Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but no GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs. J. Cell. Biochem. 67:131–135, 1997. © 1997 Wiley-Liss, Inc.     

The reversal of effects of methotrexate on toxicity and glucose transport reduction by thymidine in Ehrlich ascites tumor bearing mice

Methotrexate (MTX) suppressed the growth of Ehrlich ascites tumor cells in vivo and reduced the cellular uptake of glucose and the density of glucose transporters on the tumor cell surface. MTX inhibition of tumor growth was partially prevented by concurrent administration of thymidine. At the same time, the rate of cellular glucose uptake, the density of glucose transporters on the cell as well as the extent of thymidine, uridine and leucine incorporation were significantly increased.     

Interrelationship of number of glucose carriers and intracellular phosphoribosyl diphosphate level of Ehrlich ascites tumor cells in vitro

The intracellular phosphoribosyl diphosphate (prpp) levels of Ehrlich ascites tumor cells increased during glucose supplementation and decreased during glucose deprivation, while the numbers of glucose carriers as determined by glucose-reversible cytochalasin-B binding changed in an opposite manner relating to the extracellular glucose concentrations and the intracellular prpp levels of Ehrlich ascites tumor cells in vitro. Incubation of cells with hypoxanthine or 2,4-dinitrophenol lowered the intracellular prpp levels and resulted in an increase in numbers of glucose carriers.     

Human tumour necrosis factor-α inhibits glucose transport in cultured ehrlich ascites tumour cells

Human recombinant tumour necrosis factor-α (rhTNF-α) arrested the growth of Ehrlich ascites tumour (EAT) cells in vitro. It suppressed cellular glucose uptake and decreased the membrane density of glucose transporters as measured by glucose-reversible cytochalasin B binding. The glucose transporters' affinity for substrate was also reduced. However, rhTNF-α treatment exerted no effect on the phosphoribosyl pyrophosphate level in EAT cells. The role of rhTNF-α on the inhibition of glucose transport of tumour cells is discussed.     

Inhibition of virus-induced fusion of Ehrlich ascites tumor cells by cytochalasin B and D.

Cytochalasin B and D were found to inhibit HVJ (Sendai virus)-induced fusion of Ehrlich ascites tumor cells. Nearly complete inhibition was attained by 4 uM (2 μg/ml) cytochalasin D, whereas cytochalasin B was a less effective inhibitor. The inhibition was largely reversible. Since the transport of 2-deoxy-glucose into the tumor cells was not affected by cytochalasin D (though inhibited by cytochalasin B), the observed inhibition was not related to the effect of the drugs on sugar transport. Instead, it was suggested that the inhibition was due to the action of the drugs on microfilaments. The requirement of ATP for the cell fusion could be explained at least partly by the involvement of microfilaments in the cell fusion process.     

Interaction of polynucleotides with cultured mammalian cells : II. Cell surface charge density and RNA uptake

This report describes the uptake of high molecular weight RNA by Ehrlich ascites tumor cells treated with enzymes and polycations which reduce cell net negative surface charge density. Enzyme treatment had little effect on RNA uptake, but treatment with poly- -lysine resulted in increased binding and uptake of RNA. Present data indicate that decreased cell surface charge, increased availability of positive surface sites, and cell death, all contribute to increased RNA uptake. The individual contributions of these factors has been partially resolved. A possible mechanism for polyanion uptake by cells is proposed.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

Metabolic pathways in Ehrlich ascites tumor cells recovering from a low bioenergetic status

Ehrlich ascites tumor cells were found to be in a low bioenergetic status, as evaluated by acridine orange uptake and ATP content, when resuspended in a glucose medium shortly after removal from the animal. Dye uptake as well as ATP content then increased for about 2 h at room temperature. This effect was only slightly inhibited by oligomycin. Cells resuspended in a glucose-free medium initially showed high dye uptake and ATP level, which were stable over time: in this case oligomycin caused a drop in both dye uptake and ATP level. The above findings, which are indicative of a marked Crabtree effect in Ehrlich ascites tumor cells, means that it is unlikely that limiting ADP and P_i play an important role in the glucose-induced inhibition of oxidative phosphorylation in this system.     

