Ehrlich ascites tumor cell surface membranes: an abnormality in ether lipid content

Most mammalian neoplasms have a defect in ether lipid content manifested by the presence of abnormally large quantities of 0-alkyl glyceryl ethers, in contrast to normal tissues in which the alk-1-enyl structure predominates. These lipids are for the most part structural. The manner in which tumor cell plasma membranes differ from normal may be important, and it has been hitherto unclear whether or not the 0-alkyl lipid abnormality of neoplasms includes the plasma membrane. The present investigation reveals that 0-alkyl lipids are present in the membranes of Ehrlich ascites tumor cells isolated by several different methods. The amount of 0-alkyl lipid, on a weight basis, represents 1-3 percent of the total phospholipids and 1-4 percent of the total aliphatic lipid. These quantities are the same as or greater than the amount of 0-alkyl lipid found in microsomes, mitochondria, and whole cell homogenate. As is generally the case for intact neoplastic tissues, the quantity of 0-alkyl lipids of Ehrlich ascites tumor plasma membrane is greater than the amount of alk-1-enyl lipids.     

Studies on the cellular mechanism of free fatty acid uptake using an analog, hexadecanol

Hexadecanol was employed as a fatty acid analog in an attempt to elucidate the role of the carboxyl group in free fatty acid uptake. Large quantities of albumin-bound [1-(14)C]hexadecanol were taken up by Ehrlich ascites cells during in vitro incubation. More than 90% of the (14)C that was taken up remained as hexadecanol even after 1 hr of incubation at 37 degrees C. Addition of unlabeled hexadecanol did not appreciably alter the rate of [U-(14)C]glucose oxidation or incorporation into total lipids, suggesting that the slow rate of hexadecanol metabolism was not due to a toxic effect of this analog. However, more of the labeled glucose was incorporated into phospholipids and less into glycerides, indicating that hexadecanol did exert some metabolic effect on the cells. Uptake was temperature dependent but relatively unresponsive to the presence of glucose or fluoride and cyanide. Hexadecanol was incorporated into exchangeable and nonexchangeable cellular pools as determined by its availability for release to a medium containing albumin. These results indicate that a mammalian cell can rapidly take up large amounts of a long-chain hydrocarbon derivative that does not contain a carboxyl group. Furthermore, the data are compatible with the hypothesis that free fatty acids are taken up by a nonenzymatic process such as diffusion into the lipid phase of the cell membrane.     

Production of oxygen free radicals by Ehrlich ascites tumour cells: effect of lipids

Phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 and platelet activating factor (PAF) stimulated the generation of oxygen free radicals (nitro-blue tetrazolium reduction) in Ehrlich ascites tumour (EAT) cells. PAF was effective at an optimal concentration of 4 muM, but was inhibited by BN 52021, a specific PAF antagonist. Lyso-PAF was ineffective. Inclusion of different lipids during incubation prior to the addition of PAF, resulted in the activation/inhibition of free radical generation. Among the phospholipids at a concentration of 50 mug/ml, the order of activation was phosphatidylserine > phosphatidylglycerol > phosphoinositides > phosphatidylinositol > phosphatidylethanolamine. Phosphatidylcholine was not effective, while sphingolipids were inhibitory. In addition, Ehrlich ascites tumour cells grown in mice under marginal vitamin A deficiency, showed an augmented production of free radicals compared to control cells. This was suppressed by exogenous addition of vitamin A or superoxide dismutase. These results suggest that membrane lipids and dietary factors like vitamin A probably function as physiological modulators in regulating the free radical generation.     

Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol.

1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.     

