D. Bossi

Calcium permeability of Ehrlich ascites tumour cell plasma membrane in vivo

Passive Ca2+ entry into Ehrlich ascites tumour cells has been investigated. Passive equilibrium of Ca2+ takes place in ascites tumour cells only under conditions of exhaustive energy depletion. The specific Ca2+ ionophore A23187 does not affect Ca2+ entry into ascites tumour cells under active metabolic conditions, but it increases the rate of Ca2+ equilibration in ascites tumour cells in the early stages of energy depletion. The results of the present experiments lead to the conclusion that in ascites tumour cell plasma membrane Ca2+ permeability is not a limiting step in the regulation of intracellular calcium content, while the energy-dependent Ca2+ extrusion is the main mechanism that prevents uncontrolled intracellular Ca2+ increase. The results taken together support the hypothesis that increased Ca2+ influx into the cell, caused by plasma membrane alteration, is responsible for permanently elevated mitotic activity and for deranged metabolic behavior of these neoplastic cells.

Lack of effect of the Ca2+ ionophore A23187 on tumour cells

The Ca2+ ionophore A23187 increases intracellular calcium content in normal thymic cells, while it is without effect on the corresponding neoplastic cell (Ascites thymoma) and on Ehrlich ascites tumour cells. The A23187-induced total cell calcium increase in normal thymocytes takes place both in control and energy-depleted cells, while it is lacking in neoplastic cells. In addition the ionophore stimulates aerobic glycolysis of normal thymocytes, whereas it is ineffective on neoplastic cells. The study of intracellular calcium exchange properties reveals that in normal cells the ionophore A23187 provokes a 60% increase of the exchangeable pool together with a more significant, 4-fold enlargement of the unexchangeable pool. These effects are lacking in cancer cells. The data give rise to interesting considerations concerning the regulation and compartmentalization of calcium in neoplastic cells. The results will be also discussed in relation to the models that predict altered cell calcium metabolism as a cause of cancer cell high aerobic glycolysis and uncontrolled growth.

Involvement of the Ca2+/phospholipid-dependent protein kinase in the G1 transit of T51B rat liver epithelial cells

The G0----G1 and G1----S transitions (but not the intervening events) in the G1 phase of T51B rat liver epithelial cells in serum-stimulated confluent cultures required a high concentration of extracellular Ca2+ and were accompanied or immediately preceded by increases in the amount of EDTA-extractable protein kinase C, a Ca2+/phospholipid-dependent enzyme. Involvement of this Ca2+-dependent enzyme in the two Ca2+-dependent transitions was further indicated by the facts that 12-O-tetradecanoyl phorbol-13-acetate (TPA), a compound that stimulated protein kinase C from T51B cells even in the absence of Ca2+, enabled these cells to transit G1 in Ca2+-deficient medium, while a TPA analogue (4 alpha-phorbol-12, 13-didecanoate (4 alpha-PDD) that did not stimulate the enzyme in cell-free preparations did not promote G0----G1 or G1----S transit in Ca2+-deficient medium.

Calcium metabolism in Ehrlich ascites tumour cells

Ehrlich ascites tumour cells are able, under the proper experimental conditions, to extrude a substantial amount of Ca2+ from the intracellular space. The Ca2+ extrusion mechanism, probably located at the plasma membrane level, appears to be similar to that found in red blood cells. It is energy-dependent and both respiration and glycolysis are able to drive it. The use of some inhibitors and uncouplers, besides showing that this activity is different from that linked to the mitochondrial Ca2+ pump which acts in the opposite direction, proposes some speculations on the energy compartmentation in the Ehrlich ascites tumour cells.

Intracellular calcium and magnesium content and aerobic lactate production in intact Ehrlich ascites tumour cells

The high rate of aerobic glycolysis and the low ‘Pasteur effect’ are the most consistent biochemical features of tumour cell energy metabolism ([ 11, reviewed [2]) but their causes and meaning are still under discussion [3-61. Among various hypotheses proposed to explain the high aerobic glycolysis of cancer cells one, postulating a profound alteration of cell calcium metabolism, seems very attractive as it could explain some other biological features relevant to neoplasia (viz., uncontrolled growth, invasivity ([3], reviewed [7]). This hypothesis implies the existence in cancer cells of a decreased cytosolic Ca2’ concentration resulting from both a low plasma membrane permeability to Ca2+ and a high Ca2+ accumulating capacity of tumour mitochondria. The following alteration of the Ca2’/ Mg2+ ratio would then be responsible for the increased activity of some key glycolytic enzymes [3]. Following our results on Ca2+ extrusion by intact Ehrlich ascites tumour cells (ATC) [8,9] we now report data on the relationship between calcium content and rate of aerobic glycolysis in ATC. For this purpose we utilized, as experimental model, intact ATC suspended in isosmotic, Tris-buffered, mannitol-sucrose solution, since in this medium ATC show an unusual low rate of aerobic glycolysis; in addition we took advantage of the antibiotic ionophore A23 187, which has been shown to strongly increase ATC calcium content [8,9]. The results obtained led us to the conclusion that in intact ATC the intracellular calcium concentration, and the size of its exchangeable pool, do play a

