Changes in phospholipid metabolism of SV3T3 transformed fibroblasts restricted by plating and cultivation at low serum concentration and subsequently re-stimulated by serum

We have studied 32P-phosphate incorporation, using 1 hour pulses, into phospholipids of SV3T3 transformed fibroblasts plated and cultured in a 1% serum medium. Two successive periods were observed:On the first 2 days after plating, cells grow exponentially with a lowered rate of multiplication. The percentages of incorporation of 32P-phosphate into the different phospholipids are then unchanged, but the rate of incorporation is decreased.On the 4th day, cells are almost completely arrested, and the percentages of incorporation into the different phospholipids are then sharply altered.If, during the first period, cells are subsequently re-stimulated by a medium containing 10% serum, i.e. by simply increasing the rate of exponential growth, it can be observed a response of phospholipid metabolism (for instance the “PI response”) similar to that which was previously reported as characteristic of untransformed fibroblasts.     

Phosphorylation of chicken growth hormone

The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and gamma -32P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32P-phosphate labelled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer.     

Influence of potassium and calcium ions on the phosphatidylinositol and phosphatidylcholine metabolism in rat fibroblasts after growth stimulation by calf serum.

In secondary cultures of embryonic rat fibroblasts which were arrested in G1 (G0) by serum depletion and subsequently triggered into the cell cycle by readdition of growth factors isolated from fetal calf serum the influence of the potassium and calcium concentrations in the medium on phosphatidylinositol and phosphatidylcholine metabolism was investigated. The incorporation of inorganic [32P]phosphate into phosphatidylinositol is dependent on the potassium content of the culture medium. The specific activity of 32P in phosphatidylinositol is increased at K+ concentrations between 0.1 and 1 mM. Also calcium (between 0.01 and 2 mM) slightly stimulates phosphatidylinositol metabolism. Also the incorporation of myo-[3H]inositol is increased at potassium concentrations between 0.2 and 1 mM, whereas calcium is slightly inhibitory. The labelling of phosphatidylcholine with either [32P]phosphate or [3H]choline is not dependent on the potassium and calcium concentrations of the culture medium. Moreover, the phospholipid metabolism of permanently growing epithelioid and fibroblastoid cells lines, which were investigated, is considerably less dependent on the K+ and Ca2+ ions.     

Phosphate depletion decreases mitogen-mediated stimulation of phospholipid synthesis in human peripheral lymphocytes

Concanavalin A-mediated stimulation of ³²P-phosphate incorporation into phospholipids of human peripheral lymphocytes is comparatively studied in normal and phosphate-depleted media. In the phosphate-depleted medium, 2 hours after the start of cell activation, the stimulation sharply decreases for phosphatidylinositol (6.5-fold) and for phosphatidylcholine (in the latter case, the stimulation is even replaced by a slight inhibition of the incorporation). These results must be related to the rate-limiting effect of inorganic phosphate on ATP formation and thus on phospholipid synthesis, an effect which may be particularly pronounced when there is both phosphate depletion and cell activation.     

Increased hyaluronate synthesis is required for fibroblast detachment and mitosis.

Human-embryo fibroblasts were synchronized by means of colchicine and cytochalasin, and the production of hyaluronate was determined by [3H]glucosamine incorporation and ion-exchange chromatography. Cells arrested by colchicine synthesized small amounts of hyaluronate, whereas cells blocked by cytochalasin were stimulated in hyaluronate production. When the colchicine block was released, there was an increased synthesis of hyaluronate, which appeared first in the cellular fraction and was then shed into the culture medium. After release of the cytochalasin block, the hyaluronate production declined to that found with unsynchronized cells. A comparable increase of hyaluronate synthase activity was observed during mitosis. When hyaluronate synthesis was blocked by periodate-oxidized UDP-glucuronic acid, the cells were arrested in mitosis before rounding of cells. These results suggest that hyaluronate synthesis is required for detachment and rounding of cells during mitosis.     

Biotin-deficient growth of Bacillus polymyxa

When Bacillus polymyxa, a wild-type biotin auxotroph, is grown in biotin-deficient medium, a retardation of cell division and consequential cell elongation are the initial detectable consequences of limited biotin. Subsequent events in biotin-deficient cells include, in chronological order: inhibition of net ribonucleic acid (RNA) synthesis and a simultaneous arithmetical accumulation of protein; loss of net RNA, deoxyribonucleic acid, and protein synthesis; morphological aberration, death, and lysis. Incorporation studies employing ³²P-phosphate and ¹⁴CO₂ demonstrate an initial selective inhibition of net ribosomal RNA synthesis over that of ribosomal protein or total protein. Biotin could not be replaced by various extracts from which biotin had been removed, nor could osmotic stabilizers be found which could prevent lysis of the culture.     

