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E Eigenbrodt

Publications and source records attributed to E Eigenbrodt.

At least 19 recordsLinked to original sources
PubMed1992

Cell cycle-associated expression of M2-type isozyme of pyruvate kinase in proliferating rat thymocytes.

During a complete cell cycle of rat thymocytes stimulated by concanavalin A and interleukin 2, the activity and mRNA level of pyruvate kinase reached a maximum (8-12-fold increase) 48 h after stimulation coinciding with the S-phase of the cell cycle. Increases of cellular enzyme activity, pyruvate kinase protein, and mRNA levels are correlated up to 48 h of culture. Afterwards pyruvate kinase activity and mRNA levels decrease, whereas the pyruvate kinase protein continues to increase throughout mitosis. This change of specific pyruvate kinase activity points to a posttranslational modification of the enzyme besides its transcriptional regulation. The presence of the M2-type isozyme was determined by the following methods: (a) native cellulose acetate electrophoresis and activity staining, (b) Northern blot hybridization with M1- and M2-specific cDNA probes, and (c) determination of kinetic parameters. The isozyme pattern did not change during the cell cycle progression. The induction of pyruvate kinase is completely abolished by 2-difluoromethylornithine-mediated polyamine depletion. However, the proportion of hybridizable pyruvate kinase mRNA was not affected. These data suggest the requirement of polyamines for efficient translation rather than transcription during cell growth.

Animals↗
PubMed1992

In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation.

MCF-7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP-treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increases in the enzyme activities involved in glutamate degradation and in the malate-aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6-bisphosphate and pyruvate accumulated in AMP-arrested cells. Based on the lowered NAD level in the AMP-treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P-ribose-PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented.

Adenosine Monophosphate↗
PubMed1992

Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies.

Four monoclonal antibodies (MAb) specific for the L-type isoenzyme of rat pyruvate kinase (L-PK) were produced and characterized. They detect at least two different epitopes of the isoenzyme, as shown in interference binding assay and Western blot analysis after peptide mapping. The MAb were used in immunohistology to demonstrate the L-PK isoenzyme in paraffin-embedded normal rat tissues. L-PK was found only in hepatocytes, kidney proximal tubules, islet cells of pancreas, and epithelial cells of the villi of small intestine. The content of L-PK in hepatocytes was often lower in the periportal areas as compared with the periveneous zone. In kidneys a clearcut difference in L-PK content and distribution existed between male and female rats. Male animals possessed more L-PK in the kidney cortex than females. The L-PK content in the inner cortical zone (straight proximal tubules) was higher than in the outer cortical zone (convoluted proximal tubules) in kidneys of males. In contrast, female rats displayed less L-PK in the inner than in the outer cortical zone of the kidneys. Only some of them exhibited the same amount of the isoenzyme in both parts of the kidney proximal tubules.

Animals↗
PubMed1992

Hormone-sensitive carbohydrate metabolism in rat hepatocyte-hepatoma hybrid cells.

Hepatocyte-hepatoma hybrid cells were obtained by fusion of hepatocytes from adult rats and Fao hepatoma cells in the presence of polyethylene glycol. These hybrids were called hepatocytoma cells. The preservation of liver-specific enzyme activities and metabolic functions was studied in the hybrid clone 1E3. 1) The proliferating hepatocytoma cells formed monolayers presenting morphological similarity to primary cultures of hepatocytes. 2) In contrast to Fao hepatoma cells, activities of all gluconeogenic key enzymes were preserved at normal or reduced levels. 3) Lactate-dependent glucose formation was maintained at a state reduced to 36% of the gluconeogenesis in hepatocytes; no glucose formation was detected in Fao hepatoma cells. 4) The activity of the liver-specific glucokinase was reduced in hepatocytoma cells, but it was still present in contrast to Fao cells. The liver-specific isoenzyme pyruvate kinase type L was replaced by the isoenzyme type M2. 5) Gluconeogenic and glycolytic enzyme activities were regulated in hepatocytoma cells by glucagon (0.1 microM) and by insulin (0.1 microM). 6) The genome of hepatocytoma cells and its expression were stable for at least one year, when spontaneously dedifferentiating cells were removed by recloning in hypoxanthine-aminopterine-thymidine (HAT) medium.