Manipulation of alkylglycerolipid levels in cultured cells Fatty alcohol versus alkylglycerol supplements

Ehrlich ascites cells and monolayers of L-M cell fibroblasts were grown in medium containing either long-chain fatty alcohols or alkylglycerols. The cells were then analyzed to determine the contribution of these lipid precursors to the synthesis of complex lipids for the purpose of defining the most efficient system to elevate the levels of ether phospholipids. Label from high specific activity [1-¹⁴C]hexadecanol, [1-¹⁴C]octadecanol, and [1-¹⁴Cloctadecenol was incorporated into alkyi linkages (C-18:1 >C-16:0 >C-18:0); however, similar labeling of acyl groups occurred. Increasing the amount of hexadecanol in the growth medium resulted in a higher percentage of ¹⁴C-labeled acyl groups than alkyl linkages at all concentrations of the alcohol supplement. Supplements of rac-[1-¹⁴C]hexadecylglycerol to the growth media were assimilated into phospholipids, which significantly increased as a function of the amounts added. Mass determinations of the alkyl ether phospholipid content in L-M cells incubated for 24 h with an alkylglycerol mixture (10.8 μg/ml) showed an approximate 70% increase over control levels; supplementation had only a slight effect on the alk-1-enyl content. The systems described will be useful for investigating biophysical and biochemical effects of alkyl ether enrichment in membranes.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes.

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.     

质子跨膜转运能够引起艾氏腹水癌细胞质膜表面质子化

荧光探针Ｆｌｕｏｒｅｓｃｅｉｎ－ＰＥ在８ｍｍｏｌ／Ｌ Ｃａ＾２＋促进下,能够标记于艾氏腹水癌细胞质膜表面。在不影响膜结构的条件下,利用微量ＴｒｉｔｏｎＸ－１００消除膜表面与介质之间的质子浓度差,通过酸滴定实验,能够得到膜表面ＰＨ与ＦＰＥ荧光强度之间的相关曲线。根据此曲线测定了艾氏腹水癌细胞质膜表面ＰＨ：当介质ＰＨ为７．２时,其膜表面ＰＨ为６．７,其质子浓度大约为介质中的３倍,质子跨膜转运抑制剂ＣＣ     

Tenacious binding of lipids to vimentin during its isolation and purification from Ehrlich ascites tumor cells

Vimentin enriched in cytoskeletal frameworks by Triton X-100 extraction of Ehrlich ascites tumor cells and purified from a low ionic strength extract of the cell residues by (NH4)2SO4 precipitation and DEAE-Sepharose and ssDNA-cellulose chromatography in the presence of 6 M urea was highly contaminated with lipids. Thin-layer chromatography of a chloroform-methanol extract of the purified protein revealed, besides small amounts of phospholipids, the presence of large quantities of neutral lipids.     

Lipids as integral constituents of nuclear and chromatin fractions in Ehrlich ascites tumor cells

Lipid content and composition of DNA, histone and non-histone proteins of Ehrlich ascites tumor cell chromatin were investigated. All fractions contained small amounts of lipids, mostly neutral ones, in a specific distribution. According to isotopic studies with labeled lipid precursors, incorporation took place mainly in the non-histone fraction. These findings suggest that neutral lipids attached to non-histone chromosomal proteins may also contribute to the regulatory functions ascribed to phospholipids.     

Comparative study, using fluorescent methods, of cell membranes and their reconstituted liposomes

Cell plasma membranes and proteoliposomes reconstituted from solubilized plasma membranes of thymocytes and Ehrlich ascites carcinoma cells have been studied by fluorescent methods. It has been shown that proteoliposomes are characterized by greater polarization and rigidity of microsurroundings in membrane proteins and greater microviscosity of membrane lipids. Proteoliposomes from thymocyte membranes contain less membrane proteins and express lower polarity of the lipid bilayer than proteoliposomes from Ehrlich ascites cells.     

Interactions of lipids and proteins: Some general principles

Methods to describe the binding of phospholipids to membrane proteins are described. It is shown that it is difficult to obtain estimates of the number of phospholipids bound to the surface of a membrane protein from ESR experiments in which plots of free to bound spin label (y) vs. molar ratio of lipid to protein are extrapolated toy=0. The relative advantages and disadvantages of ESR and fluorescence methods for measuring relative binding constants of phospholipids to membrane proteins are discussed. The particular problems associated with comparing binding constants of molecules of very different sizes (e.g., fatty acids and cardiolipin) are described and equations are presented to account for these problems. The possible effects of membrane viscosity and thickness on activity of membrane proteins are discussed, but it is concluded that effects of phospholipid structure on activity can only be understood in terms of a reasonably complete kinetic model for the protein.     