Studies on protein uptake by isolated tumor cells. 3. Apparent stimulations due to pH, hypertonicity, polycations, or dehydration and their relation to the enhanced penetration of infectious nucleic acids

Fixation of ¹³¹I-serum albumin by Ehrlich ascites tumor cells in suspensions and sarcoma S-180 monolayers was measured under experimental conditions. Anaerobic incubation and inhibitors of the oxidative metabolism critically restricted the range of glucose concentrations capable of supporting cell life; in glucose concentrations higher than 10^-2 M, Ehrlich cells suffered from their own acid production; in concentrations 10^-2 M, lower than they underwent damage by starvation. Both types of damage were accompanied by increased albumin fixation unrelated to pinocytosis. Different procedures recommended to enhance the uptake of infectious viral RNA by animal cells in culture were tested for their ability to increase albumin uptake. They enhanced the penetration of both albumin and vital dyes and decreased the viability of cell populations. Their effect, therefore, is related to cell damage. It was postulated that reversible damage to cells favors RNA infection by leading to abnormal uptake processes and by decreasing intracellular digestion. This abnormal uptake is different from pinocytosis and also from the massive fixation of albumin to dead cells. The latter phenomenon is due to adsorption by intracellular sites exposed by disruption of the cell membrane. Polycations are able to induce all three forms of fixation depending on the experimental conditions.     

Identification of the D-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to identify, the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [3H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable site. If 280 nM (40000 microunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Glucose regulates its own transport in Ehrlich ascites tumour cells

The capacity of Ehrlich ascites tumour cells to take up 2-deoxy-D-glucose and to bind cytochalasin B varies adaptatively with the level of glucose in the plasma or culture medium. The effect of glucose is exerted directly on the cells and does not necessarily require the participation of hormones such as insulin, glucagon or corticosterone, although glucagon and the glucocorticoids, but not insulin, can also increase the number of glucose carrier molecules administered in vitro. Cycloheximide suppresses the acute inductive effect of glucose, suggesting that protein synthesis might be required for the increased transport activity.     

Patterns in the combined action of irradiation and glucose loading on tumor cells

It was shown that incubation of Ehrlich ascites tumor cells with glucose or in buffer solutions of low pH decreases their viability. The cell survival rate depends on pH values irrespective of the protoxidation method and oxygenation conditions used. At the same time, radiosensitivity of Ehrlich ascites tumor cells is practically invariable with pH being decreased from 7.0 to 5.0. The effects of glucose and radiation are additive in conditions simulating the effect of hyperglycaemia in vivo.     

Role of the F-actin cytoskeleton in the RVD and RVI processes in Ehrlich ascites tumor cells.

The role of the F-actin cytoskeleton in cell volume regulation was studied in Ehrlich ascites tumor cells, using a quantitative rhodamine-phalloidin assay, confocal laser scanning microscopy, and electronic cell sizing. A hypotonic challenge (160 mOsm) was associated with a decrease in cellular F-actin content at 1 and 3 min and a hypertonic challenge (600 mOsm) with an increase in cellular F-actin content at 1, 3, and 5 min, respectively, compared to isotonic (310 mOsm) control cells. Confocal visualization of F-actin in fixed, intact Ehrlich cells demonstrated that osmotic challenges mainly affect the F-actin in the cortical region of the cells, with no visible changes in F-actin in other cell regions. The possible role of the F-actin cytoskeleton in RVD was studied using 0. 5 microM cytochalasin B (CB), cytochalasin D (CD), or chaetoglobosin C (ChtC), a cytochalasin analog with little or no affinity for F-actin. Recovery of cell volume after hypotonic swelling was slower in cells pretreated for 3 min with 0.5 microM CB, but not in CD- and ChtC-treated cells, compared to osmotically swollen control cells. Moreover, the maximal cell volume after swelling was decreased in CB-treated, but not in CD- or Chtc-treated cells. Following a hypertonic challenge imposed using the RVD/RVI protocol, recovery from cell shrinkage was slower in CB-treated, but not in CD- or Chtc-treated cells, whereas the minimal cell volume after shrinkage was unaltered by either of these treatments. It is concluded that osmotic cell swelling and shrinkage elicit a decrease and an increase in the F-actin content in Ehrlich cells, respectively. The RVD and RVI processes are inhibited by 0.5 microM CB, but not by 0.5 microM CD, which is more specific for actin.     

