l-Glutamine transport in native vesicles isolated from Ehrlich ascites tumor cell membranes

Native vesicles isolated from Ehrlich ascites tumor cells accumulate glutamine by means of Na⁺-dependent transport systems; thiocyanate seems to be the more effective anion. The apparent affinity constant for the process was 0.38 mM. The Arrhenius plot gave an apparent activation energy of 12.3 kJ/mol. The structural analogs of glutamine, acivicin (2.5 mM) and azaserine (2.5 mM), inhibited the net uptake by 67 and 70%, respectively. The sulfhydryl reagents mersalyl, PCMBS, NEM, and DTNB also inhibited net uptake, suggesting that sulfhydryl groups may be involved in the activity of the carrier protein. A strong inhibition was detected when the vesicles were incubated in the presence of alanine, cysteine, or serine; in addition, histidine, but not glutamate or leucine, had a negative effect on glutamine transport.     

Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release

Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4′-diisothiocyano-1,2′-dihenylethane-2,2′-disulfonic acid (³H₂DIDS) at 4°C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of ³H₂DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the ³H₂DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23°C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (τ_½ = 360 min) component representing 33% of the radioactivity, and a fast (τ_½ = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 μ/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinectics of release to that of a single component (τ_½ = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously los in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither ³H₂DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.     

The rate of formation of surface membrane ether lipids in Ehrlich ascites tumor cells: kinetic considerations

A previous investigation has shown that O-alkyl phospholipids are present in the surface membrane of Ehrlich ascites tumor cells. In the present investigation it was shown that 90% or more of [1-3H]hexadecanol injected intraperitoneally into mice bearing Ehrlich ascites tumors is taken up by the neoplastic cells in less than 15 min. Near maximum formation of surface membrane O-alkyl phospholipids requires approximately 8 h. The rate of accumulation of O-alkyl phospholipids is very similar both for the whole cell and for the surface membrane. Further examination of the data revealed that the conversion of hexadecanol into O-alkyl glycerophospholipids can be described by a simple model in which O-alkyl lipids appear at a single rate constant of 0.25 to 0.35 per hour and disappear at a rate of 0.02 per hour or less. These rate constants were obtained initially by stochastic analysis and validated both by deterministic methods and by compartmental analysis using the SAAM computer program. The method of kinetic analysis described may find broader application in providing comparative rate constants for the in vivo turnover of O-alkyl lipids in both normal and neoplastic tissues. The advantage of a stochastic approach is that kinetic data may be obtained with fewer assumptions relating to pool structure or specific models.     

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.     

Kinetic analysis of cell size and DNA content distributions during tumor cell proliferation: Ehrlich ascites tumor study.

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distibution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G1-G0 and G2-G0 transitions of non-cycling cells as revealed by the variation of cell size distribution.     

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.     

Leukotriene-D4 induced cell shrinkage in Ehrlich ascites tumor cells

Summary The nature of the leukotriene-D₄ (LTD₄) induced cell shrinkage in Ehrlich ascites tumor cells has been investigated. LTD₄ treatment of Ehrlich cells induces net loss of cellular KCl and cell shrinkage independent of the initial cell volume. LTD₄ also produces water loss and reduction in cell volume when all extracellular and all intracellular Cl has been replaced by NO₃. On the other hand, LTD₄ fails to produce any significant changes in cell volume in the presence of the K-channel blocker quinine, suggesting that LTD₄ in Ehrlich cells induces Cl-independent K loss through the Ca²⁺-dependent K channels. However, the effect of physiological doses of LTD₄ on cell volume seems not to be as potent in Cl-free, NO₃ cells when compared to Cl-containing cells, indicating that LTD₄ in Ehrlich cells also provokes Cl-dependent K loss. LTD₄ seems not to produce K loss through an electroneutral K⁺/H⁺ exchange system. LTD₄ still produces Cl-independent K loss and cell shrinkage in the presence of the anticalmodulin drug pimozide but not in the presence of the LTD₄ receptor antagonist L-649,923 or the 5-lipoxygenase inhibitor NDGA. Pretreatment of the cells with pertussis toxin, which inactivates inhibitory guanine nucleotide binding proteins (G-proteins), leads to partial inhibition of the LTD₄-induced shrinkage. It is suggested that the LTD₄-induced activation of K and Cl transporting systems in Ehrlich ascites tumor cells is mediated via a G-protein coupled receptor and that LTD₄ might exert its effect through another lipoxygenase product. The Ca²⁺-calmodulin complex is not involved in the LTD₄-induced activation of K and Cl transporting systems.     

Experimentally induced modifications in surface morphology of Ehrlich ascites tumor cells

Observations with scanning electron microscopy /SEM/ were carried out on Ehrlich ascites tumor /EAT/ cells incubated 2 to 4 h at 37 degrees C in hyperosmotic media in the presence and in the absence of serum. It was found that the cells even if maintain spherical shape show significant differences in the architecture of their surface. A need for control observations with SEM of cell surface morphology in biochemical and biophysical research concerning plasma membrane functions and properties is pointed out.     