Characterization of aconitate hydratase from mitochondria and cytoplasm of ascites tumor cells

This paper describes the characterization of aconitate hydratase (EC 4.2.1.3) in cytoplasmic and mitochondrial extracts from Ehrlich ascites tumor cells carried by BALB/C mice. The results show a similar distribution of aconitate hydratase in both extracts, with specific activities much lower than those found in pig and mouse tissues. Mitochondrial aconitate hydratase shows a substrate inhibition by citrate with a Km similar to that found in cytoplasm (Km = 1.0 mM and 0.9 mM, respectively). Oxalacetate produces a mixed type of inhibition in both cytoplasmic and mitochondrial aconitate hydratases with different inhibition constants (Ki = 0.3 mM and 1.0 mM, respectively). Moreover, the specific activities of aconitate hydratase in both cytoplasm and mitochondria decrease when the tumor progresses in the peritoneum of BALB/C mice, as well as the percentage of aconitate hydratase activity in the presence of oxalacetate as the inhibitor. These results indicate that the activity and kinetics of aconitate hydratase are markedly altered by neoplastic transformation as occurs in Ehrlich ascites tumor cells. Since aconitate hydratase is not a key enzyme, these unexpected data are of interest in the study of cancer biochemistry.     

Interaction in vitro of nonepithelial intermediate filament proteins with total cellular lipids, individual phospholipids, and a phospholipid mixture

The interaction of nonepithelial intermediate filament (IF) proteins with vesicles produced from total Ehrlich ascites tumor cell lipids results in the formation of complexes which in sucrose density gradient centrifugation attain positions distinctly different from those of the original reactants. In KBr density gradient equilibrium centrifugation, the IF protein-lipid adducts accumulate as thin proteolipid films on top of the KBr gradients, whereas in the absence of lipids the proteins remain distributed within the density gradients. Similar results were obtained with vesicles derived from individual phospholipids and a mixture thereof. The affinity of IF proteins for negatively charged phospholipids is greater than that for vesicles derived from uncharged phospholipids. Limited digestion of IF proteins with various proteinases demonstrated that for optimal association of the reactants IF proteins must carry an intact N terminus and that the isolated N-terminal polypeptide itself shows strong reactivity with lipid vesicles. Arginine-phosphate interactions between the N terminus and phospholipids seem to be partly responsible for this association. However, as shown by hydrophobic interaction chromatography on phenyl- and octyl-Sepharose 4B, IF proteins and their proteolytic derivatives also appear to have high affinities for aromatic and aliphatic substructures of biologically important molecules. The results are discussed in terms of a possible functional role of IF protein-lipid interactions in the association of nonepithelial intermediate filaments with intracellular membrane systems.     

Regulation of ether lipids and their precursors in relation to glycolysis in cultured neoplastic cells

Tumors typically show high rates of glycolysis and elevated levels of ether lipids, particularly the alkyldiacylglycerols; thus, we investigated the relationship between ether lipid accumulation and glucose metabolism in a neoplastic cell line (B2-1). The B2-1 cells grown in 5.5 mM galactose in the absence of glucose produced very low levels of alkyldiacylglycerols, triacylglycerols, lactic acid, and dihydroxyacetone-P. Increasing concentrations of glucose caused a progressive increase in lactic acid, dihydroxyacetone-P, and up to a ten-fold increase in alkyldiacylglycerols and triacylglycerols. Glucose supplements also caused an increased incorporation of [9,10-3H]palmitic acid into alkyldiacylglycerols and triacylglycerols. These metabolic changes appeared to be independent of altered growth rates of the cells. The addition of hexadecanol along with glucose to the cultures resulted in a shorter lag and a more rapid rate of accumulation of alkyldiacylglycerols; hexadecanol supplements alone had no effect. The extent of uptake and oxidation of hexadecanol was similar in both the glucose and galactose-grown cells. These results indicate that the levels of alkyldiacylglycerols in neoplastic cells can be regulated by the extent their precursors are formed from glucose.     

