glutaminase and Carcinoma--Ehrlich-Tumor

Topics: Animals; Arginase; Asparaginase; Carcinoma 256, Walker; Carcinoma, Brown-Pearce; Carcinoma, Ehrlich Tumor; Carcinoma, Krebs 2; Chymotrypsin; Deoxyribonucleases; Enzyme Therapy; Glutaminase; Hyaluronoglucosaminidase; Leukemia, Experimental; Leukemia, Radiation-Induced; Lyases; Lymphoma, Non-Hodgkin; Mammary Neoplasms, Experimental; Mice; Neoplasms, Experimental; Pyridoxal Phosphate; Rats; Ribonucleases; Salts; Sarcoma, Avian; Sarcoma, Experimental; Trypsin; Vanadium

Topics: Amino Acids; Animals; Asparaginase; Carcinoma, Ehrlich Tumor; Child; Clinical Trials as Topic; Diet; Dietary Proteins; Female; Glutaminase; Humans; Leukemia, Lymphoid; Mice; Mice, Inbred Strains; Neoplasms; Nitrogen

Ehrlich ascites tumor cells (EATC) is a highly proliferative malignant cell line derived from mouse mammary epithelia, whereas their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, show a less transformed phenotype and loss of their tumorigenic capacity in vivo correlated with an inhibition of glutaminase expression. The mRNA differential display technique was applied to these two cell lines for the identification and isolation of genes whose transcription was altered. Side-by-side comparisons of cDNA patterns among relevant RNA samples revealed four genes significantly downregulated in 0.28AS-2 cells: high-mobility group Hmga2 protein, Fmnl3 or formin-like protein 3, Nedd-4 ubiquitin-protein ligase, and ubiquitin carboxyl-terminal hydrolase Usp-15. These positives were confirmed by Northern analysis. The four targeted genes have relevant functions in cell growth and proliferation. Our results show the validity of mRNA differential display technique to get insights into the molecular mechanisms underlying the acquisition of a more differentiated phenotype by tumor cells after inhibition of glutaminase expression.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Cell Proliferation; DNA, Complementary; Down-Regulation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glutaminase; Mice; Rats; RNA, Antisense; RNA, Messenger

Tumor cells expressing antisense glutaminase RNA show a drastic inhibition of glutaminase activity and they acquire a more differentiated phenotype. We have studied the expression of Sp1 and Sp3 transcription factors in both Ehrlich tumor cells and their derivative 0.28AS-2 antisense glutaminase expressing cells. The expression of phosphorylated Sp1 in 0.28AS-2 cells was 3-fold the expression in EATC. Full length Sp3 was also incremented in 0.28AS-2 cells. Sp1 and Sp3 binding to a consensus Sp1 probe was higher in 0.28AS-2 nuclear extracts, as determined by supershift assays. Sp1-DNA binding was inhibited by phosphatase treatment, demonstrating that phosphorylation of Sp1 is critical for its DNA binding capacity. The Sp1 and Sp3 DNA binding found in 0.28AS-2 cells was also correlated with an increased Sp1 activity, as shown in transient transfections assays carried out with a luciferase reporter plasmid. Incubation of Ehrlich tumor cells with the differentiation agent PMA could not totally reproduce the Sp1/Sp3 changes observed in 0.28AS-2 cells. However, it was demonstrated that the intracellular concentration of glutamine, but not glutamate or aspartate, is increased in 0.28AS-2 cells. In conclusion, the antisense inhibition of glutaminase leads to an increased expression of phosphorylated Sp1 and that correlates with an increase in Sp1 activity.

Topics: Animals; Carcinoma, Ehrlich Tumor; DNA-Binding Proteins; Glutaminase; Phosphorylation; RNA, Antisense; Sp1 Transcription Factor; Sp3 Transcription Factor; Transcription Factors; Transcription, Genetic

Glutamine is an essential amino acid in cancer cells and is required for the growth of many other cell types. Glutaminase activity is positively correlated with malignancy in tumours and with growth rate in normal cells. In the present work, Ehrlich ascites tumour cells, and their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, were assayed for apoptosis induced by methotrexate and hydrogen peroxide. It is shown that Ehrlich ascites tumour cells, expressing antisense mRNA for glutaminase, contain lower levels of glutathione than normal ascites cells. In addition, 0.28AS-2 cells contain a higher number of apoptotic cells and are more sensitive to both methotrexate and hydrogen peroxide toxicity than normal cells. Taken together, these results provide insights into the role of glutaminase in apoptosis by demonstrating that the expression of antisense mRNA for glutaminase alters apoptosis and glutathione antioxidant capacity.

