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• W2051219431 abstract "In differentiated tissues, such as muscle and brain, increased adenosine monophosphate (AMP) levels stimulate glycolytic flux rates. In the breast cancer cell line MCF-7, which characteristically has a constantly high glycolytic flux rate, AMP induces a strong inhibition of glycolysis. The human breast cancer cell line MDA-MB-453, on the other hand, is characterized by a more differentiated metabolic phenotype. MDA-MB-453 cells have a lower glycolytic flux rate and higher pyruvate consumption than MCF-7 cells. In addition, they have an active glycerol 3-phosphate shuttle. AMP inhibits cell proliferation as well as NAD and NADH synthesis in both MCF-7 and MDA-MB-453 cells. However, in MDA-MB-453 cells glycolysis is slightly activated by AMP. This disparate response of glycolytic flux rate to AMP treatment is presumably caused by the fact that the reduced NAD and NADH levels in AMP-treated MDA-MB-453 cells reduce lactate dehydrogenase but not cytosolic glycerol-3-phosphate dehydrogenase reaction. Due to the different enzymatic complement in MCF-7 cells, proliferation is inhibited under glucose starvation, whereas MDA-MB-453 cells grow under these conditions. The inhibition of cell proliferation correlates with a reduction in glycolytic carbon flow to synthetic processes and a decrease in phosphotyrosine content of several proteins in both cell lines. In differentiated tissues, such as muscle and brain, increased adenosine monophosphate (AMP) levels stimulate glycolytic flux rates. In the breast cancer cell line MCF-7, which characteristically has a constantly high glycolytic flux rate, AMP induces a strong inhibition of glycolysis. The human breast cancer cell line MDA-MB-453, on the other hand, is characterized by a more differentiated metabolic phenotype. MDA-MB-453 cells have a lower glycolytic flux rate and higher pyruvate consumption than MCF-7 cells. In addition, they have an active glycerol 3-phosphate shuttle. AMP inhibits cell proliferation as well as NAD and NADH synthesis in both MCF-7 and MDA-MB-453 cells. However, in MDA-MB-453 cells glycolysis is slightly activated by AMP. This disparate response of glycolytic flux rate to AMP treatment is presumably caused by the fact that the reduced NAD and NADH levels in AMP-treated MDA-MB-453 cells reduce lactate dehydrogenase but not cytosolic glycerol-3-phosphate dehydrogenase reaction. Due to the different enzymatic complement in MCF-7 cells, proliferation is inhibited under glucose starvation, whereas MDA-MB-453 cells grow under these conditions. The inhibition of cell proliferation correlates with a reduction in glycolytic carbon flow to synthetic processes and a decrease in phosphotyrosine content of several proteins in both cell lines. Both proliferating cells and tumor cells maintain a high glycolytic rate even under aerobic conditions, a process referred to as aerobic glycolysis. Observations on aerobic glycolysis in tumor cells prompted Warburg (1Warburg O. Science. 1956; 124: 269-270Crossref Google Scholar) to postulate an altered respiratory function leading to an increased glycolytic capacity and a high rate of lactate formation from glucose in the presence of oxygen. Data from former reports suggest that there are many factors contributing to the origin of aerobic glycolysis (2Eigenbrodt E. Gerbracht U. Mazurek S. Presek P. Friis R. Biochemical and Molecular Aspects of Selected Cancers. 