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.     

The role of hypoxia in the effect of glucose load on irradiated tumor cells

In experiments on Ehrlich ascites tumor cells it was shown that hypoxia, which reduces the lethal effect of gamma-rays, can considerably enhance the injury of cells by glucose. Treatment of tumor cells with glucose in hypoxic conditions followed by exposure to ionizing radiation under both hypoxia and normal aeration causes a 6-7-fold increase in cell injury as compared to irradiation alone. Moreover, the glucose treatment in hypoxic conditions (without concomitant irradiation) may cause approximately 99% death of tumor cells. The data obtained permit to consider the glucose treatment as an effective means by breaking the tumor radioresistance conditioned by a pool of hypoxic cells.     

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 problem of enhancing the biological action of ionizing radiations. Glucose as an agent for increasing the radiosensitivity of Ehrlich ascitic carcinoma cells in vitro

The effect of short-term incubation of Ehrlich ascites tumor cells in a medium containing excess glucose on their radiosensitivity was studied with a reference to the growth of tumors of ascitic and solid forms. It was shown that the incubation of cells with glucose, being accompanied by a change in pH of the suspension, caused, by itself, only a slight increase in the duration of the latent period of solid tumor formation and in the life-span of mice with ascitic tumors but increased considerably the lethal effect of radiation, as was estimated by the above mentioned criteria and the percentage of inoculated tumors.     

Glucose transport in developing Ehrlich ascites tumor cells: parallel changes in the rate of glucose uptake and cytochalasin B binding activity during tumor development and methotrexate treatment

The ability of Ehrlich ascites tumor cells to take up glucose increased progressively during the course of tumor development. Simultaneously as the rate of uptake rose, the density of a class of glucose-reversible binding sites for cytochalasin B on the cell surface also increased. In its stereospecificity requirement toward competing sugars and in its sensitivity to phloretin and diethylstilbestrol, this class of binding sites resembled the putative glucose carriers identified in various other cell systems and may represent the glucose transporter in Ehrlich ascites cells. Work with methotrexate (MTX) substantiated this view. Methotrexate arrested tumor growth, inhibited glucose uptake, and reduced the number of cytochalasin B binding sites. In both MTX-treated and untreated cells, the magnitude of changes in number of cytochalasin B binding sites closely paralleled and sufficiently accounted for the magnitude of changes in glucose uptake. Qualitative changes in the turnover and affinity for substrate of the putative glucose carrier need not be invoked.     

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.     

用~（31）P磁共振谱对艾氏腹水癌和肉瘤S－180细胞代谢的研究

艾氏腹水癌细胞和肉瘤Ｓ－１８０细胞是抗肿瘤药物筛选常用细胞株．实验采用取自小鼠腹腔的第７～８天的艾氏腹水癌和Ｓ－１８０细胞,用３１Ｐ磁共振谱测得了细胞内小分子含磷代谢成分;计算了细胞内ｐＨ值;还用３１Ｐ谱探讨了作用机制不同的三种抗代谢物：碘乙酸、２,４－二硝基苯酚及棉酚对艾氏腹水癌细胞代谢的影响     

Shutoff of histone synthesis by Mengovirus infection of Ehrlich ascites tumor cells.

The histone synthesizing capacity of mengovirus-infected Ehrlich ascites tumor cells and of their corresponding postnuclear supernatants was investigated as a funcion of time post-infection. In addition, histone synthesis was compared with the synthesis of other basic host proteins under identical conditions. In the scope of mengovirus infection of Ehrlich ascites tumor cells the less complex fraction comprising basic protein, separated from the acidic proteins by carboxymethyl cellulose chromatography, can be regarded as a representative of total host protein. Histones and the remaining basic host proteins therefore are well suited as easily identifiable indicators of the host protein synthesizing potential of mengovirus-infected Ehrlich ascites tumor cells. The cessation of histone synthesis proceeds faster than the arrest of the synthesis of other basic host protein.     

Radiosensitivity of clonogenic cells of Ehrlich ascites carcinoma forming colonies in semiliquid agar in diffusion chambers

A study was made of the dependence of survival of Ehrlich ascites tumor clonogenic cells on the 3d and 7th days following inoculation upon radiation dose (60Co-gamma-rays) delivered under well oxygenated in vitro conditions. No differences were detected in radiosensitivity of 3- and 7-day Ehrlich ascites tumor cells: in both cases, the "dose-effect" curves were S-shaped with a small shoulder and close D0 values.     

Change in energy metabolism of ascites cancer cells with a decrease in pH]

In Hank's balanced salt solution EL-4 ascites thymoma cells possessed endogenous respiration which was sufficient for the maintenance of their ATP level: pH decrease down to 6.0 had no effect either on endogenous respiration or the ATP level. Glucose had no influence on the respiration of EL-4 cells but inhibited that of Ehrlich ascites carcinoma (EAC) cells by 40% (Crabtree effect); respiration of the both cell lines was strongly (4-fold) inhibited after simultaneous addition of glucose, lactate and pH decrease. EL-4 cells had no endogenous glycolysis; EAC cells showed a low level of glycolysis only after pH decrease. Glucose addition led to activation of glycolysis (both inhibited 2-fold after a decrease of pH down to 6.0. The respiration inhibition at pH 7.3 and 6.0 caused no decrease of ATP depletion when glucose was present in the medium; this result may be due to suppression of ATP consumption. Incubation of EL-4 cells under respiration and glycolysis deficiency conditions resulted in a sharp ATP depletion; pH decrease delayed this depletion.     

Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins

Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate sensitivity. Group A hybrids had diphtheria toxin-binding sites but Group B and C had no detectable binding sites. Elongation factor-2 of all the hybrids was susceptible to ADP-ribosylation by fragment A of diphtheria toxin. Cells of Group A and B became more sensitive to CRM 45 (cross-reacting material 45 of diphtheria toxin) after they were exposed to low pH (pH = 4.5). The resistance of Group C to CRM 45 was not affected by the same treatment. Group A and B hybrids and human fibroblasts had similar sensitivities to a hybrid toxin composed of wheat germ agglutinin and fragment A of diphtheria toxin, but Group C and Ehrlich tumor cells were resistant to this hybrid toxin. All the hybrids and Ehrlich tumor cells were more sensitive to a hybrid toxin composed of wheat germ agglutinin and subunit A of ricin than were human fibroblasts. On subcloning of Group B hybrids, one Group C hybrid was obtained, but no Group A hybrid. These facts suggest that Ehrlich ascites tumor cells differ from human fibroblasts in the expression of a factor(s) that is involved in entry of fragment A of diphtheria toxin into the cytoplasm after the toxin binds to its surface receptors.     

Measurement of 'in situ' mitochondrial membrane potential in Ehrlich ascites tumor cells during aerobic glycolysis

(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented.     

Phosphocreatine in Ehrlich ascites tumor cells detected by noninvasive 31P NMR spectroscopy

³¹P NMR spectra of intact Ehrlich ascites tumor cells of high phosphorylation potential reveal a well-defined resonance peak assignable to phosphocreatine, corresponding to 2.3 μmoles/ml cell H₂O in adenosine-treated cells containing 5.2 μmoles ATP/ml. The NMR spectrum of Ehrlich cells incubated with iodoacetate and glucose indicates depletion of phosphocreatine and ATP to undetectable levels and substantial accumulation of fructose-1,6-bisphosphate. From the difference between the chemical shifts of internal P_i and phosphocreatine resonances, the intracellular pH was estimated to be 7.1 ± 0.1 in protein-synthesizing cells suspended in a medium of pH 7.4 at 10°C. Ehrlich cells are unable to transfer the labeled amidine group from L-(guanidino-¹⁴C)-arginine to the large intracellular glycine pool to form labeled guanidinoacetate, the demethylated precursor of creatine. These results imply that the synthesis of phosphocreatine in ATP-rich Ehrlich cells is limited primarily by the extracellular free creatine supply, the extent of which depends upon the degree of cachectic perturbation of energy and nitrogen metabolism of the tumor-bearing host.     

Glutamine and glucose as energy substrates for Ehrlich ascites tumour cells

Energy metabolism of freshly harvested Ehrlich ascites tumour cells in the presence of 5 mM glucose and/or 0.5 mM glutamine was studied. The rate of oxygen utilization was not altered by the addition of 0.5 mM glutamine; 5 mM glucose induced an inhibition of respiration. In the presence of both glucose and glutamine, the Crabtree effect decreased. In these conditions, the rates of oxygen uptake, the CO2 evolution and the changes in the redox states of cytochromes indicate that glucose is preferred by Ehrlich ascites tumour cells as energy substrate. Glucose decreased the rate of glutamine utilization by 34%. On the other hand, glutaminolysis did not inhibit glycolysis.     

Effect of palmitate, acetate and glucose on glutamine metabolism in Ehrlich ascites tumor cells

Acetate and the long chain free fatty acid palmitate provoked a decrease in the rates of glutamine utilization and glutamate production in Ehrlich ascites tumor cells incubated with 0.5 mM glutamine. There was a cumulative effect with glucose on glutamine metabolism.     

ESR studies of the interaction of copper(II)GHK, histidine, and Ehrlich cells

The tridentate complex CuGHK does not form ESR detectable adducts upon addition to either glutathione or Ehrlich ascites cells under our conditions. The absence of adducts is consistent with the poor uptake of CuGHK by cells. ESR spectra are used to characterize adduct formation between CuGHK and histidine. The CuGHK-histidine adduct is not stable in the presence of Ehrlich ascites tumor cells. It is argued that a Cu(His)2 complex is formed as a consequence of the interaction of GHK with cells.     

Role of orthophosphate concentration in the regulation of ribose phosphate synthesis and purine metabolism in Ehrlich ascites tumor cells

Concentrations of intracellular orthophosphate were determined in Ehrlich ascites tumor cells incubated with glucose, inosine, or uridine in media of different orthophosphate concentration. The effects of orthophosphate concentration on the accumulation of lactate and of phosphoribosyl pyrophosphate and on concentrations of ribose 1-phosphate and ribose 5-phosphate in tumor cells incubated with glucose were also determined. Both the phosphorolysis of inosine and the rate of catabolism of ATP in cells incubated with 2-deoxyglucose were also influenced by the orthophosphate concentration of the medium.     

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.     

The interphase death of dividing cells. The relation of interphase death to cell energy requirements

In experiments with irradiated cells of Chinese hamster and Ehrlich ascites tumor a study was made of the influence of energy provision on their interphase death rate. The presence of the uncoupler of respiration and oxidative phosphorylation--carbonyl cyanide-3-chlorophenylhydrazone--in a medium without glucose was shown to drastically increase the interphase death rate of cells of both types, whereas this effect was not observed in a medium with glucose.     

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.