Topics: Animals; Antioxidants; Apoptosis; Carcinoma, Ehrlich Tumor; Caspase 3; Caspases; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Cell Separation; Cell Survival; DNA; DNA Fragmentation; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Flow Cytometry; Glutaminase; Glutathione; Glutathione Reductase; Hydrogen Peroxide; Methotrexate; Mice; Oligonucleotides, Antisense; Phosphatidylserines; Rats; Reactive Oxygen Species; RNA, Messenger; Time Factors

In this study the enzyme glutaminase, purified from the ascites fluid of ovarian cancer patients, was analysed for its antiangiogenic activity. Intraperitoneal administration of this enzyme reduces the number of tumor directed capillaries in solid and ascites tumor bearing Swiss mice induced by transplantation of Ehrlich ascites cells. The enzyme has a critical role in regulating the secretion of vascular endothelial growth factor (VEGF) from tumor cell and in turn tumor growth. Glutamine analogue like 6-diazo, 5- oxo L-norleucine (DON) is also found to be effective in regulating vascular endothelial growth factor (VEGF) secretion from tumor cells in vitro. Treatment with enzyme reduced serum VEGF levels of the tumor induced animals. In vitro VEGF production by EAC cells was reduced in a concentration dependent manner in presence of glutamine analogue.

Topics: Animals; Antineoplastic Agents; Body Weight; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Mice; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Ovarian Neoplasms; Time Factors; Vascular Endothelial Growth Factor A

Glutaminase (EC 3.5.1.2) is a key enzyme in rapidly proliferating cells. Using anti-sense technology, an Ehrlich ascites tumor cell line (0.28AS-2) with reduced glutaminase activity has been obtained. We investigated the in vivo growth characteristics of the 0.28AS-2 cells. When injected i.p. into normal Swiss albino mice, the 0.28AS-2 cells were unable to grow. On the contrary, when injected into nude mice, they developed into solid tumors. Mice inoculated with 0.28AS-2 cells kept immunologic memory and rejected a second inoculation with parental Ehrlich ascites tumor cells. Expression of both polymorphic epithelial mucin-1 (MUC-1) and the enzyme N-acetyl-alpha-D-galactosaminidase, proteins implicated in host immune system escape, were markedly diminished in 0.28AS-2 cells. Study of the immune system response in mice inoculated with 0.28AS-2 cells revealed an increase in splenic CD18 cells and the presence of a large number of activated F4/80+ macrophages in the ascites cavity. These features, not observed in mice inoculated with parental Ehrlich ascites tumor cells, indicate that a distinctive, strong immune response occurred in animals inoculated with 0.28AS-2 cells. Our results suggest that inhibition of glutaminase expression using anti-sense technology induces phenotypic changes in Ehrlich ascites tumor cells that allow the development of an effective anti-tumor immune response, which makes the cells unable to develop in vivo tumors.

Topics: alpha-N-Acetylgalactosaminidase; Animals; Carcinoma, Ehrlich Tumor; Female; Glutaminase; Hexosaminidases; Immunity, Cellular; Immunologic Memory; Mice; Mice, Nude; Mucin-1; Neoplasm Proteins; RNA, Antisense; Transforming Growth Factor beta; Tumor Cells, Cultured