2. Academic Press Inc., San Diego, CA1994: 311-385Google Scholar). The altered control of glycolysis by expression of certain isoenzymes is one important factor (2Eigenbrodt E. Gerbracht U. Mazurek S. Presek P. Friis R. Biochemical and Molecular Aspects of Selected Cancers. 2. Academic Press Inc., San Diego, CA1994: 311-385Google Scholar, 3Arora K.K. Pedersen P.L. J. Biol. Chem. 1988; 263: 17422-17428Google Scholar, 4Staal G.E.J. Kalff A. Heesbeen E.C. van Veelen C.W.M. Rijksen G. Cancer Res. 1987; 47: 5047-5051Google Scholar, 5Vora S. Halper J.P. Knowles D.M. Cancer Res. 1985; 45: 2993-3001Google Scholar, 6Hue L. Rider M.H. Biochem. J. 1987; 245: 313-324Google Scholar, 7Skala H. Vibert M. Lamas E. Maire P. Schweighoffer F. Kahn A. Eur. J. Biochem. 1987; 163: 513-518Google Scholar, 8Carney D.N. Marangos P.J. Ihde D.C. Bunn Jr., P.A. Cohen M.H. Minna J.D. Gazdar A.F. Lancet. 1982; 3: 583-585Google Scholar, 9Steinberg P. Weiße G. Eigenbrodt E. Oesch F. Carcinogenesis. 1994; 15: 125-127Google Scholar, 10Schwartz M.K. Clin. Biochem. 1990; 23: 395-398Google Scholar, 11Freitas I. Bertone V. Griffini P. Accossato P. Baronzio G.F. Pontiggia P. Stoward P.J. Anticancer Res. 1991; 11: 1293-1300Google Scholar, 12Board M. Humm S. Newsholme E.A. Biochem. J. 1990; 265: 503-509Google Scholar). Furthermore, the glycerol 3-phosphate shuttle and the malate-aspartate shuttle are altered in such a way that transport of cytosolic hydrogen into the mitochondria is reduced, requiring tumor cells to reoxidize NADH cytosolically by lactate dehydrogenase (13Sanchez-Jimenez F. Martinez P. Nunez de Castro I. Olavarria J.S. Biochimie (Paris). 1985; 67: 259-264Google Scholar, 14Lopez-Alarcon L. Eboli M.-L. Cancer Res. 1986; 46: 5589-5591Google Scholar, 15Lopez-Alarcon L. Eboli M.L. De Liberali E. Palombini G. Galeotti T. Arch. Biochem. Biophys. 1979; 192: 391-395Google Scholar). Additionally, oxidation of pyruvate is reduced in favor of glutamine oxidation (16Singer S. Souza K. Thilly W.G. Cancer Res. 1995; 55: 5140-5145Google Scholar, 17Marchok A.C. Huang S.F. Martin D.H. Carcinogenesis. 1984; 5: 789-796Google Scholar, 18McKeehan W.L. McKeehan K.A. Calkins D. J. Biol. Chem. 1981; 256: 2973-2981Google Scholar, 19Reitzer L.J. Wice B.M. Kennell D. J. Biol. Chem. 1979; 254: 2669-2676Google Scholar, 20Wice B.M. Reitzer L.J. Kennell D. J. Biol. Chem. 1981; 256: 7812-7819Google Scholar, 21Lanks K.W. J. Biol. Chem. 1987; 262: 10093-10097Google Scholar, 22Sauer L.A. Dauchy R.T. Nagel W.O. Morris H.P. J. Biol. Chem. 1980; 255: 3844-3848Google Scholar, 23Moreadith R.W. Lehninger A.L. J. Biol. Chem. 1984; 259: 6215-6221Google Scholar, 24Moreadith R.W. Lehninger A.L. J. Biol. Chem. 1984; 259: 6222-6227Google Scholar, 25McKeehan W.L. Cell Biol. Int. Rep. 1982; 6: 635-650Google Scholar). Due to the expression of the mitochondrial, NAD-dependent malate decarboxylase, malate is converted to pyruvate and lactate (22Sauer L.A. Dauchy R.T. Nagel W.O. Morris H.P. J. Biol. Chem. 1980; 255: 3844-3848Google Scholar–24Moreadith R.W. Lehninger A.L. J. Biol. Chem. 1984; 259: 6222-6227Google Scholar). The conversion of glutamine to lactate is called, in analogy to glycolysis, glutaminolysis (25McKeehan W.L. Cell Biol. Int. Rep. 1982; 6: 635-650Google Scholar). In tumor cells the glycolytic capacity can be so great that all of the cell's energy requirements are derived from glycolysis (2Eigenbrodt E. Gerbracht U. Mazurek S. Presek P. Friis R. Biochemical and Molecular Aspects of Selected Cancers. 