A study of hyperlipidaemia induced by ascites tumours.

In mice four stages of hyperlipidaemia induced by Ehrlich ascites tumour could be distinguished. Hyperlipidaemia is characterized mainly by increased serum VLDL content accompanied by high triglyceride concentration. The only exception was the regressive stage III where the serum lipid level (VLDL) has temporarily decreased. From the results obtained with the simultaneous examination of the liver, mesenteric fat tissue, tumour cell and ascites plasma lipids, it may be concluded that the endogeneous fat mobilization induced by tumour cells via increased VLDL synthesis and secretion of liver will lead to hyperlipidaemia and to the total depletion of the lipid stores. Rapid and reversible fall in lipid level following the withdrawal of ascites fluid in tumorous animals demonstrated clearly the direct effect of the tumour cells on lipid-lipoprotein metabolism of the host organism.     

The modelling of the growth of Ehrlich carcinoma and teratoma T-36 with ascitic fluid and tumor-cell dialysate]

The study of the effect of ascitic fluid and dialysate of Ehrlich ascites tumor cells (M.m. less than 15 kDa) on the growth of Ehrlich carcinoma and teratoma T-36 has shown that both the ascitic fluid and dialysate can protect tumor cells in vivo. The number of animals with tumors increased from 0% in control animals to 60 and 20%, respectively, in experimental ones after transplantation i.m. of 20 x 10(3) Ehrlich tumor cells into mice. Compared to control, ascitic fluid and dialysate of Ehrlich ascites tumor cells increased the rate of tumor growth to 195 and 153%, respectively. It is suggested that this test-system simulates the effect of tumor humoral factors in vivo.     

Chalone-like inhibition of Ehrlich ascites cell proliferation in vitro by an ultrafiltrate obtained from the ascitic fluid.

An aqueous ultrafiltrate (10 000-50 000 dalton) prepared from the cell-free ascitic fluid of mice bearing Ehrlich ascites tumour (EAT) in the plateau phase of growth (12-16 days after transplantation) was investigated with regard to its inhibitory effects on the proliferation of EAT cells in a 24-hr suspension culture. The following results were obtained: (1) The in vitro proliferation of cells obtained from the plateau phase of in vivo growth was reversibly inhibited. (2) The dose-response curves show a plateau with a maximum inhibition of about 50%, which suggests that not all cells can be affected. (3) Young cells (4-6 days after transplantation) were not inhibited. (4) Preincubation of plateau phase cells in the culture medium before treatment abolishes the inhibitory effect of the ultrafiltrate. This effect of preincubation is dependent on time and serum concentration. It provides the possibility to differentiate between true "chalone-like" and cytotoxic effects. (5) the inhibitory properties of the ultrafiltrate are destroyed by heating or trypsin treatment. (6) Extracts prepared in the same way from ascitic fluid of mice bearing lymphocytic leukemia L1210 do not inhibit the proliferation of EAT cells. Corresponding extracts from ascitic fluid of mice bearing myelocytic leukemia YM were found to be inhibitory; however, the inhibitory effect was also found on preincubated cells and is therefore considered to be due to an unspecific cytotoxicity. In conclusion, evidence was obtained for a factor from the ascitic fluid of mice bearing EAT, which prevents EAT cells from entering the proliferating state.     

CELL PROLIFERATION DURING the COURSE of IMMUNOLOGICAL REJECTION of EHRLICH ASCITES TUMOUR CELLS

Ehrlich ascites tumours were transplanted from their normal hosts (mice) into rats, and studies made of the timing of cell cycle 120-170 hr after transplantation, when tumour regression was under way. There was no significant change in the duration of the cell cycle. Some increase was found in the average and spread of G₂ durations, largely compensated by a decrease in S duration. When taken together with previously published work the results suggest that the immunological pressure of transplantation into a heterologous host prolongs the G₂ period.     

Mitoxantrone toxicity on Ehrlich ascites tumour cells: inhibition of the transplasma membrane redox activity

This study focused on the potent cytotoxic effect that mitoxantrone produces on Ehrlich ascites tumour cells. Host mice treated with mitoxantrone showed a life span three times higher than control non-treated host mice. Mitoxantrone also showed a potent cytotoxic effect on Ehrlich cells incubated in vitro for only a few hours. Studies on the effect of mitoxantrone on a plasma membrane redox system showed that mitoxantrone inhibits this activity, which is apparently related to cell proliferation.     