Animals↗
PubMed1992

Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells.

As a common characteristic of tumor cells, as well as of normal proliferating cells in the G1-phase of cell cycle, one finds constitutive high levels of all the glycolytic metabolites arising between glucose 6-phosphate and phosphoenolpyruvate. Thus, it is that the phosphometabolites fructose 1,6-bisphosphate, ribose 5-P, P-ribose-PP, NAD, GTP, CTO, UTP, UDP-glucose, glycerol 3-P, glycerol phosphocholine and glycerol phosphoethanolamine are useful in the 31P-nuclear magnetic resonance (NMR) detection of solid tumors in animals and man. This expansion of phosphometabolites is achieved during tumor formation as a result of reductions in levels of enzymes degrading phosphometabolites, owing to the decline in the glycerol 3-P hydrogen shuttle, and as a consequence of alterations in the glycolytic isoenzyme equipment. Tumor cells typically express a particular isoenzyme of pyruvate kinase called type M2 (K) at high levels. This isoenzyme is subject to a complex regulation by amino acids, by fructose 1,6-bisphosphate, and by hormonal- and oncogene-dependent phosphorylation. Pyruvate kinase type M2 is a substrate for the oncogene encoded PP60v-src-tyrosine kinase. A drastic decrease in the affinity for its substrate phosphoenolpyruvate found after transformation by the src-oncogene can be explained as a consequence of the phosphorylation of pyruvate kinase in serine and tyrosine. These phosphorylations induce the breakdown of tetrameric pyruvate kinase to the trimeric and dimeric forms. Unlike the tetrameric form, the dimeric form as a low affinity for phosphoenolpyruvate. Partial inactivation of pyruvate kinase and enolase on the one hand, and a hyperactivation of hexokinase and phosphofructokinase on the other hand, lead to an expansion of all metabolites. Only when these metabolites attain high levels, thereby assuring a sufficient supply of metabolites for RNA, DNA, lipid, and complex carbohydrate synthesis, can cell proliferation proceed. This accumulation of metabolites in the G1-phase cells has been termed a "metabolic budget system" because it senses not only the actual nutrient levels, but also the supply over a period of time. Monoclonal antibodies specific for the dimeric form of pyruvate kinase type M2 can be used for the immunohistological detection of tumor cells. The amount of the dimeric form in tumor cells closely correlates with the degree of malignancy and can be used for a nonspecific detection of tumors based on assays performed with patient's plasma.

Adenosine Triphosphate↗
PubMed1990

Di(2-ethylhexyl)phthalate alters carbohydrate enzyme activities and foci incidence in rat liver.

The effect of di(2-ethylhexyl)phthalate (DEHP) on diethylnitrosamine (DEN)-initiated preneoplastic liver lesions with expression of gamma-glutamyltranspeptidase (GGTase) and loss of adenosine triphosphatase (ATPase) as well as alterations of hepatic carbohydrate metabolism in male and female Sprague-Dawley rats have been investigated. Two treatment schedules have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic islands and by the biochemical determination of alterations in enzyme activities of liver homogenates and of serum, the last indicating hepatotoxicity. For initiation, a single dose of DEN was given, followed by treatment with various doses of DEHP given three times weekly by gavage for 7 or 11 consecutive weeks. As histochemical enzyme markers, the expression of positive GGTase as well as the deficiency in ATPase were used for identification of liver foci. The weanling female rats (protocol A) were found to be more sensitive to the carcinogenic effect of DEN in view of foci incidence than the mature male rats which underwent partial hepatectomy prior to DEN application. The administration of 200 mg DEHP/kg body wt increased the incidence of ATPase-deficient foci in both male and female rats; however, concentrations of 1000 and 2000 mg DEHP/kg decreased the incidence of liver foci. The number of foci with expression of GGTase was only slightly increased in female rats following a DEHP concentration of 50 mg/kg, and 200 mg/kg body wt. DEHP alone did not induce preneoplastic lesions that could be identified by these two markers. Biochemical investigations indicate that DEHP alters the metabolic pattern in liver. An increase of the NADP-linked enzymes glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme, extra-mitochondrial ICDH as well as an enhancement of NAD-dependent alpha-G3PDH and lactate dehydrogenase were found following DEHP administration. On the other hand the glycolytic enzymes pyruvate kinase (PK) and enolase as well as the gluconeogenetic enzyme fructose-1,6-bisphosphatase (FBPase) were significantly reduced. In protocol B (male rats) the reactions of PK, FBPase and malic enzyme were more altered after DEHP exposure than in protocol A, while the activity of G6PDH was more increased in protocol A. Most enzymes being involved in the carbohydrate metabolism are influenced by DEHP in a dose-dependent manner. There was no increase in serum FBPase activity in both male and female rats after DEHP treatment but a reduction of glutamate-oxalate-transaminase and glutamate-pyruvate-transaminase activities was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1990