The effect of Mg2+ upon 6-phosphofructokinase activity in ehrlich ascites tumor cells in vivo

The effect of Mg2+ addition to intact Ehrlich ascites tumor cells (EATC) has been investigated. A decrease of glucose 6-phosphate (G6P) content and an increase of fructose 1,6-diphosphate (FDP) content are detected in glucose utilizing EATC incubated with increasing Mg2+ concentrations (from 0 to 5.0 mM). The strong enhancement of FDP/G6P ratio is taken as evidence for in vivo stimulation of phosphofructokinase 1 (PFK) (ATP:D-fructose-6-phosphate 1-phosphotransferase; EC 2.7.1.11). A similar effect can be observed when glucose is replaced by fructose as the glycolytic substrate. Stimulation of PFK is paralleled by substantial depletion of ATP. Cytochalasin B prevents the observed phenomena. Cell total Mg increases by about 15% when EATC are incubated with 5 mM Mg2+. The overall data show that extracellular Mg2+ may modulate glycolytic flux in EATC in vivo. Implications and significance of these phenomena in the regulation of cancer cell metabolic features are discussed.

Further observations on the effect of calcium ionophores on ascites tumor cells.

The effect of the Ca2+ ionophore ionomycin on neoplastic thymocytes in comparison to its effect on normal thymus cells was studied. Ionomycin increases intracellular Ca2+ in normal lymphocytes but fails to increase Ca2+ in neoplastic thymocytes. In these cells the ionophore causes a transient increase in cytosolic free Ca2+. The lack of effect of ionomycin reproduces that of A23187, but it does not depend on reduced availability of intracellular Mg2+ to exchange with Ca2+; it appears to depend on the strong activity of the plasma membrane Ca2+-extruding pump that counteracts ionomycin permeabilization and that can be partly inhibited by the calmodulin inhibitor R24571 (calmidazolium). Neoplastic thymocytes show a high content of magnesium, the intracellular binding of which is efficiently regulated by endogenous ATP. The data show also an interesting correlation between the regulation of energy metabolism (aerobic glycolysis) and cation homeostasis in the neoplastic cells studied.

The influence of extracellular calcium on the distribution of protein kinase C in non-neoplastic and neoplastic rat liver cells

Non-neoplastic T51B rat liver epithelial cells cannot proliferate in Ca2+-deficient medium. This proliferative inhibition in Ca2+-deficient medium is accompanied by a large reduction in the amount of cellular EDTA-extractable Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity. By contrast, tumorigenic epithelial cells from several Morris hepatomas proliferate in Ca2+-deficient medium and either maintain or greatly increase their content of EDTA-extractable protein kinase C.

The effect of magnesium on glycolysis of permeabilized Ehrlich Ascites tumor cells

We have previously observed that extracellular Mg2+ influences the phosphofructokinase (PFK) activity of intact Ehrlich Ascites tumour cells (EATC). In this study we have investigated the mechanism by which Mg2+ modulates this key glycolytic enzyme in EATC made permeable to the cation by either digitonin or dextran sulphate. Results showed that when Mg2+ is freely permeable to the cytosol, the in vivo PFK activity, calculated as FDP/G6P ratio, is not increased as it is in intact cells. We also observed that in permeabilized cells Mg2+ determines the increase of glucose 6 phosphate (G6P), fructose 1,6 bisphosphate (FDP) and lactate production. We hypothesize that extracellular Mg2+ regulates PFK and glycolysis in these neoplastic cells not by entering the cytosol but by a specific interaction with the plasma membrane.

Impairment of microsomal calcium sequestration activity upon superoxide dismutase depletion in rat liver.

We have studied the Ca2+-accumulation activity of microsomal vesicles isolated from the liver of rats held for from 2 to 8 weeks on a copper-deficient diet. With this treatment that deeply modifies fatty acid composition, microsomal membranes show progressively lower Ca2+ sequestration. The activity can be fully restored upon physiological copper supply to the depleted animals. The determination of kinetic parameters of microsomal Ca2+ uptake shows that copper deficiency affects mainly the apparent velocity, leaving unaffected the apparent affinity of the pump for Ca2+. Many similarities were found between this model and the Morris hepatomas with different growth rate. The data support the hypothesis that the oxidative stress imposed on the cell by the loss of superoxide dismutase can influence many cell features, with different implications in the regulation of several biological and biochemical functions.