Radiosensitizing and damaging effects of hyperthermia on different biological systems. Ehrlich ascites carcinoma cells

The cytotoxic and radiosensitizing effects of hyperthermia was shown on Ehrlich ascites tumor cells heated in vitro. The effect of hyperthermia resulted in the formation of local lesions in membranes of dying cells.     

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.     

Regulation of pyruvate dehydrogenase complex activity in Ehrlich ascites carcinoma cells by Ca2+ and pyruvate

The dependence of pyruvate dehydrogenase complex (PDC) activity on [Ca2+] was determined in Ehrlich ascites carcinoma cells at different pyruvate concentrations. The resulting family of curves had the following characteristics: a) bell-shaped appearance of all curves with maximum activity at 600 nM Ca2+; b) unchanged position of maxima with changes in pyruvate concentration; c) nonmonotonous changes in PDC activity with increasing pyruvate concentration at fixed [Ca2+]. Feasible mechanisms involving Ca2+-dependent phosphatase and kinase which are consistent with the experimental findings are discussed. To determine the steps in the chain of PDC reactions which determine the observed phenomena, a mathematical model is suggested which is based on the known data on the structural--functional relationships between the complex components--pyruvate dehydrogenase (E1), dihydrolipoyl acetyl transferase (E2), dihydrolipoyl dehydrogenase (E3), protein X, kinase, and phosphatase. To adequately describe the non-trivial dependence of PDC activity on [Ca2+] at different pyruvate concentrations, it was also necessary to consider the interdependence of some steps in the general chain of PDC reactions. Phenomenon (a) is shown to be due only to the involvement of protein X in the PDC reactions, phenomenon (b) to be due to changes in the activity of kinase, and phenomenon (c) to be due to dependence of acetylation and transacetylation rates on pyruvate concentration.     

Relationship between fatty acid and glucose utilization in Ehrlich ascites tumor cells

Glucose greatly increased total free fatty acid (FFA) esterification by Ehrlich ascites tumor cells. However, the FFA concentration of the cells was not altered. Less exogenous FFA was oxidized to CO(2) at any given extracellular FFA:albumin molar ratio when glucose was available, but increasing amounts of radioactive CO(2) were produced as the FFA:albumin molar ratio was raised, even in the presence of glucose. It is suggested that glucose, by providing either energy or an excess of triose acceptor for fatty acid esterification, stimulated FFA uptake only indirectly, by increasing the utilization of FFA subsequent to initial uptake from the medium, i.e., by increasing the turnover rate of the cellular FFA pool. Availability of glucose decreased the oxidation of endogenous lipid radioactivity and the depletion of endogenous lipid ester radioactivity. Most of the radioactivity utilized was derived from phospholipids, and depletion of phospholipid radio-activity was spared when glucose was available. Depletion of cellular total lipid ester also was spared in the presence of glucose. Availability of FFA did not decrease total glucose uptake or its oxidation to CO(2). Glucose utilization by these cells appears not to be regulated by FFA availability in the manner that Randle and coworkers described for muscle.     

Effect of hyperthermia on growth of Ehrlich ascites tumor cells

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.     

Neutral glycosphingolipids in Ehrlich ascites carcinoma cells

The structure of the neutral glycosphingolipids of the Ehrlich ascite carcinoma (EAC) cells was studied. The main four components were identified as glycosylceramide, lastosylceramide, N-acetylgalactosyllactosylceramide and galactosyl-N-acetyllactosylceramide (asialo-GM1). The neutral glycolipid pattern of the cells was found to depend on their density. Dilution of the cell suspension resulted in an increased content of asia-lo-GM1, whereas the content of the other neutral glycolipids remained unchanged. The possible connection between these changes and the earlier disclosed cell density dependence of the gangliosides in EAC cells is discussed.     

Regulation of putrescine metabolism in Ehrlich ascites carcinoma cells exposed to hypotonic medium

When exposed to hypotonic growth medium, Ehrlich ascites carcinoma cells showed a rapid stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in 4 h, followed by a rise in their putrescine content. This effect was totally abolished by addition of a slightly hypertonic concentration of sodium chloride or sucrose to the medium. The general protein synthesis was unaffected by the hypotonic treatment. The uptake of putrescine and, to a lesser extent, spermidine was enhanced, and the conversion of the radioactive putrescine into spermidine appeared partially inhibited during later stages of the hypotonic treatment. As a result, the half-life of putrescine increased from 2.8 h under isoosmotic conditions to 7.3 h in hypoosmotic medium. Both exogenous ([¹⁴C]-putrescine-derived) and endogenous ([¹⁴C]ornithine-derived) putrescine degraded at similar rates in control and hypotonic cells, yet the putrescine taken from the medium degraded preferably to nonpolyamine products, while the putrescine synthesized in the cell was converted evenly to spermidine and to other metabolites. Adenosylmethionine decarboxylase activity (EC 4.1.1.50), which provides the second precursor for spermidine and spermine synthesis, was distinctly inhibited in the hypotonic medium. Inhibition was likewise observed in spermidine synthase activity, while spermine synthase was marginally stimulated. It appears that the hypotonic treatment serves a special condition under which not only the formation of putrescine is enhanced dramatically but the cells also attempt to conserve the diamine by preventing its further metabolism to higher polyamines.     

Inhibition of lactate transport and glycolysis in Ehrlich ascites tumor cells by bioflavonoids.

