term culture of hepatocytes: effect of hormones on enzyme activities and metabolic capacity.

(i) Hepatocytes isolated from adult rats were cultured for 2 to 3 weeks on collagen in a modified, serum-free Waymouth medium containing fatty acids and varying concentrations of glucocorticoid, insulin and glucagon. (ii) In the presence of all three hormones, it was possible to maintain the content of DNA, the activity of glucokinase, pyruvate kinase, hexokinase and lactate dehydrogenase at initial levels for 2 to 3 weeks. The activity of glucokinase and pyruvate kinase was affected by the concentration of insulin. (iii) The activity of alcohol dehydrogenase was stable for 3 days and declined to about 25% of the initial level after 2 weeks of culture, irrespective of the presence of hormones. (iv) Maintenance of albumin secretion was dependent on the presence of glucocorticoid, and glucocorticoid and insulin showed an additive or, at some time points, a synergistic effect on its secretion. (v) The content of cytochrome P-450 could be kept at 65% of the initial level, provided that a relatively high concentration of dexamethasone was present (10(-6) M). (vi) In the absence of hormones, urea synthesis was 70% of initial levels throughout the experimental period. With insulin and glucocorticoid present, a high concentration of glucagon (10(-8) M) was required to maintain the synthesis of urea at this level. (vii) It is concluded that hepatocyte cultures as described in the present study may be a useful, well-defined system for long-term metabolic, pharmacologic and toxicologic studies.

Glucocorticoids and thyroid hormones stimulate biochemical and morphological differentiation of human fetal lung in organ culture.

We characterized the stimulatory effects of both glucocorticoids and thyroid hormones on the surfactant system in human fetal lung. Synthesis of phosphatidylcholine (PC) and morphology were examined in explant cultures (15-24 weeks gestation) maintained 1-7 days in serum-free Waymouth's medium in a 95%-air-5% CO2 atmosphere. Control explants (no hormones) had the same rate of choline incorporation into PC between 1 and 7 days, but a significant increase in tissue PC content [82 +/- 21%, (+/- SEM), day 6 vs. 1], consistent with slow turnover of PC. [3H]Choline incorporation was stimulated 36%, 137%, and 192% by T3 (2 nM), dexamethasone (Dex; 10 nM), and T3 plus Dex, respectively, after 6 days of exposure (optimal response) compared to 19%, 38%, and 84% after 2 days of exposure. Thus, a supra-additive response occurred in the presence of both hormones and was greater at a shorter exposure time. Dex increased the percent saturation of newly synthesized PC (28.9 +/- 0.9% vs. 17.8 +/- 0.8% for control), but T3 did not, whereas both hormones increased tissue PC content (74.4 +/- 7.3% and 18.7 +/- 7.8% increase vs. control, respectively). Pulse-chase experiments with [3H]choline suggest that remodeling of unsaturated PC to saturated PC occurred during culture and was stimulated by Dex. Incorporation of [3H]acetate and [3H]glycerol into PC was stimulated by Dex (830% and 77%, respectively), but not by T3; the distribution of incorporated radioactivity among phospholipids was changed by Dex (increased counts per min into PC and phosphatidylglycerol with acetate and glycerol, respectively), but not by T3. Half-maximal stimulation of choline incorporation occurred at concentrations of Dex (2.1 nM) and T3 (0.03 nM) that are similar to the Kd values for receptor binding (5 and 0.05 nM, respectively). The relative potencies of thyroid hormones were T3 greater than T4 greater than rT3, and for corticosteroids, Dex much greater than corticosterone greater than 11-dehydrocorticosterone = cortisol greater than cortisone. Stimulation by either T3 or cortisol was reversed within 24-48 h of hormone removal. Initial treatment of explants with Dex enhanced the subsequent response to T3, but not vice versa. Culture for 4-5 days in the absence of hormones produced some morphological maturation of the epithelial cells, whereas treatment with T3 plus Dex markedly increased the number and size of lamellar bodies in epithelial cells, caused extensive proliferation of apical microvilli, and reduced glycogen deposits. Our findings are consistent with receptor-mediated stimulation of surfactant synthesis in human lung by both glucocorticoids and thyroid hormones.(ABSTRACT TRUNCATED AT 400 WORDS)

1395 HORMONES AND SURFACTANT SYNTHESIS IN EXPLANTS OF HUMAN FETAL LUNG

To further study the effects of glucocorticoids and thyroid hormones in fetal lung, we cultured human lung (16-22wk) for 4-8d as explants in serum-free Waymouth's medium with 95% air/5% CO2. Effects of dexamethasone (Dex, 10 nM) and T3 (2 nM) on phosphatidylcholine (PC) synthesis varied with precursor (Table).The additive hormonal effect occurred over a range of choline concentrations, did not alter the distribution of label among acid-soluble precursors, and correlated with tissue saturated PC content. Dex, but not T3, altered the distribution of precursor among phospholipids; compared to controls, glycerol incorporated more into PG (13.5 vs 4.9%) and less into PI (13.1 vs 18.9%), and acetate incorporated more into PC (81.5 vs 73.1%) and less into sphingomyelin (2.1 vs 6.3%). By electron microscopy, epithelial cells of treated explants showed less glycogen, many mo amellar bodies, and proliferation of microvilli. We conclude that low concentrations of glucocorticoids and thyroid hormone stimulate surfactant production in fetal lung in the absence of serum or other hormones. The two hormones appear to act at different biochemical sites to produce a synergistic response.

