Phosphatidylethanolamine synthesis in ethanolamine-responsive and -nonresponsive cells in culture

Mammalian cells can be classified into two types based upon whether or not they show growth response to ethanolamine (Etn) in culture. The content of phosphatidylethanolamine (PE) in phospholipid and incorporation of radioactive Etn into the cells were examined in the Etn-responsive and -nonresponsive cells in order to elucidate the mechanisms of growth stimulation by Etn. In all Etn-responsive cells tested, 5 microM Etn significantly altered the composition of cellular phospholipid compared to that grown without Etn, while Etn-nonresponsive cells had a similar phospholipid composition whether the growth medium contained Etn or not. Using two rat mammary carcinoma cell lines, 64-24 (responsive type) and 22-1 (nonresponsive type), further studies were carried out. In 64-24 cells there was a proportional increase in PE content as the dosage of Etn in the medium was increased. The increase in PE content leveled off at 10 microM. Further, the increase in PE content was correlated with increased rate of growth. In contrast, PE content or growth rate did not change at all in 22-1 cells. In 64-24 cells radioactive Etn (0.1-50 microM) was incorporated four- to five-fold more efficiently into phospholipid, and the aqueous pool of precursors of PE was ten times less as compared to 22-1 cells, indicating that Etn-responsive cells utilize Etn supplied in the medium to synthesize PE far more efficiently than Etn-nonresponsive cells. De novo synthesis of PE must not be sufficient to support optimum growth in Etn-responsive cells.     

Incorporation of [3H]ethanolamine into acetylcholine by a human cholinergic neuroblastoma clone

Human neuroblastoma cholinergic LA-N-2 cells were used as an experimental model to test the possibility that the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) and free choline (Cho) (Andriamampandry et al. 1989) could contribute to acetylcholine (AcCho) synthesis. LA-N-2 cells were incubated with [³H]Cho for 90 min and 22.7% of the radioactivity was present in PCho, 18.5% in free Cho and 4.8% as AcCho. The ratio of Cho/AcCho, however, was of about 1 after 16 hours of incubation. The incorporation of 10M [³H]ethanolamine (Etn) into MeEtn, PMeEtn, PMe₂Etn and their corresponding phospholipids was reduced in cells incubated in medium containing 7.2M choline as compared to cells incubated in medium devoid of choline indicating that the lack of Cho from the incubation medium stimulated the conversion of PEtn to Cho water soluble derivatives. Incubation of LA-N-2 cells with [³H]Etn led to the labelling of [³H]AcCho. Cultures incubated in parallel with [³H]Cho showed that roughly 10% of [³H]AcCho obtained after 16 hrs of incubation with the Cho label derived from [³H]Etn. The synthesis of Cho and AcCho from Etn may be enhanced after cellular differentiation induced by the growth of the cells in the presence of retinoic acid (RA). The results indicate that the methylation of [³H]Etn and/or of [³H]PEtn may be used by cholinergic neurons as precursor for AcCho.Abbreviations Etn ethanolamine - MeEtn monomethylethanolamine - Me₂Etn dimethylethanolamine - P- phosphoryl - AcCho acetylcholine - Ptd phosphatidyl - LPtd lysophosphatidyl - RA retinoic acid     

The conversion of ethanolamine and of its metabolites to choline in human neuroblastoma clones: Effect of differentiation induced by retinoic acid

The sequential methylation of ethanolamine (Etn) or phosphorylethanolamine to the corresponding choline (Cho) derivatives was studied in both undifferentiated and retinoic acid (RA) differentiated human neuroblastoma clones LA-N-1 and LA-N-2. Conversion of Etn derivatives to the respective Cho metabolites was low in both cell types. However, after treatment of the cultures with ethanol or RA, the methylation of phosphoryl-Etn was stimulated while that of phosphatidyl-Etn was severely reduced in both cholinergic LA-N-2 and catecholaminergic LA-N-1 cells.Abbreviations Etn ethanolamine - MeEtn monomethylethanolamine - Me₂Etn dimethylethanolamine - Cho choline - PEtn phosphorylethanolamine - PtdEtn phosphatidylethanolamine - LPtdEtn lysophosphatidylethanolamine - RA retinoic acid     

Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine.