Bioflavonoids are potent inhibitors of lactate transport in Ehrlich ascites tumor cells. The most effective bioflavonoids have four to five hydroxyl groups. Sugar substitution at carbon three, or reduction of the double bond between carbons two and three, decreases their inhibitory activity. Quercetin, the most extensively studied of these compounds, inhibits lactate efflux by 50% at 0.1 micrograms/mg of protein. On addition of quercetin to glycolyzing Ehrlich ascites tumor cells, lactate accumulates inside the cell and the intracellular pH drops. Total lactate production is also inhibited. Nigericin prevents the internal acidification that occurs in the presence of quercetin and also reduces the inhibition of glycolysis. Thus, it appears that inhibition of lactate efflux can affect glycolysis through a lowering of the intracellular pH. The inhibitory effect of quercetin on glycolysis can be explained by its effect on lactate efflux and its previously reported effect on the Na+--K+ ATPase [Suolinna, E.--M., et al. (1974) J. Natl. Cancer Inst. 53, 1515].     

Fluorescence study of Ehrlich ascites carcinoma cells after X-irradiation.

The mechanism of the increase in ultra-violet fluorescence (U.V.F.) intensity of Ehrlich ascites carcinoma cells after X-irradiation were investigated. It was shown that there were two mechanisms: size-dependent (mechanism I) and size-dependent (mechanism II). The basis of mechanism I is most probably an increase in protein content. Mechanism II is related to an increase in the U.V.F. quantum yield, though its biological nature is still unknown.     

Amino acid supply and protein metabolism in Ehrlich ascites tumour cells. 2. Incorporation of 14C-tyrosine

The incorporation of 14C-tyrosine into protein of incubated Ehrlich ascites tumour cells was measured over a 10-fold range of external amino acid concentrations, the composition of which simulated that of mouse intraperitoneal fluid. Incorporation was linear with time and the rate of incorporation increased with increasing external amino acid concentration up to at least six times intraperitoneal concentrations. Further increases in external amino acid supply did not yield a concomitant increase in the rate of tyrosine incorporation, a maximum fractional protein synthetic rate of approximately 100% d-1 being achieved. The specific activity of tyrosine in intracellular fluid was approximately 95% of that of extracellular fluid, irrespective of the external amino acid concentration.     

Cytokinetic studies on the switch from glucose to uridine metabolism, and vice versa, of Ehrlich ascites tumour cells in vitro

Glucose is normally required as the energy source and for the proliferation of neoplastic cells. For Ehrlich ascites tumour cells, kept under glucose-free culture conditions, this requirement was alleviated by uridine, indicating that the supply of ribose is obligatory for sustaining growth capacity. In a 96-hr culture experiment with mouse-derived cells, the increase in cell number from cultures supplemented with 5 mM uridine was 50-70%, whilst lactate production was 5% that of controls. An increase in the number of multinucleate cells was observed from cell-smears; DNA histograms indicated the presence of cells with a DNA content higher than 4c and an increased portion of cells in G2 phase. For precise determination of changes in cell cycle distribution on transfer of cells from glucose-supplemented to glucose-free conditions, the progression of phase-accumulated cells (by centrifugal elutriation) was monitored by DNA distribution analysis; G2 cells continued the cycle at a rate comparable to controls but were delayed, in the following cycle, predominantly in S and G2 phases. This was also observed with G1 cells from a G1-accumulated fraction in the first cycle. The addition of glucose to cells kept for some hours in glucose-free, uridine-supplemented medium resulted in an immediate increase in mitotic index (amplification by the colcemid method). The results are interpreted and support our concept that the delivery of compounds, necessary for normal growth, i.e. hexoses for glycoproteins and glycolipids, are limited as a consequence of the 'metabolic channelling' of pentose from uridine in Ehrlich ascites tumour cells. Therefore, the constantly lowered growth-rate in uridine-supplemented cells observed with long-term culture experiments could reflect an adaptation of growth-cycle to these limitations.     

Cytokinetic studies on the switch from glucose to uridine metabolism, and vice versa, of Ehrlich ascites tumour cells in vitro

Abstract. Glucose is normally required as the energy source and for the proliferation of neoplastic cells. For Ehrlich ascites tumour cells, kept under glucose-free culture conditions, this requirement was alleviated by uridine, indicating that the supply of ribose is obligatory for sustaining growth capacity.
In a 96-hr culture experiment with mouse-derived cells, the increase in cell number from cultures supplemented with 5 mM uridine was 50–70%, whilst lactate production was 5% that of controls. An increase in the number of multinucleate cells was observed from cell-smears; DNA histograms indicated the presence of cells with a DNA content higher than 4c and an increased portion of cells in G₂ phase. For precise determination of changes in cell cycle distribution on transfer of cells from glucose-supplemented to glucose-free conditions, the progression of phase-accumulated cells (by centrifugal elutriation) was monitored by DNA distribution analysis; G₂ cells continued the cycle at a rate comparable to controls but were delayed, in the following cycle, predominantly in S and G₂ phases. This was also observed with G₁ cells from a G₁-accumulated fraction in the first cycle.
The addition of glucose to cells kept for some hours in glucose-free, uridine-supplemented medium resulted in an immediate increase in mitotic index (amplification by the colcemid method).
The results are interpreted and support our concept that the delivery of compounds, necessary for normal growth, i.e. hexoses for glycoproteins and glycolipids, are limited as a consequence of the 'metabolic channelling' of pentose from uridine in Ehrlich ascites tumour cells. Therefore, the constantly lowered growth-rate in uridine-supplemented cells observed with long-term culture experiments could reflect an adaption of growth-cycle to these limitations.