Phosphate-activated glutaminase has a critical role in tumours and rapidly dividing cells and its activity is correlated with malignancy. Ehrlich ascites tumour cells transfected with the pcDNA3 vector containing an antisense segment (0.28 kb) of rat kidney glutaminase showed impairment in the growth rate and plating efficiency, as well as a shortage in the glutaminase protein and activity. The C-terminal segment used is well conserved in all glutaminase sequences known. The transfected cells, named 0.28AS-2, displayed remarkable changes in their morphology compared with the parental cell line. The 0.28AS-2 cells also lost their tumourigenic capacity in vivo. Control mice developed an ascitic tumour, with a lifespan of 16+/-1 days, when inoculated with 10(7) cells/mouse; on the contrary, animals inoculated with transfected cells up to 2.5 times the cell numbers of control mice did not develop tumours and behaved as healthy animals. The ability to revert the transformed phenotype of antisense-transfected cells confirms the relevance of glutaminase in the transformation process and could provide new ways for the study of gene therapy.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Division; Gene Expression Regulation, Enzymologic; Glutaminase; Kidney; Kinetics; Mice; Rats; RNA, Antisense; RNA, Messenger; Transfection; Tumor Cells, Cultured; Tumor Stem Cell Assay

Distribution of glutamine level in different tissues of tumor bearing mice such as brain, liver, kidney, spleen, large and small intestine and the tumor itself were studied in three solid tumor models, viz, Ehrlich ascites carcinoma, Sarcoma-180 and methylcholanthrene induced carcinoma. Tumor bearing mice were subjected to therapy for 7 days with the glutaminase purified from malignant S-180 cell. The results exhibit a significant decrease in tumor burden after enzyme therapy. Host tissue glutamine levels were significantly elevated in tumor bearing untreated mice in comparison to the normal ones, while significant lower values were obtained after enzyme therapy. It therefore appears that elevated levels of glutamine in host tissue are associated with the tumor burden.

Topics: Animals; Brain; Carcinoma, Ehrlich Tumor; Glutaminase; Glutamine; Intestinal Mucosa; Kidney; Liver; Male; Methylcholanthrene; Mice; Sarcoma 180; Skin Neoplasms; Spleen; Tissue Distribution

Angiogenesis or the generation of new blood vessel, is an important factor in the growth of a solid tumor. Hence, it becomes a necessary parameter of any kind of therapeutic study. Glutamine is an essential nutrient of tumor tissue and glutamine related therapy involves clearance of circulatory glutamine by glutaminase. Therefore, using different murine solid tumor models, the present study was undertaken to find out whether the S-180 cell glutaminase has any effect on angiogenesis of solid tumor, or not. Result indicates that the purified S-180 cell glutaminase reduces tumor volume and restrict the generation of neo blood vessels. Therefore, it can be concluded that this enzyme may be an effective device against the cancer metastasis.

Topics: Angiogenesis Inhibitors; Animals; Carcinogens; Carcinoma, Ehrlich Tumor; Drug Screening Assays, Antitumor; Glutaminase; Glutamine; Injections, Intraperitoneal; Male; Methylcholanthrene; Mice; Neoplasm Proteins; Neoplasms, Experimental; Neovascularization, Pathologic; Sarcoma 180

Angiogenesis or the generation of new blood vessels, is an important factor regarding the growth of a tumor. Hence, it becomes a necessary parameter of any kind in therapeutic studies. Glutamine is an essential nutrient of tumor tissue and glutamine related therapy involves clearance of circulatory glutamine by glutaminase. So, whether this enzyme has any effect on angiogenesis of a tumor or not becomes an obvious question. To address this question, this study has been carried out with different murine tumor models. The results indicate that purified glutaminase reduces tumor volume as well as restricts the generation of new blood vessels. Glutaminase is effective in the case of solid as well as ascites tumor models. In the case of induced cancer, the host exhibits delayed onset of neoplasia following enzyme treatment and tumor host interactions determine the intensity of the neovascularisation process. Therefore, it can be concluded that this enzyme might be an effective agent against cancer metastasis.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cervix Uteri; Female; Glutaminase; Glutamine; Liver; Male; Methylcholanthrene; Mice; Neovascularization, Pathologic; Neovascularization, Physiologic; Sarcoma 180; Uterine Cervical Dysplasia

High rate of glutamine use is a characteristic of tumor cell both in vivo and in vitro and experimental cancer therapies have developed by depriving tumor cells of glutamine. In several investigations, bacterial glutaminase was found to be a potent therapeutic agent against varieties of tumor, but it showed suppressive effects on haematopoietic systems and inhibitory effects on normal lymphocytic blastogenesis. No antineoplastic study has nevertheless been undertaken with glutaminase enzyme purified from mammalian source. In the present study we report the purification of glutaminase enzyme from mitochondria of highly malignant S-180 cell using ion exchange chromatography and affinity column chromatography of glutamine. Purified enzyme is a kidney type phosphate dependent glutaminase with Mr 64 KD. Effect of enzyme therapy has been investigated in transplantable as well as induced tumor model in both ascites and solid form. It has been observed that the enzyme at the total dose of 10 unit/mouse successfully inhibited the tumor burden both in ascitic and solid tumor and subsequently increases the host's life span. There was no significant toxic effect on the peripheral blood cells.