Pantothenic acid and its derivatives protect ehrlich ascites tumor cells against lipid peroxidation

Preincubation of Ehrlich ascites tumor cells at 22 or 32°C, but not at 0°C, with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine reduced lipid peroxidation (measured by production of thiobarbituric acid-reactive compounds) induced by the Fenton reaction (Fe²⁺ + H₂O₂) and partly protected the plasma membrane against the leakiness to cytoplasmic proteins produced by the same reagent. Pantothenic acid and its derivatives did not inhibit (Fe²⁺ + H₂O₂)-induced peroxidation of phospholipid multilamellar vesicles, thus indicating that their effect on the cells was not due to the scavenging mechanism. Homopantothenic acid and its 4′-phosphate ester (which are not precursors of CoA) neither protected Ehrlich ascites tumor cells against lipid peroxidation nor prevented plasma membrane leakiness under the same conditions. Incubation of the cells with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine significantly increased the amount of cellular CoA and potentiated incorporation of added palmitate into phospholipids and cholesterol esters. It is concluded that pantothenic acid and its related compounds protect the plasma membrane of Ehrlich ascites tumor cells against the damage by oxygen free radicals due to increasing cellular level of CoA. The latter compound may act by diminishing propagation of lipid peroxidation and promoting repair mechanisms, mainly the synthesis of phospholipids.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat.

The rates of synthesis, turnover, and half-lives were determined for brain microsomal ether phospholipids in the awake adult unanesthetized rat. A multicompartmental kinetic model of phospholipid metabolism, based on known pathways of synthesis, was applied to data generated by a 5 min intravenous infusion of [1,1-(3)H]hexadecanol. At 2 h post-infusion, 29%, 33%, and 31% of the total labeled brain phospholipid was found in the 1-O-alkyl-2-acyl-sn-glycero-3-phosphate, ethanolamine, and choline ether phospholipid fractions, respectively. Autoradiography and membrane fractionation showed that 3% of the net incorporated radiotracer was in myelin at 2 h, compared to 97% in gray matter microsomal and synaptosomal fractions. Based on evidence that ether phospholipid synthesis occurs in the microsomal membrane fraction, we calculated the synthesis rates of plasmanylcholine, plasmanylethanolamine, plasmenylethanolamine, and plasmenylcholine equal to 1.2, 9.3, 27.6, and 21.5 nmol. g(-1). min(-1), respectively. Therefore, 8% of the total brain ether phospholipids have half-lives of about 36.5, 26.7, 23.1, and 15.1 min, respectively. Furthermore, we clearly demonstrate that there are at least two pools of ether phospholipids in the adult rat brain. One is the static myelin pool with a slow rate of tracer incorporation and the other is a dynamic pool found in gray matter.The short half-lives of microsomal ether phospholipids and the rapid transfer to synaptosomes are consistent with evidence of the marked involvement of these lipids in brain signal transduction and synaptic function.     

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180 000 sites/cell but this fell to 125 000 (p <0.001) in cells enriched in monounsaturated fatty acids and rose to 386 000 (p <0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants ( and ) of 0.03 and 0.01 nM⁻¹. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM⁻¹ whereas values of 0.01 and 0.005 nM⁻¹ were obtained in the cells enriched in polyunsaturated fatty acids (all comparison p <0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.     

A model for the asymmetric lipid distribution in the human erythrocyte membrane

The asymmetric transverse distribution of phospholipids in the human erythrocyte membrane can be explained by differences between the rate constants of flip and flop motion of the lipids. A selective interaction between aminophospholipids and spectrin does not need to be assumed for creating and maintaining the asymmetric localization of these lipids. Shape transformation of red cells could be caused by alterations of the flip-flop rate constants leading to a change of the lipid distribution and, consequently, to a differential area expansion of the outer and inner membrane leaflet.