Serum inhibition of proliferation of serum-free mouse embryo cells

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominantly diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.     

Isolated Coxiella burnetii synthesizes DNA during acid activation in the absence of host cells

Two populations of Coxiella burnetii were isolated from fibroblast tissue cultures and examined for their ability to synthesize DNA when incubated in a defined medium. Both the populations released by mechanical lysis of heavily infected host cells, as well as those recovered from the tissue culture medium, incorporated H3 32PO4 into DNA. Incorporation occurred at pH 4.5 but not at pH 7.0, and proceeded for 12-15 h. When incorporation of [3H]thymidine was studied, only the organisms obtained by mechanical lysis of host cells were active. Those which had been released by natural means into the tissue culture medium, and then recovered for study, did not incorporate precursor thymidine but were extremely active in protein biosynthesis. In mechanically released organisms, thymidine incorporation was inhibited immediately by rifamycin (40 microM) and hydroxyurea (10 mM), but it was not affected by chloramphenicol (310 microM) until 4 h after addition of the drug. Incorporation of H3 32PO4 by both populations of organisms was also inhibited by rifamycin, chloramphenicol and hydroxyurea, but the time sequence of inhibition differed. Southern hybridization utilizing 32P-labelled DNA suggested that both populations synthesized authentic chromosomal DNA sequences, as well as QpH1 plasmid DNA, during acid activation of metabolism.     

Persistent biologic actions of insulin on cultured fetal human fibroblasts

Summary Monolayer cultures of fetal human fibroblasts, preincubated in serum-free culture medium overnight (about 18 hr), were incubated with insulin (0.1 to 100 mU per ml), then washed and incubated in an insulin-free medium. The effect of insulin on glucose utilization, uridine incorporation into RNA and leucine incorporation into protein was maintained after removal of insulin and washing. For both glucose utilization and uridine incorporation into RNA, this effect was demonstrated at physiologic levels of insulin (0.1 mU per ml). When anti-insulin serum was added to the cultures after the cell preincubated with insulin were washed, this effect was greatly attenuated. This lasting effect of insulin was probably not due to nonspecifically bound insulin becoming available to the cells. Binding of¹²⁵I-monoiodoinsulin was examined in monolayer cultures of fetal human fibroblasts. When unlabeled insulin was present at about 1 mU per ml concentration, 50% displacement of monoiodoinsulin occured. When fibroblasts were incubated with monoiodoinsulin and then removed from the radioactive medium, initial dissociation of the bound hormone occurred rapidly but then reached a plateau. This prolonged insulin effect appears to result from persistent binding of insulin to its receptor. Supported in part by PHS Grants AM-02456, AM-05020 and AM-15312, by the Kroc Foundation and by the Diabetes Center (AM-17047). Supported in part by Research Career Development Award AM-47142 from NIAMDD.     

Incorporation of tritium into cell materials of Rhodopseudomonas spheroides from tritiated water in the medium under aerobic conditions.

When Rhodopseudomonas spheroides cells grown aerobically in the dark were incubated in medium containing tritiated water (THO), incorporation of T into the bacterial cell materials occurred under growth and no-growth conditions. The overall T incorporation under no-growth conditions was stimulated by vigorous aeration and was suppressed strongly in the presence of either 10(-3) M KCN or 0.3% HgCl2, indicating that the bulk of the incorporation might depend upon bacterial cell metabolism or respiration. 10 mug/ml chloramphenicol and 20 mug/ml rifamipicin slightly suppressed the T incorporation. The extent of T incorporation was proportional to the concentration of T in the medium. Accordingly, regardless of differences in the concentration of T in the medium, the maximum ratio of T content per hydrogen atom in the cell materials to that of THO in the medium was approximately 0.2 in non-growing cells and 0.5 in growing cells, whereas the value was 0.02-0.03 in cells incubated in medium containing KCN or HgCl2. The non-growing cells aerated in THO medium were lyophilized and fractionated by the modified method of Schneider. More than 40% of the total T incorporated into the cell materials was recovered in the cold PCA-soluble fraction, whereas the distribution of T into fractions solbule in ether-ethanol, hot PCA and alkali was 10 to 20% each. More than 75% of the T extracted in the cold PCA-soluble fraction was volatile. While the amounts of RNA and protein in the non-growing cells decreased on adding chloramphenicol or rifampicin, the distribution of T in these fractions did not change much. Our results on T incorporation into non-growing cells indicate that the major T incorporation into bacterial cell materials is independent of biosynthetic reactions using labeled precursors produced by the assimilation of T into metabolites, but presumably depends on energy-linked conformational changes of macromolecules.     