The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immunologically distinct from the mouse NAD-dependent [corrected] bifunctional enzyme

Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2',5'-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase-cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.     

The antitumor effect of bleomycin combined with bestatin against Ehrlich ascites carcinoma in mice

The effect of bleomycin against Ehrlich ascites carcinoma transplanted subcutaneously to mice used in combination with bestatin was investigated. Male Balb/c mice weighting approximately 20 g and bred in our laboratories were used in this study. Each mouse was injected in its left lateral abdominal region subcutaneously with 7 X 10(6) tumor cells in 0.2 ml of ascites fluid. The mice were divided into four groups: control, bestatin alone (5 mg/kg intraperitoneally on Days 9-14), bleomycin alone (10 mg/kg intraperitoneally on Days 7 and 8), and bestatin plus bleomycin. Our results show that bestatin enhances the antitumor effect of bleomycin against Ehrlich ascites carcinoma as measured by the increased survival rates. Being an agent of very low toxicity, bestatin should be considered as a part of the chemoimmunotherapy protocol.     

Microspectrofluorometry of ultraviolet-inducible fluorescence in supravitally-stained ascites tumour cells

Synopsis Ultraviolet irradiation of tumour cells (Ehrlich tetraploid ascites tumour of mice, TO strain), supravitally stained with thiazine dyes (Azure II, Azure A, Methylene Blue, Toluidine Blue) or an oxazine dye (Brilliant Cresyl Blue), induces blue fluorescence in cytoplasmic bodies believed to be lipid droplets or lysosome-like bodies. Microspectrofluorometry of the inducible fluorescence in Ehrlich tumour cells gives bimodal excitation (340/394 nm) and emission (443/700 nm) curves.     

Effects of dietary fat composition on the Ehrlich ascites tumor fluid lipoproteins.

Mice bearing the Ehrlich ascites tumor were fed diets rich in either coconut oil or sunflower oil. From 20 to 40% less lipid was present in the ascites tumor fluid when the mice were fed the sunflower oil diet. This was associated with a reduction in the amount of very low density lipoproteins (VLDL) and high density lipoproteins (HDL), the main lipoprotein fractions present in the ascites tumor fluid. The VLDL from the mice fed sunflower oil contained more cholesteryl esters and a lower free to esterified cholesterol ratio than those from the mice fed coconut oil. Very little change occurred in the composition of the HDL. All of the lipids contained in both lipoprotein fractions exhibited appreciable differences in fatty acid composition. Much more monoenoic and less polyenoic fatty acid were present in the lipids from the mice fed the coconut oil diet, but no appreciable change in saturated fatty acid content occurred. Similar changes in fatty acid composition were observed in the blood plasma of the tumor-bearing mice. There was no qualitative difference in the apolipoprotein patterns of either the ascites fluid VLDL or HDL. Pyrene fluorescence studies indicated that the fluidity of the VLDL was increased when the mice were fed the sunflower oil diets. No difference in HDL fluidity, however, was observed by this technique. These results indicate that the amount, composition, and physical properties of certain of the lipoproteins contained in the ascites tumor fluid can be modified by changing the composition of the dietary fat fed to mice bearing the Ehrlich ascites tumor.     

The effect of insulin on the growth of transplanted tumors in mice

The growth of four murine transplantable tumours, Ehrlich ascites or solid tumour, an aplastic carcinoma, B-16 melanoma, and a thymoma, were suppressed in mice treated daily with insulin (2 IU). Since insulin increased the number of plaque-forming cells and the phagocytic activity of the liver and spleen cells, the retardation of the tumour growth was ascribed to immunological mechanisms.     

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.     

