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.     

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.     

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     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 × 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

Distribution of anti-RNA antibody-binding sites on the surfaces of Ehrlich ascites tumor cells

The distribution of anti-RNA antibody-binding sites on the surfaces of Ehrlich ascites tumor cells was studied by membrane immunofluorescence after binding anti-RNA antibody on the cell surfaces. Results showed that the cells formed caps after incubation with anti-RNA antibody at 4 degrees C and the elevation of their temperature to 37 degrees C. Pronase treatment of the cell surfaces completely inhibited reactivity with anti-RNA antibody. These results suggest that the RNAs on the surfaces of Ehrlich ascites tumor cells are linked to membrane protein membrane-bound cytoskeleton complexes.     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 X 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Direct measurement of the membrane potential of ehrlich ascites tumor cells: Lack of effect of valinomycin and ouabain

The Journal of Membrane Biology - The membrane potential of Ehrlich ascites tumor cells and the effects of valinomycin and ouabain upon it have been determined. The membrane potential in control...     

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.     

VARIATION OF CELL KINETIC PARAMETERS IN RELATION TO THE GROWTH RATE OF THE EHRLICH ASCITES TUMOR

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Tumoricidal adsorption of Staphylococcus aureus organisms on Ehrlich ascites tumor cells sensitized with rabbit antibody

A tumoricidal effect was observed when protein A-bearing Staphylococcus aureus organisms were adsorbed on Ehrlich ascites tumor cells previously sensitized with antiserum from a rabbit immunized with Ehrlich ascites tumor cells. Electron micrographs showed that staphylococci were firmly attached to the tumor cells, which might explain how effectively the attached cocci killed the tumor cells. The tumoricidal effect was confirmed not only by an in vitro experiment but also by an in vivo one. The possible applications of the tumoricidal adsorption as an indicator for staphylococcal virulence or for selective anti-tumor action was discussed.     

Variation of cell kinetic parameters in relation to the growth rate of the Ehrlich ascites tumor.

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Negatively charged RNase-susceptible molecules on the surface of ascites tumor cells

RNase-susceptible ionogenic groups on the cell surface membranes of two leukemic and two nonleukemic strains of ascites tumor cells were studied by cell electrophoresis, DEAE-Sephadex A-25 column and paper chromatography, and indirect membrane immunofluorescence. RNase treatment of the nonleukemic ascites tumor cells (Ehrlich ascites tumor and Sarcoma 180) produced a significant reduction in their electrophoretic mobilities. When the cells were labeled with [3H]uridine then incubated with RNase, there was a marked increased in the radioactive nucleotides present in the incubation medium as compared to the results of the experiment with RNase-untreated controls. Indirect membrane immunofluorescence studies of nonleukemic ascites tumor cells suggest that the sites that react with anti-RNA antibody are distributed diffusely on their surfaces. RNase treatment of these cells markedly reduced their ability to react with the antibody. It thus appears that RNAs are present on the surface membrane of nonleukemic ascites tumor cells and that RNase digests these RNAs, removing negatively charged nucleotides from their electrophoretic surfaces. This results in a reduction in mobility. In contrast, leukemic ascites cells (L1210 and C1498) incubated with RNase showed no significant change in mobility or in the amount of nucleotides released into the incubation medium. Moreover, no fluorescence was found on the surface of cells examined by indirect membrane immunofluorescence. This suggests that leukemic ascites cells are devoid of RNAs on their surface.     

Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

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.     

精氨酸对艾氏腹水癌细胞体外作用的机制探讨

本研究采用小鼠艾氏腹水癌细胞探讨了精氨酸对一些肿瘤细胞体外作用的可能机制。结果表明精氨酸对艾氏腹水癌细胞体外蛋白质合成有显著的抑制作用,其作用受培养介质中一些氨基酸的影响;细胞内游离氨基酸浓度分析结果提示精氨酸的作用可能并不是通过干扰细胞内游离氨基酸池所引起,其具体作用机制尚待进一步实验的揭示。     

On the mechanism of glycolysis stimulation by neutral detergents in 3T3 and Ehrlich ascites tumor cells

Glycolysis of 3T3 and Ehrlich ascites tumor cells was greatly enhanced by Nonidet P-40 or Triton X-100 at about 100 micrograms/mg cell protein. This enhanced glycolysis was partly sensitive to rutamycin and partly sensitive to ouabain, suggesting that the detergent released the control of the ATPase of the mitochondria and of the plasma membrane Na+K+-ATPase. Nonidet P-40 had no effect on glycolysis in cell-free extracts from Ehrlich ascites tumor cells to which soluble mitochondrial ATPase was added. Measuring ouabain-sensitive 22Na efflux and using ouabain-sensitive lactate production as a measure of ATP hydrolysis by the Na+K+ pump, it was shown that Nonidet P-40 greatly decreased the efficiency of the Na+K+ pump. Quercetin increased the efficiency of pumping in EAT cells both in the absence and presence of the detergent.     

Studies on cell surface conformation following injury. II. Scanning and transmission electron microscopy of cell surface changes following anoxic injury in Ehrlich ascites tumor cells.

Exposure of Ehrlich ascites tumor cells to anoxia resulted in rapid and characteristic conformational changes of cell surface topography. Combined scanning and transmission E/M studies revealed rapid alterations including simplification of the cell surface configuration with disappearance of microvilli which were replaced with formation of blebs and recesses at the cell periphery. These surface changes were accompanied by characteristic organelle alterations inside the cells which in this and other cellular systems have been shown to be reversible. Later, the cell surface topography became smoother and monotonic with small blebs and cribriform invaginations in addition to larger eruptions of the cell periphery. Combined transmission E/M studies revealed fragmentation of cellular membrane systems and lysis of organelles indicating the irreversible phase of anoxic injury. The rapid conformational surface changes encountered in Ehrlich ascites tumor cells following anoxia suggest the important role of the plasma membrane and its unfolding as a virtually instantaneous response of the cells to this injury.     

Dependence of the radioprotective effect of beta-mercaptoethylamine on the thiol level of the outer cell membrane

In experiments with Ehrlich ascites tumor cells with the use of a specific thiol blocker, 6,6'-dithiodinicotinic acid, it was shown that thiols of a cell plasmic membrane are necessary for the radioprotective effect of mercaptoethylamine to be realized.     

Antigenicity and Immunogenicity of Cell Surface Fraction of Ehrlich Ascites Tumor Cells

A membrane fraction corresponding to one-tenth of the dry weight of the whole cell was prepared from Ehrlich ascites tumor cells. Such a fraction retained antigenicity in immune adherence inhibition test using antisera taken from mice hyperimmunized to Ehrlich tumors. The antigenicity was impaired by freezing and thawing, and disappeared after such treatment was repeated more than five times, but was stable to heating up to 60°. The above fraction retained immunogenicity to induce resistance of mice against Ehrlich tumors and retained considerable immunogenicity after drying with acetone-ether.