2. Academic Press Inc., San Diego, CA1994: 311-385Google Scholar, 26Weber G. Stubbs M. Morris H.P. Cancer Res. 1971; 31: 2177-2183Google Scholar). Therefore, high glycolytic activity ensures the survival and the migration of tumor cells in hypoxic areas (2Eigenbrodt E. Gerbracht U. Mazurek S. Presek P. Friis R. Biochemical and Molecular Aspects of Selected Cancers. 2. Academic Press Inc., San Diego, CA1994: 311-385Google Scholar, 26Weber G. Stubbs M. Morris H.P. Cancer Res. 1971; 31: 2177-2183Google Scholar, 27Epner D.E. Partin A.W. Schalken J.A. Isaacs J.T. Coffey D.S. Cancer Res. 1993; 53: 1995-1997Google Scholar). The main role of the glutaminolytic pathway is the generation of energy (2Eigenbrodt E. Gerbracht U. Mazurek S. Presek P. Friis R. Biochemical and Molecular Aspects of Selected Cancers. 2. Academic Press Inc., San Diego, CA1994: 311-385Google Scholar, 25McKeehan W.L. Cell Biol. Int. Rep. 1982; 6: 635-650Google Scholar). However, a high glycolytic rate is not always linked to cell proliferation or tumor formation. There are several cell lines that are able to grow in a medium with 5 mM galactose or with low glucose supply (0.5 mM) without producing lactate via glycolysis (19Reitzer L.J. Wice B.M. Kennell D. J. Biol. Chem. 1979; 254: 2669-2676Google Scholar, 20Wice B.M. Reitzer L.J. Kennell D. J. Biol. Chem. 1981; 256: 7812-7819Google Scholar, 21Lanks K.W. J. Biol. Chem. 1987; 262: 10093-10097Google Scholar, 28Zielke H.R. Ozand P.T. Tildon J.T. Sevdalian D.A. Cornblath M. J. Cell. Physiol. 1978; 95: 41-48Google Scholar, 29Zielke H.R. Sumbilla C.M. Sevdalian D.A. Hawkins R.L. Ozand P.T. J. Cell. Physiol. 1980; 104: 433-441Google Scholar, 30Wolfrom C. Kadhom N. Polini G. Poggi J. Moatti N. Gautier M. Exp. Cell Res. 1989; 183: 303-318Google Scholar, 31Henderson J.F. Khoo M.K.Y. J. Biol. Chem. 1965; 240: 2349-2357Google Scholar, 32Henderson J.F. Khoo M.K.Y. J. Biol. Chem. 1965; 240: 2363-2366Google Scholar, 33Renner E.D. Plagemann P.G.W. Bernlohr R.W. J. Biol. Chem. 1972; 247: 5765-5776Google Scholar). Investigations with labeled glucose and galactose have shown that the carbons of the two carbohydrates can either be used to synthesize nucleotides, phospholipids, and complex carbohydrates or can flow through pyruvate kinase to pyruvate and lactate for energy production (2Eigenbrodt E. Gerbracht U. Mazurek S. Presek P. Friis R. Biochemical and Molecular Aspects of Selected Cancers. 2. Academic Press Inc., San Diego, CA1994: 311-385Google Scholar, 19Reitzer L.J. Wice B.M. Kennell D. J. Biol. Chem. 1979; 254: 2669-2676Google Scholar, 20Wice B.M. Reitzer L.J. Kennell D. J. Biol. Chem. 1981; 256: 7812-7819Google Scholar, 29Zielke H.R. Sumbilla C.M. Sevdalian D.A. Hawkins R.L. Ozand P.T. J. Cell. Physiol. 1980; 104: 433-441Google Scholar, 30Wolfrom C. Kadhom N. Polini G. Poggi J. Moatti N. Gautier M. Exp. Cell Res. 1989; 183: 303-318Google Scholar, 31Henderson J.F. Khoo M.K.Y. J. Biol. Chem. 1965; 240: 2349-2357Google Scholar, 32Henderson J.F. Khoo M.K.Y. J. Biol. Chem. 1965; 240: 2363-2366Google Scholar, 33Renner E.D. Plagemann P.G.W. Bernlohr R.W. J. Biol. Chem. 1972; 247: 5765-5776Google Scholar). Under glucose starvation, energy is not produced by glycolysis but by pyruvate oxidation or by conversion of glutamine to lactate (18McKeehan W.L. McKeehan K.A. Calkins D. J. Biol. Chem. 1981; 256: 2973-2981Google Scholar, 