[Immunohistologic photometric quantification of pyruvate kinase content of rat tumors].

86 benign and malignant tumours were investigated histologically and immunohistologically. Staining intensity was measured by scanning-photometry. The computed optical density was digitalized and evaluated with an image analysis system. The results indicate a significantly higher amount of M2-PK in malignant tumours. In the adenocarcinomas of the mammary gland, the content of the M2-PK increased parallel with malignancy (p less than 0.01; r = 0.743). The M2-PK can serve as an indicator of the grade of malignancy in mammary tumours of the rat.

Animals↗
PubMed1989

Comparison of three rat liver foci bioassays--incidence of preneoplastic foci initiated by diethylnitrosamine.

Three rat liver foci bioassays have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic foci, and by the biochemical determination of alterations in enzyme activities of serum indicating hepatotoxicity. We studied the initiation/promotion schedules according to Oesterle and Deml (A), and according to Pereira (B, Broad Spectrum Protocol), and the initiation/selection protocol according to Tatematsu et al. (C), with diethylnitrosamine (DEN), given as a single initiating dose of 10 and 30 mg/kg body wt respectively. With all schedules Sprague-Dawley rats, either females, 3 weeks old (A), or males, 6 weeks old (B, C) were used. For promotion polychlorinated biphenyls (A) or phenobarbital (B) were administered. Selection was performed with 2-acetylaminofluorene (C). The rats in schemes (B) and (C) underwent partial hepatectomy one day prior to initiation. The number and total area of foci deficient in adenosine-5'-triphosphatase (ATPase) and positive in gamma-glutamyltranspeptidase (GGTase) was evaluated. In the complete schedule with 30 mg of DEN in system (A) foci incidence exceeded that of the other systems by about 7-fold (ATPase) and 2-fold (GGTase) respectively. The lower dose of DEN and all control experiments resulted in a respective lower foci yield. With scheme (C), but not with schemes (A) and (B), e.g. serum fructose-1.6-bisphosphatase and alkaline phosphatase were increased, suggesting liver cell damage. Thus tested with DEN, scheme (A) is most sensitive and causes a low impairment of animals' welfare.

2-Acetylaminofluorene↗
PubMed1989

[Immunohistochemical demonstration of L- and M2-pyruvate kinase in primary renal cell carcinomas and their metastases].

Seventeen renal cell carcinomas (RCC) were classified histologically and investigated for their expression of L- and M2-pyruvate kinase (PK) immunohistochemically. Using monoclonal antibodies we were able to demonstrate L-PK within 15 RCC and two metastases (in thyroid gland and bone) after fixation with aceton and paraffin embedding. In normal renal tissue the enzyme was localized within proximal tubules selectively. No enzyme reaction of L-PK could be demonstrated in renal oncocytoma, thyroid carcinoma, and in carcinomas of renal pelvis and lung. In contrast to this the M2-PK was detectable in all tumors and metastases investigated in this study. The results presented in this paper are able to show that (1) RCC derive from the proximal renal tubules, but not the renal oncocytoma, (2) the detection of L-PK may be helpful for identification of metastases of RCC even if they are undifferentiated and (3) there is an enzyme shift from L-PK to M2-PK within tumor cells of RCC resulting in alteration of glucose utilization from energy supply to syntheses of substrates essential for tumor cells.

Carcinoma, Renal Cell↗
PubMed1988

Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus.

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2-PK can be phosphorylated at tyrosine residues by pp60v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2-PK revealed that M2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.