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)-deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane-associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well-known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn-requiring rat mammary cell line 64-24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12,13-dibutyrate (PDBu) under conditions where cells have either normal or PE-deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (approximately fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE-deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP-choline pathway of PC synthesis indicated that the regulatory enzyme, CTP:phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE-deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation.     

Partial purification of a phosphoethanolamine methyltransferase from rat brain cytosol

The conversion of phosphoethanolamine to phosphocholine requires 3 separate N-methyltransferases. We had previously purified the enzyme catalyzing the last methylation, phosphodimethylethanolamine N-methyltransferase. We have successfully purified the enzyme catalyzing the initial methylation of phosphoethanolamine. A 434 fold purified enzyme from rat brain was obtained by the sequential use of ammonium sulfate fractionation, Q-Sepharose fast flow column chromatography and a -aminoethyl agarose column chromatography. The pH optimum was 11 or greater, the Km value for phosphoethanolamine was 167.8±41.7 M and the Vmax was 487.3±85 mmoles/mg/hr. The kinetics for S-adenosyl-methionine, the methyldonor, has characteristics of cooperative binding with a Km of 1.805±0.59 mM and a Vmax of 16.9±3.6 moles/mg/hr. The activity was stimulated 6 fold by 2.5 mM MnCl₂ and inhibited by DZA and S-adenosylhomocysteine. These results reinforce the early in vivo observations which had provided suggestive evidence for the existence of a pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain.Abbreviations used Adomet S-adenosylmethionine - AdoHcy S-adenosyl-homocysteine - CAPS 3-(cyclohexyl)amino-1-propanesulphonic acid - Cho choline - 3-DZA 3-deazaadenosine - Etn ethanolamine - N-MT N-methyltransferase - PEG polyethyleneglycol - PMSF phenylmethanesulphonyl fluoride - PEtn phosphoethanolamine - PCho phosphocholine - PMe₂Etn phosphodimethylethanolamine - PtdCho phosphatidylcholine - PtdEtn phosphatidylethanolamine     

Ethanolamine is a co-mitogenic factor for proliferation of primary hepatocytes.

Mature adult parenchymal hepatocytes can enter the S phase in the presence of growth factors such as HGF and EGF, but rarely proliferate in culture. We hypothesized that the cell cycle of hepatocytes in culture is restricted before G(2)/M phase and we attempted to identify the factor that induces cell cycle progression. We found that the conditioned medium from long-term cultured hepatocytes contained co-mitogenic activity with other growth factors, which was attributed to ethanolamine (Etn). Etn induced not only DNA synthesis but also cell replication of cultured hepatocytes with various other growth factors. Etn and HGF synergistically induced cyclin D(1), A and B expression, however, only cyclin B but not cyclin A formed a complex with Cdc2. In addition, Etn combined with HGF enhanced PKCbetaII expression and translocated PKCbetaII to the plasma membrane, and induced filopodia formation, which was inhibited by an antisense oligonucleotide against PKCbetaII. In addition, blocking the cytoskeleton rearrangement with inhibitors (colchicine, cytochalasin D, or chlerythrine (a specific PKC inhibitor)) inhibited cyclin expression and cell proliferation. Although Etn enhanced the downstream product, cellular phosphatidylethanolamine (PE), PE itself did not show any Etn-like activities on hepatocytes. Taken together, our results indicate that Etn functions as a co-replication factor to promote the cell cycle of mature hepatocytes to G(2)/M phase in the presence of growth factors. The activity is thought to be mediated by PKCbetaII-dependent cyclin B expression.     

31P and 1H NMR studies of ethanolamine-linked phosphoglycerides metabolism in human T lymphocytes.

Aqueous and organic extracts of peripheral human T lymphocytes and of T lymphoblastoid cell lines have been examinated by 31P and 1H NMR spectroscopy in order to study the metabolism of ethanolamine (Etn) linked phosphoglycerides. The results show that the Etn concentration in the culture medium determines the composition of Etn-containing metabolites and phospholipids. The effect of phorbol esters, stimulating the synthesis and the breakdown of choline-containing phospholipids has been also studied. A phorbol 12-myristate 13-acetate (PMA) dependent membrane phosphatidylethanolamine hydrolysis, presumably mediated by protein kinase C activity, has been demonstrated.     