Resumption of cell cycle in BALB/c-3T3 fibroblasts arrested by polyamine depletion: relation with "competence" gene expression

Serum deprivation arrests BALB/c-3T3 fibroblasts (clone A31) in G0 phase, where resumption of the cell division cycle can be induced by addition of serum or of specific growth factors in a defined sequence: PDGF (inducer of a state of "competence," characterized by the expression of a family of genes including c-myc), epidermal growth factor EGF and IGF1 (Leof et al., 1982, 1983). When exponentially growing A31 cells are placed for greater than or equal to 2 days in a medium containing the alpha-difluoromethylornithine (alpha DFMO), an irreversible inhibitor of ornithine decarboxylase, they become arrested in G1 phase as a consequence of polyamine depletion (Medrano et al., 1983). In the alpha DFMO-arrested cells, addition of putrescine (60 microM) in a culture medium containing 6% fetal calf serum (FCS), but not in serum-free medium, is sufficient to induce G1 progression and entry into S phase (as determined by 3H-thymidine incorporation). The level of "competence" mRNAs is high in alpha DFMO-arrested cells. After addition of putrescine in FCS-containing medium, these mRNAs continue to be present for at least 3 h. A large proportion of alpha DFMO-arrested cells can be induced to progress to S phase by insulin (1 microM, acting via IGF1 receptor) plus putrescine in a serum-free medium (greater than or equal to 50% of FCS effect). In this case, the levels of "competence" mRNAs become low or undetectable within 3 h, EGF (10 nM) plus insulin had only slightly greater effect than insulin alone on the progression of alpha DFMO-arrested cells. When the alpha DFMO-arrested cells are subsequently incubated during 3 days in a low-serum-containing medium (0.25% FCS), they do not replenish their supply of polyamines, and then continue to express the c-myc gene. The recruitment of the polyamine-depleted, serum-deprived cells into the cell division cycle does not require PDGF and can be induced by addition of EGF and insulin plus putrescine. These data indicate that alpha DFMO arrests majority of the cells at a point situated beyond the PDGF- and EGF-dependent portion of G1 phase. During the subsequent serum deprivation, the alpha DFMO-arrested cells remain "competent" (PDGF-independent), probably as a consequence of their continued expression of c-myc (and possibly other PDGF-inducible genes).     

Quiescent SV40 virus transformed 3T3 cells in culture

Serum counteracts low nutrient concentrations in the culture medium in SV40 virus transformed 3T3 (SV3T3) cells. The transport of [³H]-leucine into TCA soluble material in SV3T3 cells is stimulated by serum and inhibited by But₂-cAMP. When SV3T3 cells are cultured in low leucine concentrations (? 8 × 10^?6 M), the cell's morphology is similar to the one of cells incubated in complete medium in the presence of But₂-cAMP and cells become quiescent. Cells become arrested throughout the cell cycle. The results suggest that the mechanism by which But₂-cAMP inhibits growth of SV3T3 cells is by inhibiting the transport of leucine in SV3T3 cells.     

Effect of vitamin K depletion and restoration on sphingolipid metabolism in Bacteroides melaninogenicus

Bacteroides melaninogenicus requires vitamin K for normal growth. Cells incubated in a vitamin K-free medium form defective cell envelopes. Studies with vitamin K-grown "K(+)" and vitamin K-depleted "K(-)" cells showed that [(14)C]choline and [(14)C]glycerol were not taken up, but several amino acids and acetate were incorporated to the same degree by both types of cultures. However, K(-) cells incorporated succinate to a greater degree than did K(+) cultures. The relative incorporation of succinate into ceramide phosphorylethanolamine and ceramide phosphorylglycerol was depressed compared with incorporation into phosphatidylethanolamine in K(-) cultures. B. melaninogenicus can be grown in serial subculture in the absence of vitamin K in the presence of 2.5 mg/ml of succinate. Under these conditions the relative incorporation of [2,3-(14)C]succinate and (32)P into ceramide phosphorylethanolamine and ceramide phosphorylglycerol is markedly depressed. Stimulation of phosphosphingolipid synthesis by vitamin K was shown by comparing the uptake of (32)P and lipid phosphorus levels of a succinate-grown, vitamin K-depleted culture supplemented with (32)P plus 0.1 micro g/ml vitamin K(1) with a similar culture supplemented with (32)P only. The phosphosphingolipids from the vitamin K-supplemented cells incorporated greater amounts of (32)P and had higher levels of phosphorus than did the ceramide phosphorylethanolamine and ceramide phosphorylglycerol of the culture without added vitamin K. It was further shown that vitamin K added to a vitamin K-depleted culture stimulated synthesis of ceramide phosphorylethanolamine and ceramide phosphorylglycerol 38 min and 60 min, respectively, following the addition of the vitamin; incorporation of (32)P into other phospholipids was unaffected.     