Interaction between daunorubicin and chromatin from Ehrlich ascites tumor cells.

Interaction of daunorubicin with chromatin from Ehrlich ascites tumor cells has been studied by spectrofluorimetry. Daunorubicin interacts with chromatin and displays at least two types of binding. The number of binding sites is reduced when compared to deoxyribonucleic acid. There is no difference in the overall structure of chromatins extracted from cells sensitive or resistant to daunorubicin.     

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.     

Malate-citrate cycle during glycolysis and glutaminolysis in Ehrlich ascites tumor cells

The malate-citrate cycle was studied during aerobic glycolysis and glutaminolysis in a strain of Ehrlich ascites tumor cells which showed a very low malate-aspartate shuttle system activity. The experimental approach includes: estimation of mitochondrial NAD[P]+-dependent malic enzyme activity; respiratory activity of freshly harvested or fasted cells, and of isolated mitochondria; and determination of the metabolites involved in the glycolytic and glutaminolytic pathways. The results suggest that in this strain, the malate-citrate shuttle is not an effective pathway for transferring glycolytic reducing equivalents from cytosol to mitochondria. Less than 15% of the glucose uptake was affected by the 1,2,3-benzenetricarboxylate inhibition of the malate-citrate shuttle. Moreover, in the presence of glucose, the malate-citrate cycle did not appear to play an important role in the glutaminolytic process. The present work supports and extends the finding of previous studies, since the results showed that the glucose metabolism depressed the oxidative processes in Ehrlich ascites tumor mitochondria, not only alone, but also in the presence of glutamine. Interestingly, the high glutamine uptake was maintained in the presence of glucose.     

A model system for the study of heteroexchange diffusion: Methotrexate-folate interactions in L1210 leukemia and Ehrlich ascites tumor cells

A unique interaction between the folate analog, methotrexate (4-amino-4-deoxy-10-methylpteroylglutamic acid), and the naturally occurring folates in L1210 leukemia and Ehrlich ascites tumor cells provides a useful model for the study of heteroexchange diffusion. The presence of intracellular binding sites with a high affinity for methotrexate but a low affinity for folic acid and its tetrahydrofolate derivatives permit the measurement of true unidirectional influx rates for methotrexate and assure that the trans-stimulation of methotrexate uptake by the intracellular presence of the other folates is due solely to a primary augmentation of this carrier influx mechanism. Further, since free methotrexate does not appear prior to saturation of the binding sites, the reaction between the folates and carrier at the inner cell membrane is undisturbed by methotrexate released from carrier as the complex enters the cell during heteroexchange, facilitating quantitation of the kinetic alterations which occur for methotrexate influx during trans-stimulation.     

Changes in the morphology of Ehrlich ascites tumour cells caused by hyperosmotic media

It was found that Ehrlich ascites tumour cells undergo significant morphology changes in media in which osmolarity is increased by NaCl or Hanks' balanced salt solution. The morphology changes include formation of filopodia- and lamellipodia-like cell surface protrusions. Their formation is enhanced by an addition of cytochalasin B. The data obtained suggest that both changes in plasma membrane properties and changes in activity of contractile apparatus participate in the formation of cell surface protrusions.     

Activation of the Na+/K+/Cl- cotransport system by reorganization of the actin filaments in Ehrlich ascites tumor cells.

Reorganization (disassembly) of the actin filaments in Ehrlich ascites tumor cells, either by hypotonic treatment in the presence of Ca2+ or by addition of cytochalasin B, results in activation of the Na+/K+/Cl- cotransport system. However, other regulatory processes, some of which may be dependent on an intact filament system, are responsible for the activation of the Na+/K+/Cl- cotransport system after cell shrinkage.