Ethanolamine modulates DNA synthesis through epidermal growth factor receptor in rat primary hepatocytes

Summary Ethanolamine (Etn) stimulates hepatocyte proliferation in vivo and in vitro; however, the physiological function of Etn in hepatocytes has yet to be elucidated. In the present study, we examined the effect of Etn using a primary culture of rat hepatocytes. The level of membrane phosphatidylethanolamine (PE) significantly decreased when the hepatocytes were cultured without Etn but increased to the level found in the liver when the culture medium was supplemented with 20–50 μM Etn. Moreover, Etn stimulated DNA synthesis in a dose-dependent manner and had a synergistic effect with epidermal growth factor (EGF). A binding assay and Western blotting showed that the number of EGF receptors was 22–30% lower in cells grown in the absence of Etn compared to those grown in its presence, but the respective Kd values were almost the same. Furthermore, tyrosine phosphorylation of the EGF receptor was significantly lower in cells grown without Etn. Phosphatidylcholine (PC) synthesis in the liver is unique in that it occurs via stepwise methylation of PE. We found that without Etn supplementation, bezafibrate-induced inhibition of PE methylation increased the level of PE by decreasing its conversion to PC and stimulated DNA synthesis. Moreover, the function of EGF in stimulating DNA synthesis was significantly enhanced under Etn-sufficient conditions. These data suggest that Etn is a nutritional factor required for synthesis of adequate PE, levels of which are important for hepatocyte proliferation.     

Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth

Toxoplasma gondii is an intracellular protozoan parasite capable of causing devastating infections in immunocompromised and immunologically immature individuals. In this report, we demonstrate the relative independence of T. gondii from its host cell for aminoglycerophospholipid synthesis. The parasite can acquire the lipid precursors serine, ethanolamine, and choline from its environment and use them for the synthesis of its major lipids, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), respectively. Dimethylethanolamine (Etn(Me)(2)), a choline analog, dramatically interfered with the PtdCho metabolism of T. gondii and caused a marked inhibition of its growth within human foreskin fibroblasts. In tissue culture medium supplemented with 2 mm Etn(Me)(2), the parasite-induced lysis of the host cells was dramatically attenuated, and the production of parasites was inhibited by more than 99%. The disruption of parasite growth was paralleled by structural abnormalities in its membranes. In contrast, no negative effect on host cell growth and morphology was observed. The data also reveal that the Etn(Me)(2)-supplemented parasite had a time-dependent decrease in its PtdCho content and an equivalent increase in phosphatidyldimethylethanolamine, whereas other major lipids, PtdSer, PtdEtn, and PtdIns, remained largely unchanged. Relative to host cells, the parasites incorporated more than 7 times as much Etn(Me)(2) into their phospholipid. These findings reveal that Etn(Me)(2) selectively alters parasite lipid metabolism and demonstrate how selective inhibition of PtdCho synthesis is a powerful approach to arresting parasite growth.     

Phosphatidylethanolamine deficiency in membrane lipids inhibits keratinocyte intercellular networks formation

Ethanolamine (Etn) is required for the growth of epithelial cells in culture. Without Etn, the amount of phosphatidylethanolamine (PE) in membrane lipids is reduced, and cell proliferation stops. When the membrane lipids are deficient of PE, some extracellular signaling processes become impaired. In this study, we examined the effect of Etn deprivation on the formation of intercellular networks in immortalized human oral keratinocytes. Keratinocytes proliferate with undifferentiated morphologies in a low-calcium medium, whereas they undergo differentiation to form intercellular networks in a high-calcium medium. The cells were first cultured with or without Etn supplement in a low-calcium (0.07 mM) medium, and then the calcium concentration was raised to 1.8 mM. The localization and organization of the following proteins were examined: (1) desmogleins and plakoglobin in desmosomes, (2) E-cadherin and beta-catenin in adherens junctions and (3) actin and keratin filaments in cytoskeletons. As expected, in the Etn-supplemented cells, the elevated level of calcium induced the junctional localization of the proteins associated with desmosomes and adherens junctions and also induced the formation of keratin and actin networks. On the contrary, in the Etn-deprived cells, the elevated level of calcium induced none of the above processes. The results suggest that having a sufficient amount of PE or proper phospholipid composition in the membranes is crucial for differentiation in epithelial cells.     