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Chromatography, Affinity; Chromatography, Ion Exchange; Glutaminase; Male; Mice; Mice, Inbred Strains; Mitochondria; Sarcoma 180; Ultracentrifugation

The pH dependence of the phosphate-activated glutaminase isolated from Ehrlich tumour cells suggests a functional role for two prototropic groups with apparent pKa of 9.3 and 7.7 at the active site of the protein; these pKa values are compatible with cysteine and histidine residues, respectively. This possibility was investigated by chemical modification studies of the purified enzyme. N-Ethylmaleimide fully inactivated the purified glutaminase; the reaction order was very close to 1.0, suggesting that N-ethylmaleimide modifies glutaminase at a single essential site. Spectrophotometric studies of the isolated protein treated with diethyl pyrocarbonate indicate that two histidine residues are modified. Since glutaminase is loosely associated to the inner mitochondrial membrane, modification experiments were also carried out using mitochondrial membrane fractions. N-Ethylmaleimide and diethyl pyrocarbonate gave similar results in mitochondria membrane-bound enzyme to those obtained with purified enzyme. Glutamate, which behaves as a competitive inhibitor of the enzyme, partially protected the inactivation caused by N-ethylmaleimide in membrane-bound experiments. The results suggest the existence of a critical histidine residue(s) in the tumour glutaminase, and strongly support the notion that a cysteine residue, which is located at (or near) the active site, is involved in the catalytic mechanism as well.

Topics: Animals; Binding Sites; Carcinoma, Ehrlich Tumor; Cysteine; Diethyl Pyrocarbonate; Enzyme Inhibitors; Ethylmaleimide; Glutaminase; Histidine; Hydrogen-Ion Concentration; Intracellular Membranes

The influence of progressive tumor growth on phosphate-activated glutaminase (PAG) expression in splenocytes from mice bearing Ehrlich ascites carcinoma cells was investigated. Implantation of Ehrlich ascites tumor cells in the peritoneal cavity of mice led to a 2.3-fold stimulation of spleen PAG activity 48 h later. Four days after tumor implantation the glutaminase activity had returned to nearly basal value and remained at this level throughout the tumor development. Northern blot analysis indicated that two species of glutaminase mRNA were expressed in the spleen, which showed a differential expression pattern during the first 2 days after tumor implantation. The abundance of the transcript of higher electrophoretic mobility (approximately 3 kb) constantly increased over the first 2 days of tumor growth. The mRNA of lower electrophoretic mobility (approximately 6 kb) peaked at 12 h after tumor implantation and returned to control values at 48 h. These results demonstrate that tumor has the capability of altering glutaminase expression in the host spleen.

Topics: Animals; Carcinoma, Ehrlich Tumor; Female; Gene Expression Regulation, Enzymologic; Glutaminase; Mice; RNA, Messenger; Spleen

The intramitocondrial localization of the phosphate-activated glutaminase from Ehrlich cells has been examined by a combination of techniques, including: mitochondria subfractionation studies, chemical modification with sulfhydryl group reagents of different permeability, enzymatic digestion in both sides of the inner mitochondrial membrane, and immunological studies. Using alkaline extraction at high ionic strength, hypoosmotic shock and freezing-thawing cycle techniques, the enzyme was found in the particulate fraction. On the contrary, glutaminase activity was labile when subfractionation was carried out by digitonin/lubrol method; Western blot analysis localized the inactive enzyme in the matrix fraction. In addition, glutaminase was fully inactivated when mitoplasts were incubated with phospholipase A2 and phospholipase C. The enzyme also showed a non-linear Arrhenius plot with a break at 24 degrees C. The membrane-impermeant thiol reagents mersalyl and p-chloromercuriphenylsulfonic acid do not inhibit glutaminase activity in freeze-thawed mitochondria and mitoplasts, but N-ethylmaleimide, which is membrane permeant, strongly inhibited the enzyme. However, mersalyl and p-chloromercuriphenylsulfonic acid were effective inhibitors when the alkylation was performed on the matrix side of mitoplasts or using detergent-solubilized enzyme. Furthermore, trypsin digestion of mitoplasts was only effective inactivating glutaminase when the proteolysis was carried out on the matrix side of the vesicles. Enzyme-linked immunosorbent assay of the soluble and membrane fractions obtained in the preparation of submitochondrial particles, revealed that most of the enzyme was solubilized, but in the inactive form. Phase separation with Triton X-114 rendered most of the protein in the aqueous phase. These results taken together discard a transmembrane localization for the protein, whereas they are consistent with anchorage of glutaminase on the matrix side of the inner mitochondrial membrane, the matrix portion of the enzyme being relevant for its function.