Absence of anthracycline-induced degradation of nuclear DNA in Ehrlich ascites tumour cells

The in vivo effects of anthracycline antibiotics on the integrity of Ehrlich ascites tumour cell DNA have been studied by sedimentation analysis of nuclear structures containing superhelical DNA in neutral sucrose gradients. These fast-sedimenting protein-DNA complexes may be released by gently lysing cells in solution containing non-ionic detergents and high NaCl concentrations (1.95 M). The supercoiled structure of DNA in these protein-DNA complexes is suggested by the characteristic sedimentation in the presence of intercalating agents. Apparently, no DNA damage could be detected in Ehrlich cells from 7-day-old tumours within 3 h after various doses of daunomycin (0.5–10 mg/kg of body wt.) were administered i.p. to mice. Sedimentation anomalies could not be observed even 15 or 30 h after administration of therapeutic doses of daunomycin or adriamycin. In contrast, at 30 min after administration to mice, therapeutic doses of bleomycin (2–8 mg/kg) caused extensive fragmentation of tumour cell DNA, which could be monitored as slowly sedimenting DNA structures (compared with the control). Similarly, DNA damage could be induced by procarbazine at therapeutic doses. Exposure to bleomycin or procarbazine abolished the characteristic biphasic response to ethidium bromide. The absence of anthra-cycline-induced degradation of Ehrlich ascites tumour cell DNA is apparently in contrast with the DNA damage observed in L1210 tumour cells. These observations suggest that DNA damage is not a necessary condition for antitumour activity.     

Interaction andin vivo growth inhibition of Ehrlich ascites tumor cells by jacalin

Jacalin has been found to agglutinate Ehrlich ascites cells. The agglutination was inhibited by α-glycosides of D-Gal and β -D-Gal(1 → 3)-D-GalNAc suggesting that the lectin-ascites interaction was carbohydrate-specific. There was 21.8% inhibition of tumour (ascites) cell growthin vivo in mice administered 50μg of jacalin by injection for 6 days following intraperitoneal injection of ascites cells. Administration of 100, 150 and 200μg jacalin resulted in 40.2, 57.5 and 83% inhibition respectively. Thein vivo inhibition of tumour cells growth by jacalin was due to its preferential binding with D-Gal-α -(1 → 6) present as terminal residues in the glycoprotein on tumour cell surface.     

APOPTOSIS OF EHRLICH MOUSE ASCITES CELLS INDUCED BY LONG-TERM EXPOSURE OF WEAK ELECTROMAGNETIC FIELDS IN VIVO

To study the possibility of apoptosis of tumor cells induced by weak electromagnetic fields (EMFs) in vivo, mice were inoculated with Ehrlich ascites cells and exposed to a long-term electromagnetic field (1 mT, 700 KHz). During the treatment, growth curves of mice were measured and compared between exposed and sham-exposed mice. The results show that the growth curves of healthy controls agree well with the ideal curve of logistic growth, but the growth curves of cancer mice deviate from the ideal curve. There is no difference in growth curves between exposed, and sham-exposed healthy mice, and they both agree with the ideal curve. However, a notable difference in growth curves between exposed and sham-exposed cancer mice was obtained. Moreover, the curves of sham-exposed mice deviate even more than those of the exposed mice; in other words, the growth curves of Ehrlich ascites mice deviate from the ideal curve of healthy mice but are shifted toward it by the EMF treatments. After the treatment, apoptosis of Ehrlich ascites cells from inoculated mice was analyzed by several methods, including flow cytometry, fluorescence microscopy, and DNA gel electrophoresis. Statistical analysis from flow cytometry shows that the apoptotic ratio of cells from exposed Ehrlich ascites mice was significantly higher than that from sham-exposed treated mice. Microscopic observation of Ehrlich ascites cells stained with acridine orange (AO) and propidium iodide (PI) showed typical apoptotic changes in exposed animals whose cell nuclei were highly condensed or fragmented and uniformly stained green by the AO, whereas cell nuclei from sham-exposed mice were stained green and showed a fine reticular pattern. Agarose gel electrophoresis of DNA from exposed mice showed that the chromatin DNA exhibited ladders, a characteristic feature of internucleosomal degradation of DNA by EMF treatments. For interactions between external electromagnetic fields and DNA, the mechanism of apoptosis of tumor cells induced by weak EMFs is discussed.     