Rat and human mammary tissue can synthesize choline moiety via the methylation of phosphatidylethanolamine.

The normal mammal requires large amounts of choline for maintenance and growth of tissue mass. Since milk, the only food for neonates, has many-fold higher free choline concentration than does maternal plasma, it is possible that mammary gland can synthesize choline molecules. The only known mammalian pathway for the synthesis de novo of choline molecules is catalysed by phosphatidylethanolamine N-methyltransferase (PeMT), which synthesizes phosphatidylcholine (PtdCho) via sequential methylation of phosphatidylethanolamine (PtdEtn) using S-adenosylmethionine (AdoMet) as a methyl donor. We identified PeMT activity in rat mammary tissue, and differences in affinities for substrate, as well as in activities as a function of pH, suggest that at least two distinct enzyme activities are involved [i.e. one catalysing the methylation of PtdEtn to form phosphatidyl-N-methylethanolamine (PtdMeEtn) and the other catalysing the methylation of PtdMeEtn and phosphatidyl-NN-dimethylethanolamine (PtdMe2Etn) to form PtdMe2Etn and PtdCho, respectively]. The relationships between AdoMet concentrations and PtdCho formation from endogenous PtdEtn in rat mammary homogenate were complex: a sigmoidal component (with a Hill coefficient of 2.2), requiring 55 microM-AdoMet for half saturation (Vmax. = 9 pmol/h per mg of protein), and a high affinity component (Kapparent = 8.7 microM and Vmax. = 3.8 pmol/h per mg of protein) were identified. When exogenous PtdMe2Etn was added as substrate, PtdCho formation exhibited Michaelis-Menten kinetics for AdoMet, and its affinity for AdoMet was high (Kapparent = 9 microM, Vmax. = 85 pmol/h per mg of protein). In the presence of endogenous substrates, the rates of PeMT-catalysed PtdCho formation within homogenates of rat mammary tissue were similar in tissue from lactating and non-lactating animals. When exogenous PtdMe2Etn was added to homogenates of rat mammary tissue, tissue from lactating rats made twice as much PtdCho as did tissue from non-lactating rats. Isolated mammary epithelial cells also exhibited PeMT activity; the rate of formation of PtdCho was much greater in intact versus broken cells. We also identified PeMT activity in homogenates of mammary tissue from non-lactating humans. The rate of PtdCho formation was of similar magnitude to that seen in rat tissue. This evidence supports the hypothesis that some of the choline found in milk could have been synthesized de novo in the mammary gland.     

Conversion of ethanolamine, monomethylethanolamine and dimethylethanolamine to choline-containing compounds by neurons in culture and by the rat brain.

The incubation of neurons from chick embryos in primary culture with [3H]ethanolamine revealed the conversion of this base into monomethyl, dimethyl and choline derivatives, including the corresponding free bases. Labelling with [methyl-3H]monomethylethanolamine and [methyl-3H]dimethylethanolamine supported the conclusion that in chick neuron cultures, phosphoethanolamine appears to be the preferential substrate for methylation, rather than ethanolamine or phosphatidylethanolamine. The methylation of the latter two compounds, in particular that of phosphatidylethanolamine, was seemingly stopped at the level of their monomethyl derivatives. Fetal rat neurons in primary culture incubated with [3H]ethanolamine showed similar results to those observed with chick neurones. However, phosphoethanolamine and phosphatidylethanolamine and, to a lesser extent, free ethanolamine, appeared to be possible substrates for methylation reactions. The methylation of water-soluble ethanolamine compounds de novo was further confirmed by experiments performed in vivo by intraventricular injection of [3H]ethanolamine. Phosphocholine and the monomethyl and dimethyl derivatives of ethanolamine were detected in the brain 15 min after injection.     