Thyroid hormone stimulation of phosphatidylcholine synthesis in cultured fetal rabbit lung.

To investigate the mechanism of thyroid hormone action on pulmonary surfactant synthesis, we characterized the effect of triiodothyronine on phosphatidylcholine synthesis in cultured fetal rabbit lung. Since glucocorticoids stimulate surfactant synthesis and reduce the incidence of Respiratory Distress Syndrome in premature infants, we also examined the interaction of triiodothyronine and dexamethasone. The rate of choline incorporation into phosphatidylcholine was determined in organ cultures of rabbit lung maintained in serum-free Waymouth's medium. In 23-d lung cultured for 72 h, the increase in choline incorporation with triiodothyronine alone, dexamethasone alone, and triiodothyronine plus dexamethasone was 50, 62, and 161%, respectively. Both triiodothyronine and dexamethasone also increased incorporation rates with glucose, glycerol, and acetate as precursors, and stimulation with triiodothyronine plus dexamethasone was at least additive. Dexamethasone, but not triiodothyronine, affected distribution of radioactivity from [3H] acetate among phospholipids. Stimulation was first detected 8-12 h after addition of triiodothyronine, and then increased in a linear fashion. With triiodothyronine plus dexamethasone, stimulation was maximal at 48-72 h, and was supra-additive at all times. Exposure of cultured lung to dexamethasone enhanced the subsequent response to triiodothyronine, but not vice versa. When triiodothyronine was removed from cultures, there was no further stimulation and the triiodothyronine effect was partially reversed within 24 h. Half-maximal stimulation of choline incorporation occurred at a triiodothyronine concentration (0.10 nM) very similar to the dissociation constant for triiodothyronine binding to nuclear receptor (0.11 nM). The relative potencies of thyroid hormone analogs for nuclear binding and stimulation of phosphatidylcholine synthesis were also similar: triiodothyroacetic acid greater than triiodothyronine-proprionic acid greater than L-triiodothyronine approximately D-triiodothyronine much greater than thyroxine much greater than 3,5-diethyl-3'-isopropyl-DL-thyronine approximately 3,5-dimethyl-3'-isopropyl-L-thyronine approximately reverse triiodothyronine. The effect of triiodothyronine was blocked by the presence of either actinomycin D or cycloheximide, inhibitors of ribonucleic acid and protein synthesis, respectively. We conclude that triiodothyronine stimulates phosphatidylcholine synthesis by a process involving nuclear receptors and de novo ribonucleic acid and protein synthesis. These findings support the concept that endogenous triiodothyronine has a physiologic role in lung maturation and suggest that a combined antenatal therapy with thyroid hormone and glucocorticoid may be useful for prevention of Respiratory Distress Syndrome in the premature infant.

GLUCOCORTICOID AND THYROID HORMONE STIMULATION OF PHOSPHATIDYLCHOLINE (PC) SYNTHESIS IN CULTURED HUMAN FETAL LUNG

We examined effects of dexamethasone (DEX) and T3 on PC synthesis in 40 specimens of human fetal lung (15-22 wk gestation) cultured for 4-8 d in serum-free Waymouth's medium with 95% air/5% CO2 on a rocker platform. In the absence of hormones, there was no consistent change in choline incorporation into PC and saturated PC during culture; PC content was 18.3 ± 2.3 μg PC-Pi/mg DNA in preculture tissue and 39.4 ± 3.4 (n=13) after 7 d culture. Choline into PC was stimulated by T3, DEX, and T3+DEX 11.9 ± 4.8, 36.6 ± 11.7, and 107 ± 36.5% (n=7), respectively, after 2 d exposure, and 51.6 ± 12.6, 103.4 ± 14.2, and 165 ± 16.8% after 6 d exposure. The content of PC increased 37.0 ± 7.6, 90.3 ± 16, and 125 ± 22% after 6 d with T3, DEX, and T3+DEX, respectively. The % saturation of newly synthesized PC was 19.9 ± 1.8, 20.9 ± 2.1, 26.4 ± 1.5 (P < .05), and 26.0 ± 1.8 (P < .05) in control, T3-, DEX-, and T3+DEX-treated cultures, respectively. Stimulation occurred by 24 h and was optimal after 4-6 d of exposure to the hormones. DEX was optimal at ˜10 nM and T3 at ˜1 nM. Cortisol was about 10% as potent as DEX and estriol was inactive. T4 and rT3 were about 10% and 1% as potent as T3. We conclude that both DEX and T3 increase saturated PC synthesis in cultured human lung, apparently acting through receptor-mediated mechanisms. We suggest that combined hormonal treatment may be more effective than glucocorticoid alone in stimulating surfactant synthesis.