Animals↗
PubMed1987

Histology and histochemistry of the liver of chickens after DENA induced hepatocarcinogenesis and ingestion of low chlorinated biphenyls.

Studies are presented which demonstrate the pathological effects of diethylnitrosamine (DENA) and polychlorinated biphenyls (PCB) in the liver of chickens. DENA, which has been shown to cause tumors in rats and chickens, was tested on 80 laying hens to determine whether changes in the liver during hepatocarcinogenesis were similar to those observed in rats. In addition, the hepatocarcinogenic and cocarcinogenic properties of Clophen C (CC), a technical mixture of low chlorinated biphenyls was tested on chickens. The livers of test animals were examined histologically for preneoplastic and neoplastic lesions. Histochemical methods were used to determine lipid and glycogen contents as well as changes in activity of alkaline phosphatase and gamma-glutamyl-transpeptidase. Putative preneoplastic lesions in the liver were first observed according to the substance and dosage applied as follows: DENA 1 X weekly (group II), beginning on day 258; DENA 2 X weekly (group III), beginning on day 183; and CC + DENA 2 X weekly (group V), beginning on day 231. Application of CC alone did not induce lesions. The concept that CC has cocarcinogenic capability was substantiated by the fact that animals from group V (CC + DENA 2 X weekly) developed twice as many tumors as did animals from group III (DENA 2 X weekly). Additionally, tumors developed earlier in the former group than in the latter.

Animals↗
PubMed1987

Decrease in glucokinase and glucose-6-phosphatase and increase in hexokinase in putative preneoplastic lesions of rat liver.

Preneoplastic liver lesions were produced in female Wistar rats by oral administration of 2-acetylaminofluorene for 165 days succeeded by a carcinogen-free standard diet up to 420 days. During the treatment numerous altered hepatic foci (AHF) and hyperplastic nodules (HN) were detected histochemically by a focal decrease or lack of adenosine-5-triphosphatase and glucose-6-phosphatase (G-6-Pase) activities. In addition, the immunohistochemically demonstrable amount of L-type pyruvate kinase was clearly reduced. The histochemically demonstrated decrease of G-6-Pase was substantiated by microbiochemical determination of the enzyme activity in microdissected material. Moreover, during the experimental period a continuous decrease in glucokinase and an increase in hexokinase was detected microbiochemically within AHF and HN. These alterations indicate a shift in the carbohydrate metabolism from gluconeogenesis to glucose utilization and pentose-phosphate-pathway for biosynthesis of nucleic acids. Beside other oncofetal markers, HK may be used as indicator of the early stages of liver carcinogenesis.

2-Acetylaminofluorene↗
PubMed1987

Immunohistochemical demonstration of decreased L-pyruvate kinase in enzyme altered rat liver lesions produced by different carcinogens.

Preneoplastic liver lesions were produced in female Wistar rats by application of 25 mg/kg N-nitrosomorpholine (NNM), 14 mg/kg diethylnitrosamine (DENA), 0.075 mg/kg aflatoxin B1 (AFB1) or 160 mg/kg safrole. These carcinogens were administered in two equal doses 12 and 24 h after partial hepatectomy. The animals then received sodium phenobarbital (0.1% in tap water) for up to 410 days. Numerous altered hepatic foci (AHF) and hyperplastic nodules (HN) were detected enzyme histochemically by their negative ATPase reaction after application of AFB1, DENA and NNM; some AHF and HN were also caused by the weak carcinogen safrole. Immunohistochemically these lesions were also L-pyruvate kinase (L-PK)-negative with a high coincidence with regard to their number and area. These results confirm the role of L-PK, an enzyme affecting the pentose phosphate pathway, as a negative marker of preneoplastic liver lesions.

Animals↗
PubMed1987

Human erythrocyte pyruvate kinase (L'/R-PK): production and characterization of a monoclonal antibody.