Topics: 4-Chloromercuribenzenesulfonate; Alkylating Agents; Animals; Carcinoma, Ehrlich Tumor; Cell Fractionation; Enzyme Inhibitors; Ethylmaleimide; Glutaminase; Intracellular Membranes; Mersalyl; Mitochondria; Phospholipases A; Phospholipases A2; Sulfhydryl Reagents; Temperature; Trypsin; Type C Phospholipases

Two alternative purification schemes to obtain the glutaminase from Ehrlich tumor cells in a highly purified form have been developed. One experimental approach is based on conventional and high-performance liquid chromatography fractionation techniques, yielding a 37-fold higher purification than has been previously reported. The method comprises: isolation of mitochondria, solubilization with Triton X-100, ion-exchange and hydroxyapatite chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography. A second purification schedule has been optimized employing native polyacrylamide gel electrophoresis, in situ activity staining, and electroelution of the protein band. This approach resulted in a simple and rapid isolation of a 10-fold higher purified glutaminase than before, minimizing also the potential for proteolytic inactivation of the enzyme. The apparent molecular weight of the protein in native form was determined by gel filtration and sucrose density gradient ultracentrifugation. Polyclonal antibodies raised against Ehrlich glutaminase were immunopurified against the pig kidney enzyme. Immunoblot analyses employing these antibodies as well as anti-rat kidney glutaminase antibodies revealed the same pattern of bands seen with the purified enzyme.

Topics: Animals; Blotting, Western; Carcinoma, Ehrlich Tumor; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Glutaminase; Isoelectric Focusing; Mitochondria; Molecular Weight; Octoxynol

Changes in phosphate-activated glutaminase activities determined in intact cells and isolated mitochondria have been followed during mouse Ehrlich ascites carcinoma development. Glutaminase activities parallel the levels of poly(A)+ RNAs encoding for the mitochondrial phosphate activated glutaminase. During the exponential growth phase, maximum activity was observed and the relative abundance of glutaminase mRNA significantly increased with regard to the stationary growth phase. The presented results show that tumor phosphate-activated glutaminase is subject to long-term regulation by differential gene expression.

Topics: Animals; Blotting, Northern; Carcinoma, Ehrlich Tumor; Cell Division; Enzyme Activation; Glutaminase; Mice; Mitochondria; Phosphates; Tumor Cells, Cultured

Glutaminase is a hematotoxic anti-tumor agent, and copper-ATP complex (Cu-ATP) is both anti-neoplastic and hematostimulatory. Combination chemotherapy with these two agents has been performed in mice bearing Ehrlich ascites carcinoma, to elucidate whether this could result in augmented tumor inhibition with reduced hematotoxicity. Glutaminase-Cu-ATP combination (glutaminase 250 IU/kg per day intraperitoneally for 10 days and Cu-ATP 2.5 mg/kg per day intraperitoneally for 10 days) was observed to be more effective in inhibiting tumor growth and in increasing the life span of the tumor hosts, compared with the individual efficacies of these two agents. Moreover, addition of Cu-ATP successfully prevented the hematotoxic effects of glutaminase in normal and in tumor-bearing animals. Thus glutaminase in combination with Cu-ATP holds promise for an effective cancer chemotherapeutic regimen.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Combined Chemotherapy Protocols; Blood Cell Count; Bone Marrow; Carcinoma, Ehrlich Tumor; Cell Nucleus; Colony-Forming Units Assay; Copper; DNA, Neoplasm; Drug Synergism; Glutaminase; Liver; Male; Mice; Neoplasm Proteins; Neoplasm Transplantation; RNA, Neoplasm; Spleen