Drug-induced porphyrin biosynthesis—XVIII. Effect of modulation of intracellular cyclic amp levels on drug-induced porphyrin biosynthesis in chick embryo liver cells maintained in serum-free medium

Chick embryo liver cells were maintained in serum-free Waymouth medium containing insulin and thyroxine. Several agents which are reported to increase intracellular adenosine 3': 5'-monophosphate (cyclic AMP) levels, viz. glucagon, sodium fluoride, cyclic AMP or its dibutyryl derivative, 3-isobutyl-1-methylxanthine and papaverine, enhanced allylisopropylacetanide (AIA)-induced porphyrin biosynthesis. The enhancement is particularly apparent at low doses of AIA. On the other hand, agents which are reported to decrease intracellular cyclic AMP levels, viz. alloxan and imidazole, diminished the ability of AIA to induce porphyrin accumulation. Adenosine, which is reported to inhibit cyclic AMP-dependent protein kinases, diminished the ability of AIA to induce porphyrin accumulation.

Drug-induced porphyrin biosynthesis—XVI. Effects of hydrocortisone, adenosine 3′:5′-cyclic monophosphoric acid, its dibutyryl derivative and a phosphodiesterase inhibitor on allylisopropylacetamide-induced porphyrin biosynthesis in chick embryo liver cells maintained in serum-free waymouth medium

Chick embryo liver cells were maintained in serum-free Waymouth medium containing insulin and thyroxine. Hydrocortisone (20.6 μM) enhanced allylisopropylacetamide (AIA)-induced δ-aminolevulinic acid (ALA) synthetase activity, as it was previously demonstrated to do in rat liver. It is likely that previous failures to observe a hydrocortisone effect in hepatic cell culture were due to the presence of hydrocortisone in serum added to the medium. Adenosine 3′: 5′-cyclic monophosphoric acid (cAMP; 0.3 mM) and its dibutyryl derivative (61 μM), although not essential for AIA induction of porphyrin biosynthesis, were observed to enhance the accumulation of porphyrins. In contrast, cAMP has been reported to be essential for AIA induction of ALA-synthetase activity in rat liver cell suspensions. The cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (135 μM), was shown to exert a similar effect on AIA-induced porphyrin biosynthesis as cAMP. It was concluded that cAMP enhances but does not function as a “second messenger” in AIA-induced porphyrin biosynthesis.

Drug-induced porphyrin biosynthesis—XV: Induction of porphyrin biosynthesis in chick embryo liver cells maintained in serum-free waymouth medium

Several drugs were shown to induce porphyrin accumulation in chick embryo liver cells maintained in serum-free Waymouth MD 705/1 medium. In this system, allylisopropylacetamide (AIA) was shown to increase δ-aminolevulinic acid (ALA)-synthetase activity 5-fold over control values. Addition of insulin to the medium resulted in enhanced porphyrin accumulation. Porphyrin accumulation in response to increasing doses of AIA, propylisopropylacetamide (PIA) and 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (Ox-DDC) was 3- to 4-fold greater in serum-containing than in serum-free medium. With 3,5-diethoxycarbonyl-1,4-dihydro-2.4.6-trimethylpyridine (DDC), however, porphyrin accumulation in response to increasing doses was slightly greater in serum-free than in serum-containing medium. In serum-free medium coproporphyrin accumulated in response to AIA and PIA, uroporphyrin in response to Ox-DDC. and a mixture of uro-, copro-, and protoporphyrin in response to DDC. The pattern of porphyrin accumulation in response to Ox-DDC and DDC was similar in serum-free and serum-containing medium. With AIA and PIA a mixture of uro-, copro- and protoporphyrin accumulated in serum-containing medium which contrasts with the accumulation of coproporphyrin alone in serum-free medium. It appears that porphyrin-inducing drugs might inhibit a variety of steps in the heme biosynthetic pathway.

The serial cultivation of suspended BHK-21/13 cells in serum-free Waymouth medium.

A simple medium system was developed to obtain growth of BHK-21 cells in shaker cultures in the absence of serum. These cells have now undergone over 80 serial passages in serum-free Waymouth medium and have been recovered from the frozen state after storage for over 1 month in medium containing 10% dimethyl sulfoxide (DMSO) and 1% bovine serum albumin (BSA). Various amounts of exogenous lipid in the form of sodium oleate were added to cultures of cells growing in serum-free Waymouth medium. Concentrations of 10-50 mug of sodium oleate/ml had no detrimental effects on the cells as measured by trypan blue uptake. Furthermore, the cells were serially passed ten times in the presence of 10 mug sodium oleate/ml. Depletion of calf serum from the growth medium and addition of known quantities of lipids to the system provides a means of revealing subtle changes in lipid synthesis and lipid turnover during cellular growth.