A reduction in energy-dependent amino acid transport by microtubular inhibitors in ehrlich ascites tumor cells

Vincristine, other periwinkle alkaloids, and colchicine partially inhibit the energy dependent transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The properties of this phenomenon were characterized in detail for vincristine. Maximum depression of the steady-state intracellular α-aminoisobutyric acid level was achieved with a vincristine concentration of > 0.5 m̈M. The inhibitory effect of vincristine increases as the extracellular α-aminoisobutyric acid concentration is increased reaching a maximum, however, of only ∼25% at a level of 5 mM, leaving a large gradient for α-aminoisobutyric acid across the cell membrane. Vincristine produced an asymmetrical effect on the bidirectional fluxes decreasing the initial uptake rate, while increasing the efflux of α-aminoisobutyric acid. Inhibition of net α-aminoisobutyric acid transport by vincristine was partially reversible (∼40%). Colchicine (50 m̈M) reduced the steady-state α-aminoisobutyric acid level by 30%, an effect that was not reversible. Inhibition by vinleurosine and vinrosidine was comparable to that of vincristine. Addition of glucose to the medium resulted in a small, but significant, decrease in the inhibitory effects of both vincristine and colchicine. The data indicate that these agents inhibit a small component of the uphill transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The inhibitory effect of vincristine cannot be attributed to an increase in the passive permeability of the cell membrane to this agent. Rather, the data along with other studies from this laboratory suggest that vincristine reduces the energy-dependent transport of α-aminoisobutyric acid by either inhibiting cellular energy metabolism or by inhibiting the coupling of energy-metabolism to the transport of this amino acid and raises the possibility that cellular microtubules play a role in these processes.     

Role of ATP on the initial rate of amino acid uptake in ehrlich ascites cells

Incubation with graded doses of rotenone can bring about a graded lowering of the cellular ATP level. Using cells with varying ATP levels, it can be shown that the initial uptake of [¹⁴C]glycine, before the cellular concentration exceeds that of the medium, is decreased as ATP decreases. Alterations in cellular cations cannot account for the difference in glycine influx. Prolonged exposure of cells to a lowered ATP content increases the exodus of cellular glycine. Valinomycin reduces the steady-state level of glycine uptake, but its effects can to a major extent be overcome by the addition of glucose. At high extracellular K⁺ (70 mM) neither the sum of the Na⁺+K⁺ gradients, nor the electrochemical potential of Na⁺ provides sufficient energy to account for glycine and 2-aminoisobutyrate accumulation if a 1:1 coupling between Na⁺ and the amino acid occurs.     

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.     

Osmotic properties of ehrlich ascites tumor cells during the cell cycle

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9–14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a “bound” form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.     

Phosphorus incorporation in the ehrlich ascites tumor cell

Data from isotopic uptake experiments were used to measure the kinetics of labelling of cellular phosphate, ATP and ADP in the Ehrlich ascites tumor cell. The results show that steady state phosphate exchange flux was 0.333 ± 0.052 (S.E.) μmoles per 10⁷ cells per hour at 37°, and that the specific activity of phosphate was the same as P_γ ATP. Metabolic inhibition reduced the phosphate flux by 30–50%. A model, based on oxidative phosphorylation and the adenylate kinase reaction is used to interpret the labelling sequence of P_β ATP and P_β ADP, and its dependence on P_γ ATP.     

The effect of various seleno-compounds on ehrlich ascites tumor cells

The effect of various concentrations and forms of selenium on in vitro viability of Ehrlich Ascites Tumor Cells (EATC) was investigated. Sodium selenite, selenium dioxide, seleno-dl-cystine, and seleno-dl-methionine, dramatically decreased EATC viability as measured by dye exclusion. Sodium selenate only marginally decreased EATC viability. Cell viabilities decreased with increasing selenium in the incubation media and as a function of time. Viabilities determined by dye exclusion did not correlate with the inhibition of tumor growth observed after treatment with selenium. Intraperitoneal injections of selenite in mice previously inoculated with EATC significantly inhibited tumor development. Delaying intraperitoneal injections of selenite to 5 and 7 days after inoculation of mice with EATC reduced the effectiveness of this nutrient on the inhibition of EATC growth. Incubation of EATC in vitro with supplemental selenium prior to injection of mice completely inhibited EATC development in vivo before any appreciable alteration in cell viability was observed.     

Immunoprophylaxis with attenuated ehrlich ascites tumor cells in admixture with BCG

Summary During propagation in tissue culture, the Ehrlich ascites carcinoma was found to lose some of its tumor-producing capacity (oncogenicity) when implanted IP or SC into CF-1 mice. On the other hand, attenuated cells retained their immunoprotective capacity; immunization of mice with a single dose (1×10⁴) of these cells induced a high degree of resistance against a challenge 1 month later with virulent Ehrlich cells maintained by IP transplantation. The admixture of BCG (1×10⁶ viable units) with attenuated cells further improved their immunogenicity. The immunogenicity of attenuated cells was almost completely abolished by gamma-irradiation (2,500 rads), but this property was significantly restored by the addition of BCG. Some evidence is presented that suggests that attenuated cells have a higher immunoprotective capacity than the corresponding virulent cells.     