3''End labelling of RNA with 32P suitable for rapid gel sequencing.

A new general method of labelling the 2',3'-diol end of RNA with 32P has been devised suitable for gel sequencing. Poly(A) polymerase (E. coli) is incubated with the RNA and limiting amounts of alpha-32P-ATP. The mono-addition product is then cleaved with periodate and beta-eliminated with aniline, leaving the RNA terminally labelled with 3' 32P-phosphate. When applied to a model compound, tRNAPhe from E. coli, over 28 residues could be read from the 3' end.     

Characterization of endogenous phosphorylation in isolated cardiac sarcolemma

The cardiac sarcolemma contains kinases which catalyze the incorporation of 32P-phosphate into acid stable and acid precipitable membrane components of low molecular weight. The phosphorylation is not influenced by cyclic AMP or calmodulin. Analysis of phosphorylation products using proteolytic digestion, organic solvent extraction, thin layer chromatography and gel filtration reveals both polypeptides and lipids as kinase substrates. Polypeptides are phosphorylated at their serine and threonine residues, while lipid phosphorylation gives rise to 32P-labelled phosphatidylinositol phosphates and some nonidentified compounds. Phosphorylated polypeptides and phosphorylated lipids do not separate in SDS polyacrylamide gel electrophoresis. On the basis of the fast time course of 32P-phosphate incorporation, it may be supposed that endogenous phosphorylation may play a role in the short term regulation of the cardiac sarcolemmal function.     

Glucose-stimulated phosphorylation of yeast isocitrate lyase in vivo

Incorporation of 32P into Saccharomyces cerevisiae isocitrate lyase was observed after addition of glucose to a culture incubated with [32P]orthophosphoric acid. A band of 32P-labelled protein was coincident with the enzyme band when immunoprecipitates were subjected to SDS-PAGE and autoradiography. No label was found in the band corresponding to the isocitrate lyase when immunoprecipitation was done with a control pre-immune serum or in the presence of excess pure unlabelled enzyme. The incorporation of phosphate was associated with a decrease in enzyme activity. Phosphorylated isocitrate lyase was not proteolytically degraded when cells were cultured in mineral medium. The loss of protein antigenicity only took place when the yeast was grown in a complex medium containing glucose.     

Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine

The absorption of light by photoreceptor cells leads to an increased incorporation of [2-3H]inositol into phosphoinositides of horizontal cells in the retina of Xenopus laevis in vitro. We have identified several retinal neurotransmitters that are involved in regulating this response. Incubation with glycine, the neurotransmitter of an interplexiform cell that has direct synaptic input onto horizontal cells, abolishes the light effect. This inhibition is reversed by preincubation with strychnine. Acetylcholine added to the culture medium enhances the incorporation of [2-3H]inositol into phosphoinositides in horizontal cells when retinas are incubated in the dark. This effect is inhibited by preincubation with atropine. However, atropine alone does not inhibit the light-enhanced incorporation of [2-3H]inositol into phosphoinositides in the retina. gamma-Aminobutyric acid, the neurotransmitter of retinal horizontal cells in X. laevis, as well as dopamine and norepinephrine, have no effect on the incorporation of [2-3H]inositol into phosphoinositides. These studies demonstrate that the light-enhanced incorporation of [2-3H]inositol into phosphoinositides of retinal horizontal cells is regulated by specific neurotransmitters, and that there are probably several synaptic inputs into horizontal cells which control this process.     