19Reitzer L.J. Wice B.M. Kennell D. J. Biol. Chem. 1979; 254: 2669-2676Google Scholar, 20Wice B.M. Reitzer L.J. Kennell D. J. Biol. Chem. 1981; 256: 7812-7819Google Scholar, 21Lanks K.W. J. Biol. Chem. 1987; 262: 10093-10097Google Scholar, 22Sauer L.A. Dauchy R.T. Nagel W.O. Morris H.P. J. Biol. Chem. 1980; 255: 3844-3848Google Scholar, 23Moreadith R.W. Lehninger A.L. J. Biol. Chem. 1984; 259: 6215-6221Google Scholar, 24Moreadith R.W. Lehninger A.L. J. Biol. Chem. 1984; 259: 6222-6227Google Scholar, 25McKeehan W.L. Cell Biol. Int. Rep. 1982; 6: 635-650Google Scholar). When those cells are replaced in a medium with a high glucose concentration (5 mM), all phosphometabolites above pyruvate kinase accumulate until the level of fructose 1,6-bisphosphate is high enough to activate pyruvate kinase (34Glaser G. Giloh H. Kasir J. Gross M. Mager J. Biochem. J. 1980; 192: 793-800Google Scholar, 35Ashizawa K. Willingham M.C. Liang C.-M. Cheng S. J. Biol. Chem. 1991; 266: 16842-16846Google Scholar, 36Eigenbrodt E. Reinacher M. Scheefers-Borchel U. Scheefers H. Friis R. Crit. Rev. Oncog. 1992; 3: 91-113Google Scholar). The mass of lactate is then derived from glucose. As a consequence, all intermediates of glycolysis between hexokinase and pyruvate kinase increase. By this mechanism the supply of phosphometabolites for synthetic processes is ensured, although pyruvate kinase is activated (2Eigenbrodt E. Gerbracht U. Mazurek S. Presek P. Friis R. Biochemical and Molecular Aspects of Selected Cancers. 2. Academic Press Inc., San Diego, CA1994: 311-385Google Scholar, 36Eigenbrodt E. Reinacher M. Scheefers-Borchel U. Scheefers H. Friis R. Crit. Rev. Oncog. 1992; 3: 91-113Google Scholar). From these observations and the fact that growth factors and oncogene-dependent phosphorylation regulate glycolysis and phosphometabolite pools, one can assume that some phosphometabolites or synthetic products derived from the phosphometabolites, e.g. sugar phosphates, AMP, NAD, NADH and serine for sphinganine synthesis, regulate cell proliferation (2Eigenbrodt E. Gerbracht U. Mazurek S. Presek P. Friis R. Biochemical and Molecular Aspects of Selected Cancers. 2. Academic Press Inc., San Diego, CA1994: 311-385Google Scholar, 4Staal G.E.J. Kalff A. Heesbeen E.C. van Veelen C.W.M. Rijksen G. Cancer Res. 1987; 47: 5047-5051Google Scholar, 6Hue L. Rider M.H. Biochem. J. 1987; 245: 313-324Google Scholar, 36Eigenbrodt E. Reinacher M. Scheefers-Borchel U. Scheefers H. Friis R. Crit. Rev. Oncog. 1992; 3: 91-113Google Scholar, 37Cooper J.A. Reiss N.A. Schwartz R.J. Hunter T. Nature. 1983; 302: 218-223Google Scholar, 38Cooper J.A. Esch F.S. Taylor S.S. Hunter T. J. Biol. Chem. 1984; 259: 7835-7841Google Scholar, 39Oude Weernink P.A. Rijksen G. Mascini E.M. Staal G.E.J. Biochim. Biophys. Acta. 1992; 1121: 61-68Google Scholar, 40Presek P. Reinacher M. Eigenbrodt E. FEBS Lett. 1988; 242: 194-198Google Scholar, 41Presek P. Glossmann H. Eigenbrodt E. Schoner W. Rübsamen H. Friis R.R. Bauer H. Cancer Res. 1980; 40: 1733-1741Google Scholar, 42Eigenbrodt E. Fister P. Rübsamen H. Friis R.R. EMBO J. 1983; 2: 1565-1570Google Scholar, 43Reiss N. Kanety H. Schlessinger J. Biochem. J. 1986; 239: 691-697Google Scholar, 44Oude Weernink P.A. Rijksen G. van der Heijden M.C.M. Staal G.E.J. Cancer Res. 1990; 50: 4604-4610Google Scholar, 45Bosca L. Mojena M. Diaz-Guerra J.M. Marquez C. Eur. J. Biochem. 