Metabolism of ether glycolipids with potentially antineoplastic activity by Ehrlich ascites tumor cells

Ehrlich ascites tumor cells were incubated in vitro with rac-1-O-[1'-14C]octadecyl-2-O-methylglycero-3-beta-D-glucopyranosi de for 24 h. The potentially antineoplastic ether glycolipid was rapidly metabolized by the cells to radioactive 1-O-octadecyl-2-O-methylglycerol (70 pmol/10(6) cells per h) and further acylated to 1-O-octadecyl-2-O-methyl-3-acylglycerols. Incubation of Ehrlich ascites cells with synthetic rac-1-O-[1'-14C]octadecyl-2-O-methyl-3-palmitoylglycerol showed that this metabolite is reconverted by deacylation to 14C-labeled 1-O-octadecyl-2-O-methylglycerol. The latter compound or a metabolite derived therefrom may be the 'toxic principle' of both the ether glyceroglycolipids and ether glycerophospholipids having a 1-O-alkyl-2-O-methylglyceryl moiety, as suggested by Unger et al. (J. Natl. Cancer Inst. 78 (1987) 219-222).     

Solubilization of alkyldihydroxyacetone-P synthase from Ehrlich ascites cell microsomal membranes.

The enzyme, alkyldihydroxyacetone-P synthase, has been solubilized and partially purified from microsomal preparations of Ehrlich ascites cells after treatment with Triton X-100 and phospholipase C, followed by chromatography on Sepharose 4B. When the Triton X-100 was removed after solubilization the enzyme was still active but eluted in the void volume of the Sepharose 4B column, whereas in the presence of detergent it eluted much later as a single peak of activity, indicating that the solubilized enzyme tends to aggregate unless detergent is present. The lower molecular weight form of alkyldihydroxyacetone-P synthase (in detergent) had an estimated molecular mass of 250,000–300,000 daltons.     

Concanavalin A-induced alterations in sodium and potassium content of Ehrlich ascites tumor cells

Net fluxes of sodium and potassium were studied in Ehrlich mouse ascites tumor cells during contact with the agglutinating protein, concanavalin A. This lectin altered cation transport markedly at concentrations of 20–105 μg/ml (6–47 μg/mg cell protein). Whereas control cells extruded sodium and maintained or accumulated potassium against electrochemical gradients, in the presence of concanavalin A there was rapid net sodium entry and potassium loss. After 10–20 minutes in concanavalin A, sodium extrusion began and potassium loss diminished but these events were prevented by ouabain. The alterations in cation content induced by concanavalin A are unlikely to be the result only of agglutination since soybean agglutinin caused much smaller changes although it agglutinated the cells equally well.     

Potassium and sodium movements in the Ehrlich mouse ascites tumor cell

Studies have been conducted on the movements of sodium and potassium into and out of the Ehrlich ascites tumor cell. Under steady state conditions, at 22 degrees C., in the absence of an exogenous source of glucose, the cell flux for both potassium and sodium averaged 0.8 microM10(7) cells/hr, or 3.0 pM/cm.(2)/sec. The cell can accumulate potassium and extrude sodium against electrochemical gradients for both ions. It is possible under the experimental conditions reported to separate the transport systems for these two ions. Thus, it has been shown that under conditions of low temperature with a diminished metabolism, net fluxes for the two ions are different. Also, following periods of 24 hours at 2 degrees C., an exogenous source of glucose enhances the accumulation of potassium sevenfold while sodium extrusion is uninfluenced by the presence of glucose. Similarly potassium exchange rates are temperature-dependent, with Q(10) values as high as 5, while exchange rates for sodium are temperature-insensitive, with Q(10) values of 1.2 to 1.6. Glycolysis has been eliminated as an energy source for the transport processes since these processes go on in the absence of an exogenous source of glucose. It is estimated that a maximum of 0.3 per cent of the energy derived from the total oxidative metabolism of glucose would be required to support independent transport of potassium and sodium.     

Kinetic analysis of a split cell population following reinoculation of an Ehrlich ascites tumor

When Ehrlich ascites cells from old tumors are inoculated into fresh hosts, their cell cycle parameters show several unusual features when studied by means of four independent techniques. These experiments measure: Labeling Index (LI); Mitotic Index (MI); Percent Labeled Mitosis (PLM); Percent of population mitotic and initially G0, (Mu/P). The minimum modifications of the standard cell-cycle model that allow simultaneous simulation of the four sets of data were then determined. These modifications are: (1) sudden shortening of transit time for the S state immediately following reinoculation; (2) a more gradual change in the transit time for M; (3) an orderly reentry of G0 cells into the beginning of the cycle; (4) a shorter first cycle for the reentering G0 cells along with a smaller coefficient of variation for the first cycle of the G0 cells. These changes allow a simultaneous simulation of the four experiments with a single parameter set. This was not achievable without the modifications. Hence, resolution of a cell population into two components allows one to account for additional kinetic features.     

Mannose toxicity in Ehrlich ascites tumor cells

Mannosephosphate isomerase (MPI) showed a higher activity than hexokinase (HKM) in its ability to phosphorylate mannose in the spleen, thymus, brain, liver, striated muscles, kidneys, and testes from BALB/c mice. This led to a HKM/MPI ratio of less than 1 in all the organs and tissues mentioned. In contrast, Ehrlich ascites tumor cells obtained from the peritoneum of BALB/c mice had low MPI activity (half of the HKM activity and, therefore, a ratio of 2). Mannose, which is nontoxic to nontumor cells at a concentration of 0.1 M, induced marked in vitro mortality of the tumor cells. Incubation of Ehrlich ascites tumor cells with mannose resulted in a high accumulation of mannose-6-phosphate and a marked depletion of ATP which did not appear when the cells were incubated with glucose. These facts may explain the selective mortality caused by mannose in the tumor cells studied.