Phosphatidylethanolamine biosynthesis in rat mammary carcinoma cells that require and do not require ethanolamine for proliferation

Epithelial cells and some of their transformed derivatives require ethanolamine to grow normally in defined culture medium. When these cells are cultured without ethanolamine, the amount of cellular phosphatidylethanolamine is considerably reduced. Using a set of rat mammary carcinoma cell lines whose growth is responsive (64-24 cells) and not responsive (22-1 cells) to ethanolamine, the biochemical mechanism of ethanolamine responsiveness was investigated. The biosynthesis and metabolism of phospholipid, particularly of those involving phosphatidylethanolamine, were thus compared between the two types of cells. The incorporation of [3H]serine into phosphatidylserine and phosphatidylethanolamine in 64-24 cells was 60 and 37%, respectively, of those in 22-1 cells. However, the activity of phosphatidylserine decarboxylase was virtually the same in these cell lines. When these cells were cultured in the presence of [32P]phosphatidylcholine and [32P]phosphatidylethanolamine, the rate of accumulation of 32P-labeled phosphatidylserine from the radioactive phosphatidylethanolamine was considerably reduced in 64-24 cells compared to that in 22-1 cells, although the rate of synthesis of phosphatidylserine and phosphatidylethanolamine from the radioactive phosphatidylcholine was similar between the two cell lines. The rate of labeling phosphatidylcholine from the radioactive phosphatidylethanolamine was also reduced in 64-24 cells, although the difference was not as great as that of phosphatidylserine. Incorporation of 32P into phosphatidylethanolamine was correlated with the concentration of ethanolamine in the culture medium in 64-24 cells, whereas in 22-1 cells the incorporation was not influenced by ethanolamine. Enzyme activities of the CDP-ethanolamine pathway were not significantly different between the two cell lines. The rate of degradation of phosphatidylethanolamine was also similar in these cell lines. These results show that ethanolamine responsiveness of 64-24 cells, and probably other epithelial cells, is due to a limited ability to synthesize phosphatidylserine resulting from a limited base-exchange activity utilizing phosphatidylethanolamine.     

Metabolism, Macromolecular Synthesis, and Nuclear Behavior of Cryptococcus albidus at 37 C

The temperature-sensitive events which prevent Cryptococcus albidus from growing at 37 C were investigated. Cultures incubated at 37 C immediately after inoculation did not increase in optical density nor in cell numbers, and by 24 h 90% of cells in such cultures were deformed and dead. When cultures in log phase were shifted from 23 to 37 C the optical density increased but the cell numbers did not. Morphological observations revealed that the increase in turbidity at 37 C represented enlargement and distortion of cells without appreciable replication. Uptake and incorporation of (14)C-leucine were similar at 23 and 37 C. There was no difference in (14)CO(2) evolution from cells at either temperature. Uptake and incorporation of adenine-8-(14)C into RNA was slightly lower in cells incubated at 37 C. There was, however, a 60% reduction in incorporation of adenine-8-(14)C into DNA after 3 hr at 37 C. Nuclear staining revealed that nuclear migration did not occur in cells incubated at 37 C. Thus the data indicate that both adenine incorporation into DNA and nuclear migration prior to nuclear division by C. albidus are temperature sensitive.     

Effect of Modification of Membrane Phospholipid Composition on Phospholipid Methylation in Aggregating Cell Culture

The effect of the presence of nitrogenous bases in the growth medium of fetal rat brain aggregating cell cultures was investigated. The presence of either N-methylethanolamine (MME) or N,N-dimethylethanolamine (DME) in the growth medium resulted in significant increase of the corresponding phospholipid, phosphatidyl-N-monomethylethanolamine (PMME) or phosphatidyl-N,N-dimethylethanolamine (PDME). They represented 28% and 32% of the total phospholipids, respectively. The presence of the new phospholipids was accompanied by a significant decrease of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Cells grown in the presence of ethanolamine or choline had only barely detectable amounts of PMME and PDME. Intact cells previously grown with the bases were incubated with [methyl-3H]methionine. Incubation of cells previously grown in presence of the bases MME and DME resulted in a marked increase of radioactivity in the corresponding phospholipids possessing one additional methyl group, PDME and PC respectively. The incorporation of S-adenosyl[methyl-3H]methionine (AdoMet) was examined in cell homogenates incubated in presence or absence of either PMME or PDME acceptors. The addition of these exogenous phospholipids caused a three-or fourfold stimulation of radioactivity incorporated into the total phospholipids of cells grown in the absence of nitrogen bases. The cells grown in presence of either MME or DME in the culture medium did not show an increased incorporation of methyl groups from AdoMet into the total phospholipids after addition of exogenous acceptors. This work suggests that MME and DME incorporated into the corresponding phospholipids function as effective substrates for phospholipid-N-methylation.