1988; 175: 317-323Google Scholar, 46Smith E.R. Merrill Jr., A.H. J. Biol. Chem. 1995; 270: 18749-18758Google Scholar, 47Schwartz J.P. Passonneau J.V. Johnson G.S. Pastan I. J. Biol. Chem. 1974; 249: 4138-4143Google Scholar, 48Smith M.L. Buchanan J.M. J. Cell. Physiol. 1979; 101: 293-310Google Scholar, 49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar). Indeed, by searching for such metabolic signals we found that extracellular AMP inhibits DNA synthesis in MCF-7 cells and stops cell proliferation. Extracellular AMP is split to adenosine by the ecto-5′-nucleotidase. Adenosine is transported into the cells via an adenosine translocator and rephosphorylated by the cytosolic adenosine kinase to AMP (49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar, 50Henderson J.F. Scott F.W. Pharmacol. & Ther. 1981; 8: 539-571Google Scholar, 51Rapaport E. J. Cell. Physiol. 1980; 105: 267-274Google Scholar, 52Weisman G.A. De B.K. Friedberg I. Pritchard R.S. Heppel L.A. J. Cell. Physiol. 1984; 119: 211-219Google Scholar, 53Weisman G.A. Lustig K.D. Lane E. Huang N. Belzer I. Friedberg I. J. Biol. Chem. 1988; 263: 12367-12372Google Scholar). The increase in intracellular AMP inhibits P-ribosePP synthetase and reduces NAD and NADH synthesis (49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar, 50Henderson J.F. Scott F.W. Pharmacol. & Ther. 1981; 8: 539-571Google Scholar, 51Rapaport E. J. Cell. Physiol. 1980; 105: 267-274Google Scholar, 52Weisman G.A. De B.K. Friedberg I. Pritchard R.S. Heppel L.A. J. Cell. Physiol. 1984; 119: 211-219Google Scholar, 53Weisman G.A. Lustig K.D. Lane E. Huang N. Belzer I. Friedberg I. J. Biol. Chem. 1988; 263: 12367-12372Google Scholar, 54Ghose A.K. Viswanadhan V.N. Sanghvi Y.S. Nord L.D. Willis R.C. Revankar G.R. Robins R.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8242-8246Google Scholar). NADH levels drop so low that lactate dehydrogenase is no longer able to transfer the hydrogen from NADH to pyruvate. As a consequence, glycolysis is inhibited at the level of the NADH producing glyceraldehyde-3-phosphate dehydrogenase reaction (49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar). The metabolic behavior of MCF-7 cells is in complete contrast to differentiated tissues and cells where the increase of AMP under hypoxic conditions drastically activates 6-phosphofructo-1-kinase and the glycolytic flux rate (55Schultz V. Lowenstein J.M. J. Biol. Chem. 1978; 253: 1938-1943Google Scholar, 56Zeleznikar R.J. Dzeja P.P. Goldberg N.D. J. Biol. Chem. 1995; 270: 7311-7319Google Scholar, 57Ohno K. Maier P. J. Cell. Physiol. 1994; 160: 358-366Google Scholar, 58Gutierrez-Juarez R. Madrid-Marina V. Pina E. Int. J. Biochem. 1993; 25: 725-729Google Scholar, 59Tejwani G.A. Trends Biochem. Sci. 1978; 2: 30-33Google Scholar). In order to investigate the mechanisms by which AMP stimulates glycolysis in differentiated cells and inhibits glycolysis in tumor cells, we decided to study another human breast cancer cell line MDA-MB-453, which has a more differentiated metabolic phenotype (e.g. low aerobic glycolytic flux rate, high pyruvate consumption). In addition, we found that MDA-MB-453 cells grow well in a medium with a low glucose concentration and with galactose, whereas MCF-7 cells are unable to grow under these nutrient conditions. MCF-7 cells were obtained from Prof. Dr. K. Goerttler, Institute for Experimental Pathology, German Cancer Research Center, Heidelberg, Germany. MDA-MB-453 cells were from the German Collection