The turnover of molecular species of phosphatidylinositol in ehrlich ascites tumor cells

It has been shown previously that ³²P_i is incorporated into phosphatidylinositol 30 times faster than into the other phospholipid classes in Ehrlich ascites tumor cells, whereas [1-¹⁴C]glycerol is incorporated at almost the same rate (Waku, K., Nakazawa, Y. and Mori W. (1976) J. Biochem. 79, 407–411). It was therefore suggested that there is a recirculating system (phosphatidylinositol → diacylglycerol → phosphatidic acid → CDPdiacylglycerol → phosphatidylinositol) of phosphatidylinositol in Ehrlich ascites tumor cells. In this work, ³²P_i or [1-³H]glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells from which the lipids were extracted after selected periods. Phosphatidylinositol was prepared and fractionated in the form of dimethylphosphatidic acid into six molecular species by AgNO₃-impregnated TLC. The specific radioactivities of the fractionated species were determined. ³²P_i was incorporated into diene molecular species and [1-³H]glycerol into monoene species with a higher rate than the other species and both precursors were incorporated into tetraene species rather slowly. ³²P/³H values appeared to be at almost the same for each molecular species, although monoene species showed slightly lower values. These results suggest that there could be a recirculating of the phosphorylinositol moiety in each of the molecular species of phosphatidylinositol.     

Electrolyte and non-electrolyte distribution in the ehrlich ascites tumor cells during the cell cycle

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na⁺, K⁺, and C1^? in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na⁺ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na⁺ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. ¹⁴C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, ¹⁴C-urea distributes in anywhere from 86–100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.     

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Summary The intracellular pH (pH _i ) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H⁺ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH _i of cells placed in an acidic medium (pH _o below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH _o is acutely reduced to 5.5, pH _i falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H⁺. The unidirectional influx of H⁺ exhibits saturation kinetics with respect to extracellular [H⁺]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK _m is 0.74 ± 0.09 × 10^–6 m.Steady-state cells with pH _i above 6.8 continuously extrude H⁺ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH _i 6.3) when returned to pH _o 7.3 medium respond by transporting H⁺, resulting in a rapid rise in pH _i . The halftime for this process is 1.09 ± 0.22 min. The H⁺ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH _i to its steady-state value.     

Estimation of rate of synthesis of chromosomal DNA in ehrlich ascites tumor cells

An estimate is made of the rate coefficient for linear DNA synthesis with exact doubling in an exponentially growing population of Ehrlich ascites tumor cells having chromosome-number dispersion. Comparison of calculated and experimental results suggest that the assumptions used in the calculation are tenable, but further experimental evidence is needed to prove this.     

The interaction of chloride with the sulfate transport system of ehrlich ascites tumor cells

The effect of Cl^? on SO₄^?2 efflux was studied in both Cl^?-containing and Cl^?-free ascites tumor cells loaded with ³⁵SO₄^?2 to test the hypothesis that Cl^?-SO₄^?2 exchange is mediated by the same mechanism responsible for SO₄^?2-self exchange. The addition of Cl^?-free, ³⁵SO₄^?2 loaded cells to a SO₄^?2-free, Cl^? medium results in: (1) SO₄^?2 efflux that is dependent on the extracellular Cl^? concentration (Km = 4.85 mM; k_e = 0.048 min^?1 at 50 mM Cl^?) and (2) net Cl^?-uptake that exceeds SO₄^?2 loss. Both SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate) and ANS (1-anilino-8-napthalene sulfonate) inhibit SO₄^?2 efflux but are without effect on Cl^? uptake. The addition of Cl^?-containing, ³⁵SO₄^?2 loaded cells to a SO₄^?2-free, C1^? medium results in: (1) a slight gain in cellular Cl^? and (2) k efor SO₄^?2 efflux identical to that for Cl^?-free cells. The results are compatible with the suggestion that: (1) Cl^? interacts with a membrane component responsible for transmembrane SO₄^?2 movement; (2) Cl^? interaction stimulates the rate of unidirectional SO₄^?2 efflux from cells initially free of Cl^? as well as the rate of SO₄^?2 turnover in cells maintained in the steady state with respect to Cl^? and SO₄^?2; and (3) in the case of cells initially free of Cl^?, the Cl^?-SO₄^?2 pathway represents only a small fraction of the total unidirectional Cl^?-influx the remainder being compatible with the electroneutral accumulation of NaCl and KCl.