The anti-neoplastic activity of bacterial glutaminase on Ehrlich ascites tumor-bearing mice was studied by determining the reduction in the tumor cell count and extension of life span of the host after therapy. The therapeutic effect of glutaminase in relation to change in activity of glutaminolytic enzymes (glutamine amidohydrolase (GNase) and glutamine aminotransferase (GAt)) in liver and plasma were also studied. Bacterial glutaminase was shown to be effective in lowering the tumor burden with increased life span of the host. Glutamine amidohydrolase activity in the liver and plasma was raised significantly with increased tumor burden, whereas GAt activity remained unchanged. Following glutaminase therapy, this high level of GNase activity decreased in comparison to the untreated control. These changes were not seen when normal mice were treated with the same enzyme. Thus alteration in the enzyme levels, particularly GNase was observed to have some correlation with progression of the tumor growth.

Topics: Animals; Carcinoma, Ehrlich Tumor; Glutaminase; Liver; Mice; Time Factors; Transaminases

Parallel investigations of the transamination pathways of glutamine oxidation in Ehrlich ascites carcinoma (EAC) and AS 30D hepatoma revealed that hepatoma cells, unlike EAC, produce very little aspartate. This cannot be explained by differences in the activity of glutamine-metabolizing enzymes. Also, the mitochondria from the hepatoma respired at a similar rate to EAC mitochondria with glutamine as sole substrate producing substantial amounts of aspartate. Unlike their isolated mitochondria, intact hepatoma cells showed a very low rate of glutamine oxidation. Compared with EAC, the rate of L-[U-14C]glutamine consumption by AS 30D hepatoma cells was much lower, with insignificant production of 14C-labelled aspartate and CO2. This suggested that the glutamine-transporting system in the hepatoma cell plasma membrane had a very low activity. Isolated hepatoma mitochondria produced 3 times more pyruvate from malate than did EAC mitochondria, indicating a higher activity of NAD(P)-dependent malic enzyme. We postulate that an active malic enzyme may suppress the synthesis of aspartate in hepatoma cells, but further evidence is needed to confirm this assumption.

Topics: Alanine; Animals; Aspartic Acid; Carbon Radioisotopes; Carcinoma, Ehrlich Tumor; Cell Membrane; Citrates; Citric Acid; Cytosol; Glutaminase; Glutamine; Glyoxylates; Kinetics; Liver Neoplasms, Experimental; Mice; Mitochondria, Liver; Rats; Rats, Inbred Strains; Substrate Specificity; Tumor Cells, Cultured; Uranium

Phosphate-dependent glutaminase activity in spleen, kidney, brain and liver is increased after tumor cell inoculation and this activity gradually increases with the progression of the tumor. The increase in enzyme activity in the liver is significant. Studies of the response of liver glutaminase after Cu-ATP treatment reveals that Cu-ATP is capable of reducing the high glutaminase level in subjects with malignant tumors to the normal level.

Topics: Adenosine Triphosphate; Animals; Brain; Carcinoma, Ehrlich Tumor; Cell Division; Glutaminase; Kidney; Liver; Mice; Spleen

Twenty-four h after tumor transplantation increases of free glutamine in plasma, liver, and kidney occurred simultaneously with the exponential phase of tumor growth. Kidney and muscle glutamine synthetase also increased in the first 2 days following tumor transplantation, while kidney and liver glutaminases decreased. The levels of free glutamine in plasma and tissues, and the activities of glutamine synthetase and glutaminase, tended to approach normal values in the last days of life of the tumor-transplanted animals. Eleven days after transplantation, liver glutamine synthetase activity diminished. The results are discussed in terms of a glutamate/glutamine intercellular cycle which could augment the circulating glutamine, the main source of nitrogen for tumor cells.

Topics: Animals; Carcinoma, Ehrlich Tumor; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Kidney; Liver; Mice

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.