of Microorganisms and Cell Cultures in Braunschweig, Germany. For cell culture, the basic medium used was glucose-free Dulbecco's minimal essential medium, supplemented with 100 units of penicillin/ml, 100 μg of streptomycin/ml, 2 mM glutamine, and 4 mM pyruvate (all from Biochrom, Berlin, Germany). For MCF-7 cells 20% (v/v) fetal calf serum (FCS) 1The abbreviations used are: FCSfetal calf serumDMEMDulbecco's modified Eagle's medium. from Biochrom, Berlin, Germany, were added to the basic medium. For MDA-MB-453 cells the basic medium was supplemented with 10% (v/v) FCS and 10 mM HEPES, pH 7.0. Glucose and galactose from Sigma were added to the media of both cell lines as described under “Results.” MCF-7 cells were cultured at 37°C in a 5% CO2 environment. MDA-MB-453 cells were cultured in a CO2-free atmosphere. For the proliferation and flux experiments 4-cm diameter dishes (MCF-7 cells) and 25-cm2 flasks (MDA-MB-453 cells) were used (both from Nunc, Wiesbaden, Germany). For the intracellular measurements MCF-7 cells were cultured on 14-cm diameter dishes, and experiments were started with 1 million cells/dish. MDA-MB-453 cells were cultured in 83-cm2 flasks, and experiments were started with 2 million cells/flask. MCF-7 cells were passaged every 4 days and MDA-MB-453 cells every 5-6 days. AMP was derived from Boehringer Mannheim, Germany, and was added to the media at a final concentration of 3 mM. fetal calf serum Dulbecco's modified Eagle's medium. For the proliferation rate and glycolytic and glutaminolytic flux measurements, AMP was added to the medium at the beginning of each passage. For the intracellular measurements AMP was added to the media on the 2nd day of the culture period. After 2 (MCF-7 cells) or 3 days (MDA-MB-453 cells), the AMP-treated cells arrested at a cell density of 5 million cells/dish, whereas control cells without AMP treatment continued to proliferate, reaching a density of 10-15 million cells/dish (MCF-7 cells) or 15-20 million cells/flask (MDA-MB-453 cells). Every 24 h cell culture supernatants were collected and immediately frozen in liquid nitrogen. The cells were removed from the plates with trypsin/EDTA from Biochrom, Berlin, Germany, and counted in a hemocytometer. The frozen supernatants were heated for 15 min at 80°C and were subsequently centrifuged at 8000 × g for 10 min (49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar). Glucose, lactate, pyruvate, glutamine, and glutamate concentrations were determined according to Bergmeyer (60Bergmeyer H.U. Verlag Chemie.Band I and II. Weinheim/ Bergstraße 3, 1974Google Scholar). For galactose measurements a commercially available test kit from Boehringer Mannheim, Germany, was employed. For the extraction of intracellular lactate, pyruvate, and NAD, cells were treated at 80°C in aqua bidest for 15 min as described previously (49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar). The concentrations of lactate, pyruvate, and NAD were measured enzymatically (60Bergmeyer H.U. Verlag Chemie.Band I and II. Weinheim/ Bergstraße 3, 1974Google Scholar). The NADH concentration was calculated via the equation [NADH] = 1.11·;10−4·;[NAD]·;[lactate]:[pyruvate] (47Schwartz J.P. Passonneau J.V. Johnson G.S. Pastan I. J. Biol. Chem. 1974; 249: 4138-4143Google Scholar). The protein content in the pellet was determined using the commercially available Bio-Rad test-kit (Bio-Rad, Munich, Germany). For the extraction of