Administration of Myo-inositol Plus Ethanolamine Elevates Phosphatidylethanolamine Plasmalogen in the Rat Cerebellum

Plasmalogens are ether-linked phospholipids highly abundant in nervous tissue. Previously we demonstrated that acute administration of myo-inositol (myo-Ins) + [2-¹³C] ethanolamine ([2-¹³C]Etn) significantly elevated phosphatidylethanolamine plasmalogen (PlsEtn) in rat whole brain. Current experiments investigated the effects of acute myo-Ins+[2-¹³C]Etn administration on [PlsEtn] and the biosynthesis of new Etn lipids using NMR spectroscopy in rat cerebral cortex, hippocampus, brainstem, midbrain and cerebellum. Treated rats received a single dose of myo-Ins+[2-¹³C]Etn and controls received saline rather than myo-Ins. Data reveal that the cerebellum is the brain region most affected by treatment, which resulted in a 22% increase in [PlsEtn] and 89% increase in newly synthesized Etn lipids relative to controls (P 0.05). Furthermore, the cerebellar PlsEtn/phosphatidylethanolamine ratio and molar percentage of PlsEtn were significantly elevated by 12% and 8%, respectively (P 0.05). These data suggest that myo-Ins influences Etn lipid metabolism in brain, particularly in the cerebellum where there is a stimulation in the biosynthesis of new Etn lipids with a preference towards PlsEtn.     

Phosphatidylethanolamine turnover is an early event in the response of NB2 lymphoma cells to prolactin

The effect of prolactin on phospholipid metabolism in the prolactin-dependent rat lymphoma cell line Nb2 was investigated in cells prelabeled with [3H]arachidonic acid or [3H]ethanolamine. Prolactin (20 ng/ml) caused (a) a 20-60% loss of radiolabeled phosphatidylethanolamine within 0.5 to 2 min, (b) a loss of [3H]ethanolamine-labeled phosphatidylethanolamine from crude membranes, (c) a rapid accumulation of [3H]phosphoethanolamine and [3H]ethanolamine, and (d) a transient increase (15 s to 2 min) in prostaglandin F2 alpha and E2. Arachidonic acid (1-2 micrograms/ml) induced Nb2 cell growth but prostaglandin F2 alpha, E2, ethanolamine, and phosphoethanolamine did not. Prostaglandin E2 inhibited while prostaglandin F2 alpha enhanced growth in the presence of prolactin or arachidonic acid. These results suggest that stimulation of Nb2 cell growth by prolactin is linked to activation of a phosphatidylethanolamine-specific phospholipase C. Arachidonic acid and prostaglandin F2 alpha may participate in regulating the mitogenic action of prolactin.     

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.     

Insufficiency of transformation by simian virus 40, polyomavirus, EJ-ras, or v-myc oncogenes for conversion of ethanolamine-responsive mammary cells to ethanolamine-nonresponsive cells.

Normal mammary epithelial cells (ethanolamine responsive) require ethanolamine to enable them to grow in defined culture medium because they cannot synthesize de novo a sufficient amount of phosphatidylethanolamine. Mammary tumor cells which retain properties of the normal tissue are also likely to be ethanolamine responsive, whereas dedifferentiated, highly tumorigenic mammary tumor cells are ethanolamine nonresponsive. The nonresponsive tumor cells are able to synthesize the necessary amount of phosphatidylethanolamine to sustain growth. Therefore, the progression of malignancy seems to convert ethanolamine-responsive mammary cells to ethanolamine-nonresponsive ones. In an attempt to prove the above assumption and to understand the mechanism responsible for the conversion during the progression of malignant transformation, mammary tumor cell line 64-24, which is typically ethanolamine responsive, was transfected with simian virus 40, polyomavirus, EJ-ras, or v-myc oncogenes, and the resulting transfectants were examined for their growth response to ethanolamine. Many of the transfectants exhibited typical transformed phenotypes; however, none of the transfectants converted to ethanolamine-nonresponsive cells. Some of the SV40 and polyomavirus transformants were able to grow in the absence of ethanolamine, although they grew better in the presence of ethanolamine, unlike typical ethanolamine-nonresponsive cells. These cells could grow in the absence of ethanolamine, even though their membrane phospholipid was phosphatidylethanolamine deficient. The present study indicates that the expression of any one of the four oncogenes tested, which allows the cells to exhibit transformed phenotypes in 64-24 cells, is not sufficient for the conversion of ethanolamine-responsive cells to -nonresponsive cells.     