The inhibitory effect of various fatty acids on aerobic glycolysis in Ehrlich ascites tumour cells

Inhibition of glycolysis in Ehrlich ascites tumour cells by saturated fatty acids, added either in form of potassium salts or incorporated into phosphatidylcholine liposomes, increases with the increasing carbon atom chain length and is independent of the concentration within the range of 0.1 to 1.0 mM. In contrast, the inhibition of glycolysis in the cytosolic fraction from Ehrlich ascites cells depends on the concentration of fatty acids. The content of ATP in Ehrlich ascites cells incubated with fatty acids increases with increasing carbon atom chain length, which leads to a crossing-over in the concentrations of pyruvate and 2-phosphoenolpyruvate. Lowering of the sum of both these metabolites by palmitate and stearate points to the inhibition not only of pyruvate kinase but also of other enzymes of early steps of glycolysis. Fatty acids in intact Ehrlich ascites cells inhibit all three key glycolytic enzymes but added to the cytosolic fraction affect mainly the activity of phosphofructokinase. The inhibition of pyruvate kinase by fatty acids is smaller in the cytosolic fraction from tumour cells than from liver and muscles.     

Sequential elution of proteins in small volumes by preparative polyacrylamide gel electrophoresis

A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor.     

Lipid peroxidation in Ehrlich ascites cell mitochondria is not determined by the polyunsaturated fatty acid content of the membrane

Lipid peroxidation intensity is compared in Ehrlich Ascites Cell and in liver mitochondria, prepared from tumor bearing mice. Malondialdehyde formation is negligible in intact ascites tumour mitochondria, but it is significantly increased in permeabilised mitochondria and in isolated mitochondrial membranes. We suggest that the resistance against oxidative stress is a consequence of efficient protective mechanisms operating in the intact tumour mitochondria and the low level of polyunsaturated fatty acids under these circumstances cannot be the rate limiting factor in lipid peroxidation. Succinate, an effective inhibitor of mitochondrial lipid peroxidation in liver, cannot determine malondialdehyde formation in ascites tumour mitochondria.     

Tumor-Specific Transplantation Resistance in Mice after Treatment of Initial Tumors with Streptococcus thermophilus

Antitumor activity observed by treatment with Streptococcus thermophilus was further investigated. The mice cured from fibrosarcoma by treatment with heat-killed preparation of S. thermophilus, when challenged with fibrosarcoma failed to take up the tumor. However, these cured mice when challenged with sarcoma-180 or Ehrlich ascites carcinoma, did not show significant changes in tumor take and/or survival compared to their respective controls. Similarly, mice cured from sarcoma-180 were challenged with fibrosarcoma, sarcoma-180 or Ehrlich ascites carcinoma. Though there was no change in the mean survival time (MST) of the dying mice regarding sarcoma-180 or Ehrlich ascites carcinoma, there was 50 and 30% increase in the number of mice that showed total regression respectively over controls. However, there was no difference in the growth rate of fibrosarcoma. Similar observations were made with mice cured from Ehrlich ascites carcinoma, challenged with these tumors. These findings thus suggest that the antitumor response was tumor-specific and that tumor-associated antigens may have a role in imparting this specificity. Bacterial treatment non-specifically augmented this primary response.     

Cardiolipin requirement by cytochrome oxidase and the catalytic role of phospholipid

During the development of Ehrlich ascites tumour in mice, a progressive decrease in red cell count is observed which is accompanied by a transformation of erythrocytes to acanthocytes. Desialylation of the glycoproteins in the plasma membrane of the erythrocytes is suppressed. The significance of these observations are discussed.     

Host zinc metabolism and the Ehrlich ascites tumour. Zinc redistribution during tumour-related stress.

Zinc redistribution between plasma and liver has been examined in mice injected with Ehrlich-ascites-tumour cells. Within 24 h of injection plasma Zn levels decrease and Zn appears in newly synthesized liver metallothionein. This response is dependent upon the number of tumour cells injected into the host. Uptake of Zn into liver and its specific accumulation in a Zn-binding protein, identified as metallothionein, continues for a number of days and reaches a plateau as tumour growth ceases. Over this time period, plasma copper rises. This redistribution also occurs in mice pretreated with cadmium in their drinking water for 1 month at levels of 20, 50, and 100 micrograms/ml. However, in each case there is a lag of 3 days before Zn increases in the livers of these animals which already contain substantial amounts of Cd/Zn-metallothionein. When Ehrlich cells are injected into mice previously placed on a Zn-deficient diet for several days, plasma Zn is already low and no net uptake of Zn into liver metallothionein is apparent. Finally, it is shown that ascites fluid can itself stimulate a transient shift of host of Zn into liver. Heat-inactivated fluid loses this property. It is suggested that, in the peritoneum, tumour cells initiate a stress response mediated by an ascites-fluid factor.