the intracellular enzymes, a homogenization buffer, pH 7.4, containing 20 mM KH2PO4/K2HPO4, 1 mM mercaptoethanol, 1 mM EDTA/Na2, 2 mMϵ-amino-n-caproic acid, and 0.2 mM phenylmethylsulfonyl fluoride was used. For the measurements of malate dehydrogenase a homogenization buffer containing 10 mM Tris, 1 mM NaF, and 1 mM mercaptoethanol, pH 7.4, was used. The extractions and the measurements of enzyme activities were carried out as described in Ref. 49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar. Protein concentrations were measured by the Biuret method using a commercially available test kit from Boehringer Mannheim, Germany. The pyruvate kinase subunits (dimer and tetramer) were separated by gel filtration as described in Ref. 36Eigenbrodt E. Reinacher M. Scheefers-Borchel U. Scheefers H. Friis R. Crit. Rev. Oncog. 1992; 3: 91-113Google Scholar. Cells were extracted with a homogenization buffer containing 10 mM Tris, 1 mM NaF, and 1 mM mercaptoethanol, pH 7.4. Isoelectric focusing was carried out with a linear gradient of glycerine (50% to 0% (v/v)) and ampholines (pI 3.5-10.5) as described previously (61Mazurek S. Hugo F. Failing K. Eigenbrodt E. J. Cell. Physiol. 1996; 167: 238-250Google Scholar). After separation on a 10% SDS-polyacrylamide gel, the proteins were transferred onto a nitrocellulose membrane by electroblotting. For the detection of phosphotyrosine a peroxidase-conjugated monoclonal anti-phosphotyrosine antibody from ICN (Costa Mesa, CA) was used. Immunostaining without anti-phosphotyrosine antibody resulted in no detectable reaction (61Mazurek S. Hugo F. Failing K. Eigenbrodt E. J. Cell. Physiol. 1996; 167: 238-250Google Scholar). For the glycolytic and glutaminolytic flux measurements as well as for the specific enzyme activities, statistical analysis was performed by a one- or two-factor (co)variance analysis with the “statistical program package BMDP,” whereby metabolite conversions or enzyme activities were plotted versus cell densities (62Dixon, W. J., (ed)(1993) University of California Press, Berkeley, Los Angeles.Google Scholar). Possible effects of cell density were taken into consideration. In all other analyses Student's t test was employed. MCF-7 and MDA-MB-453 cells were cultured in media with different glucose and galactose concentrations. The basic medium was glucose-free Dulbecco's minimal essential medium. After addition of fetal calf serum a final glucose concentration of 0.5 mM was obtained. Galactose was not detectable. In order to achieve other glucose and galactose concentrations, corresponding carbohydrates were added to the medium. To obtain a glucose-free medium fetal calf serum was dialyzed in a dialysis bag three times (for 8 h each) against 40 volumes of phosphate-buffered saline. To ensure that no vital necessary factors were lost during dialysis, the glucose-free medium was supplemented with 0.5 mM glucose, and cell proliferation was checked. There was no difference between the proliferation rate of MDA-MB-453 cells cultured in the glucose-supplemented medium (0.5 mM glucose) and that of the cells held in medium with 0.5 mM glucose from undialyzed fetal calf serum (data not shown). For cell stock breeding MCF-7 cells were cultured in the basic DMEM supplemented with 5 mM glucose. MDA-MB-453 cells were cultured in the basic DMEM supplemented with 5 mM galactose and 0.5 mM glucose from FCS (compare “Experimental Procedures”). Henceforth, these