Role of thrombin in the proliferative response of T-47D mammary tumor cells : Mitogenic action and pleiotropic modifications induced together with epidermal growth factor and insulin

The growth of the human metastatic cell line (T-47D) in a chemically defined medium (DM) is shown to be dependent on the presence of three traditional growth factors: epidermal growth factor, insulin, and transferrin. The addition of thrombin further stimulates its growth. The mitogenic action on a human mammary tumor cell line from epithelial origin is a novel action of thrombin. Cells in the DM show striking morphological changes which are dramatically enhanced by the addition of thrombin. These observations are part of a pleiotropic response to the growth factors: the protein content of the cells increases in the defined medium; the 2DG gels of the 35S- and 32P-labeled proteins show important changes in spots, several of which are probably of cytoskeletal origin. It is also shown that cells in a semisolid growth factor-supplemented medium have growth advantages over their counterparts grown with serum. All the phenotypic changes mentioned above reveal the important role of growth factors in the growth and behavior of this mammary cell line. The results obtained with thrombin indicate a new site of action of this enzyme which may be important in the metastatic spread of human mammary tumor cells.     

Cultured proliferating rat mammary epithelial cells

Summary Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with³H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. This work was supported by the Veterans Administration, Washington, DC, and by CA grant 05388 from the U.S. Public Health Service, Washington, DC.     

cAMP levels in proliferating rat mammary epithelial cells in vitro and in vivo

Agents that elevate intracellular cAMP levels are required for growth of many cell types in culture including normal rat mammary epithelial (RME) cells. To determine if the intracellular levels of cAMP that result from stimulation by agents such as cholera toxin (CT) or prostaglandin E-1 (PGE-1) are within the physiological range, cAMP levels were determined in RME cells growing in primary culture and compared to levels measured in freshly isolated mammary epithelium. The results indicate that the cAMP levels of mammary epithelial organoids obtained from 45-day-old virgin rats are 4 to 6 pmol/10⁶ cells. Growth of RME cells in primary culture in the presence of CT results in cAMP levels of approximately 15 to 20 pmol/10⁶ cells early in culture when cells are proliferating rapidly. As cells approach confluence, cAMP concentrations decrease to levels observed in fresh organoids. CT-stimulated cAMP levels appear to be within the range of those found in pregnant mammary epithelium in vivo. Growth of RME cells in medium supplemented with PGE-1 instead of CT results in cAMP levels equivalent to those found in fresh mammary epithelial organoids and under these conditions the growth rate is approximately half that found in CT-stimulated cells. These results indicate cAMP to be a positive regulator of cell growth in vivo at levels that are within the physiological range.     

Fibroblast growth factor release by bovine endothelial cells and human astrocytoma cells in culture is density dependent

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen whose actions are mediated by binding to specific cell surface receptors on a variety of cell types. However, the amino acid sequence of bFGF does not contain a classical signal peptide sequence and the extent to which cellular stores of this mitogen are released is still a matter of some controversy. In the present study we examined the release of immunoreactive bFGF into serum-free conditioned medium of bovine corneal endothelial cells (BCE) and a human astrocytoma cell line, U87-MG. Western blotting analysis of BCE conditioned medium using N-terminal specific anti-bFGF serum revealed a single immunoreactive band of 32 kilodaltons, which was reduced to 18 kilodaltons in the presence of 8 M urea. Using a sensitive two-site immunoradiometric assay we were able to quantify the release of immunoreactive bFGF into the culture medium by BCE cells and by the human astrocytoma cell line U87-MG. In each case the release of bFGF was cell density dependent, but under all conditions the level of bFGF released was significantly greater in the transformed astrocytoma line, ranging from 15- to 50-fold higher than in the BCE cultures under various conditions. At 30% confluence the concentration of immunoreactive bFGF in the medium was maintained at a constant level for up to 24 h. However, the level of immunoreactive bFGF declined rapidly in confluent cells.(ABSTRACT TRUNCATED AT 250 WORDS)