Topics: Animals; Carcinoma, Ehrlich Tumor; Glutaminase; Glutamine; Hydrogen-Ion Concentration; Immunoblotting; Kidney Neoplasms; Kinetics; Mice; Mitochondria; Molecular Weight; Phosphates; Rats

CTP synthetase (UTP: glutamine ligase (ADP-forming), EC 6.3.4.2) was purified from Ehrlich ascites tumor cells to near homogeneity and found to be a dimer composed of two seemingly identical 66 kDa subunits. The formation of CTP was accompanied by the production of equivalent amounts of ADP from ATP and glutamate from glutamine. The reaction product, CTP, was a potent inhibitor generating sigmoidal kinetics as a function of UTP with an n value of 2.0. UTP and CTP pools in the ascites cells were elevated in an early period (12-16 h) following implantation into the intraperitoneal cavity of mice, whereas ATP, GTP and glutamine pools did not change. Kinetic data and analysis of the nucleotide pools in the cells growing in vivo suggested that the biosynthesis of CTP is regulated at the level of CTP synthetase by UTP and CTP.

Topics: Adenosine Triphosphate; Animals; Carbon-Nitrogen Ligases; Carcinoma, Ehrlich Tumor; Glutaminase; Glutamine; Kinetics; Ligases; Mice; Nucleotides; Protein Conformation; Substrate Specificity

Forty-eight tumor-free mice and 32 mice bearing Ehrlich ascites tumor were randomized into 2 treatments, Acinetobacter glutaminase-asparaginase (AGA) (600 IU/kg/day for 7 days) and 0.9% NaCl controls, and into 2 or 3 isocaloric diets, normal protein (NP) (20 g protein/100 g diet), high protein (HP) (58 g protein/100 g diet), and zero protein (ZP) (tumor-free mice only). In tumor-free, NP-fed mice, AGA caused percentage reductions (P less than 0.01) in the nitrogen content of liver (50%), intestine (42%), thymus (89%), spleen (75%), and carcass (20%), but HP prevented this effect on intestine and carcass and caused percentage increases in the nitrogen content of liver (53%), intestine (36%), thymus (122%), and carcass (25%). In Ehrlich ascites tumor mice (NP or HP fed) AGA caused markedly lower (P less than 0.01) tumor burdens and increased nitrogen content of intestine (HP), kidney (NP and HP), and spleen (NP and HP). Ehrlich ascites tumor, AGA-treated, HP-fed mice ate 31% less food (P less than 0.01) (compared to NP) but HP resulted in percentage increases in the nitrogen content of liver (18%; P = 0.05), intestine (25%; P less than 0.05), and thymus (164%; P less than 0.01). In the Ehrlich ascites tumor, AGA group the HP diet caused higher hematocrit and serum total protein (both, P less than 0.05). Adverse nutritional effects of AGA seen in normal mice were markedly diminished in tumor-bearing animals. The observed nitrogen-sparing effects of the high protein: energy ratio may be relevant to humans and to other forms of neoplasia and chemotherapy.

Topics: Acinetobacter; Animals; Asparaginase; Body Weight; Carcinoma, Ehrlich Tumor; Dietary Proteins; Eating; Female; Glutaminase; Leukocyte Count; Mice; Mice, Inbred Strains; Nitrogen

The effect of L-glutamine and L-asparagine depletion by Acinetobacter L-glutaminase-L-asparaginase on the toxicity and antitumor activity of L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (NSC-163501) was tested in mice. The LD50 of six daily doses of NSC-163501 in BDF1 female mice decreased from 7.5 to 0.3 mg/kg/day by combination treatment with the enzyme. Enzyme therapy also decreased the dose of NSC-163501 needed for maximal prolongation of survival in these mice inoculated with L1210 leukemia. Nevertheless, the combination did not prolong survival in L1210-bearing mice beyond that of higher doses of NSC-163501 alone. In contrast, the combination of enzyme plus NSC-163501 inhibited the growth of established sc implanted Ehrlich ascites carcinoma in ICRf male mice much more than either agent alone. Treatment with Acinetobacter L-glutaminase-L-asparaginase decreased the L-asparagine and L-glutamine levels in acid extracts of the Ehrlich tumor. NSC-163501 did not affect the amide levels or alter the decrease produced by enzyme therapy.