Two preparations of L'/R-type pyruvate kinase from human erythrocytes characterized by SDS-PAGE were used for immunization of BALB/c mice. Their spleen cells were fused with mouse myeloma cells by polyethylene glycol according to standard techniques. Supernatants of hybridomas resulting from two separate experiments were assayed by ELISA and further characterized by immunoblotting. Using those monoclonal antibodies reacting with L'/R-PK in immunoblotting, a major band of 62 KDa MW was recognized in both preparations employed for immunization. Additionally, four smaller bands were detected. Furthermore, the monoclonal antibodies also detected a single band of 62 KDa MW in a highly purified L-PK prepared from human liver. In contrast, they showed no reaction with muscle and brain tissues containing M1-type PK. However, they reacted strongly with a single band of approximately 62 KDa in liver and kidney homogenates which is in line with immunocytochemical studies showing immunoreactive material in hepatocytes and proximal tubules of the kidney.

Antibodies, Monoclonal↗
PubMed1986

Purification of pyruvate kinase isoenzymes type M1 and M2 from dog (Canis familiaris) and comparison of their properties with those from chicken and rat.

Pyruvate kinase isoenzymes type M1 and M2 from dog muscle, lung and tumor have been isolated. The K0.5 for phosphoenol pyruvate have been determined to be 0.04 mM for dog muscle type M1, 0.24 mM for lung type M2 and 0.28 mM for tumor type M2 isoenzymes. The activator constant ka of L-serine is 240 nM from lung and 70 nM from tumor isoenzyme. Consistent with the assumption of a special form of pyruvate kinase M2 in dog tumor cells different isoelectric points and amino acid compositions have been found for the isoenzymes of lung and tumor.

Amino Acids↗
PubMed1986

Pyruvate kinase isoenzymes in altered foci and carcinoma of rat liver.

Pyruvate kinase (PK) isoenzymes, rate limiting for the last steps of glycolysis, were studied in normal rat liver, putative preneoplastic foci, neoplastic nodules and hepatocellular carcinoma. These lesions were produced by an initiation-promotion protocol: treatment with a single dose of N-nitrosomorpholine (NNM) was followed by feeding diets containing phenobarbital (PB) or alpha-hexachlorocyclohexane (alpha-HCH), or basal diet. PK was demonstrated (i) by immunocytochemistry on histological sections with antibodies specifically directed against the L and M2 isoenzymes, (ii) by electrophoretic separation of isoenzymes in homogenates from liver and larger tumors, and (iii) by electrophoretic separation of isoenzymes in parenchymal and stromal cells isolated from liver and tumors. Immunocytochemistry showed decreases of L-PK (L-PK-) in hepatocytes of most of the foci, nodules and carcinomas. Most L-PK- foci showed increases in gamma-glutamyltransferase (gamma-GT) and epoxide hydrolase (EH). PB or alpha-HCH treatment further decreased expression of L-PK in foci, but not in normal liver. Cells and foci with enhanced L-PK (L-PK+) were also found after carcinogen treatment. These did not show increases of gamma-GT or EH or any distinct morphological alterations with the exception of some which were basophilic ('tigroid') in H and E stained sections. No L-PK+ tumors were found. We could not demonstrate the M2-type PK in parenchymal cells of liver or any of the lesions described above. This isoenzyme was restricted to stromal cells in normal rat liver and in all stages of carcinogenesis as shown by immunohistology and by electrophoresis of preparations from isolated cell populations. However, stromal cells from hepatocellular carcinomas exhibited a 3-fold increase of M2-PK compared with stromal cells from normal liver. These results do not support an isoenzyme shift from L to M2-PK in the course of malignant transformation of hepatocytes as suggested previously.

Animals↗
PubMed1986

Carbohydrate metabolism in neoplastic tissue.

The kinetic and immunological properties of purified, homogeneous pyruvate kinase type M2 from chicken lung and tumors, including that of Rous sarcoma virus transformed chicken fibroblasts, have been compared. The type M2 enzymes from both lung and tumors have immunologically distinct structures. The enzyme isolated from tumors is characterized by a low affinity for phosphoenol pyruvate, pronounced L-serine activation and a strong inhibition by L-alanine, L-proline and several other amino acids. The chicken lung enzyme is only slightly affected by serine. The cellular amino acid concentration is in such a range that the tumor enzyme is strongly inhibited whereas the lung enzyme is only partially inhibited. The characteristics of the tumor enzyme allow the optimal adaptation to lactic acid and production of energy from glucose or amino acids dependent on substrate and oxygen supply.

Amino Acids↗