Topics: Acinetobacter; Amino Acids; Animals; Asparaginase; Carcinoma, Ehrlich Tumor; Drug Synergism; Drug Therapy, Combination; Female; Glutaminase; Glutamine; Glycine; Isoxazoles; Lethal Dose 50; Leukemia L1210; Male; Mice; Oxazoles

Glutamine aminohydrolase is found to be present in microsomal and soluble supernatant in liver of EAC-bearing mice. Enzymes obtained from these two sources were characterized and found to behave differently from the mitochondrial glutaminase of both normal and tumour-bearing mice.

Topics: Animals; Carcinoma, Ehrlich Tumor; Glutaminase; Hydrogen-Ion Concentration; Liver; Lysosomes; Mice; Microsomes, Liver; Mitochondria, Liver; Neoplasm Transplantation; Phosphates

Topics: Alcaligenes; Ammonia-Lyases; Animals; Asparaginase; Carboxypeptidases; Carcinoma; Carcinoma 256, Walker; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cell Line; Enzyme Therapy; Glutaminase; Humans; Leukemia, Experimental; Liver Neoplasms; Mice; Neoplasms; Neoplasms, Experimental; Sarcoma, Experimental

Topics: Animals; Borates; Carcinoma, Ehrlich Tumor; Cell Fractionation; Centrifugation; Digitonin; Enzyme Activation; Glutamate Dehydrogenase; Glutaminase; Hydrogen-Ion Concentration; In Vitro Techniques; Isoenzymes; Kinetics; Mice; Mitochondria; Phosphates; Polyethylene Glycols; RNA, Neoplasm; Sonication

Topics: Alanine; Alcaligenes; Amino Acid Isomerases; Amino Acid Oxidoreductases; Animals; Aspartate Aminotransferases; Bacillus; Body Weight; Carcinoma, Ehrlich Tumor; Cattle; Glutamate Dehydrogenase; Glutaminase; Isoenzymes; Ketoglutaric Acids; Kinetics; Leucine; Liver; Lysine; Mice; Pseudomonas; Pseudomonas aeruginosa; Time Factors; Transaminases

Topics: Ammonia; Animals; Carbon Dioxide; Carbon Isotopes; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cyanides; Glutamates; Glutaminase; Glutamine; Hydrazones; In Vitro Techniques; Ketoglutaric Acids; Kinetics; Liver Neoplasms; Mice; Mitochondria; NADP; Neoplasms, Experimental; Oxygen Consumption; Pyruvates; Rats; Rotenone

Topics: Animals; Carcinoma, Ehrlich Tumor; Glutaminase; Injections, Intraperitoneal; Injections, Subcutaneous; Kidney Transplantation; Mice; Neoplasm Transplantation; Transferases; Transplantation, Homologous

1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of (14)CO(2) from l-[U-(14)C]glutamine was not inhibited though that from l-[U-(14)C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO(2) production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP(+)] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate-oxaloacetate transaminase compared with that of glutamate dehydrogenase.

Topics: Acetates; Animals; Carbon Dioxide; Carbon Isotopes; Carcinoma, Ehrlich Tumor; Glutamate Dehydrogenase; Glutamates; Glutaminase; Glutamine; In Vitro Techniques; Kidney; Mice; Mitochondria; Mitochondria, Liver; NADP; Oxygen Consumption; Permeability; Pyruvates; Rats; Transaminases

Topics: Acyltransferases; Animals; Asparaginase; Carcinoma, Ehrlich Tumor; Drug Synergism; Escherichia coli; Female; Glutaminase; Glutamine; Injections, Intraperitoneal; Leucine; Ligases; Mice; Neoplasm Proteins

Topics: Animals; Antineoplastic Agents; Asparaginase; Asparagine; Bacteria; Carbon Isotopes; Carcinoma, Ehrlich Tumor; Escherichia coli; Female; Glutaminase; Glutamine; Leucine; Mice; Neoplasm Proteins

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Glutaminase; Kidney; Rats; Swine

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Glutaminase; Leukemia; Leukemia, Experimental; Lymphoma; Lymphoma, Non-Hodgkin; Mercaptopurine; Mice; Neoplasms; Neoplasms, Experimental; Pharmacology; Research; Sarcoma 180; Toxicology