cells will be referred to as “proliferating” MCF-7 or MDA-MB-453 cells. For the described experiments with other glucose and galactose concentrations, cells were cultured for one passage in the new medium, and the cells of the second passage were used for the measurements. The effect of AMP on metabolites and enzymes was always determined in the culture medium in which the cells grew best. For MCF-7 cells this was basic DMEM supplemented with 5 mM glucose and for MDA-MB-453 the basic DMEM supplemented with 5 mM galactose and 0.5 mM glucose (from FCS). The concentration of AMP depends on the content of adenosine deaminase in the FCS charge (49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar). Adenosine deaminase degrades AMP to inosine monophosphate, which has no inhibitory effect on the proliferation rate of the two cell lines (49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar). For the experiments described in this paper an AMP concentration of 3 mM was chosen. Fig. 1, A and B, shows the effect of AMP and different glucose and galactose concentrations on the cell proliferation rate of MCF-7 and MDA-MB-453 cells. In MCF-7 cells the highest proliferation rate was reached at a glucose concentration of 5 mM in the medium (Fig. 1A). Reduction of the glucose concentration to 0.5 mM led to an inhibition of cell proliferation to less than half the maximal rate. In glucose-free DMEM supplemented with 5 mM galactose, the cells became adherent but ceased to proliferate. The addition of AMP to the culture medium (DMEM with 5 mM glucose) totally inhibited cell proliferation. This inhibition was reversible. After degradation of AMP in the medium or after reculture in AMP-free medium, cell proliferation began again and reached normal values (49Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Google Scholar). MDA-MB-453 cells demonstrated a totally different association between proliferation rate and availability of glucose and galactose in the medium. MDA-MB-453 cells grew best in a medium containing 5 mM galactose (Fig. 1B). In the absence of galactose, cell proliferation was inhibited in MDA-MB-453 cells. If glucose was also removed cell proliferation was totally arrested. The cells did not become confluent. In the presence of glucose in the medium, the cell proliferation rate was only about half the rate in galactose containing medium. An increase of the glucose concentration from 0.5 to 5 mM had no effect on the proliferation rate. As in MCF-7 cells, incubation of MDA-MB-453 cells with AMP led to a total inhibition of cell proliferation. After reculture of the AMP-treated MDA-MB-453 cells in AMP-free medium, cell proliferation once again reached normal rates (data not shown). For flux measurements, two different forms of calculations were chosen. The first calculation is in nmol/(h·;dish) and describes the direct correlation between the consumption of a specific carbon source (glucose, galactose, glutamine, or pyruvate) and lactate or glutamate production. The second form of calculation in nmol/(h·;105 cells) describes the consumption or production of a certain metabolite by each cell. In MCF-7 cells the measurements of the glycolytic flux in nmol/(h·;dish) showed a close linkage between glucose consumption and lactate production, independent of the glucose concentration in the medium (0.5 mM or 5 mM) (Fig. 2A and Table I). The slope of the regression line with 5 mM glucose was 1.7, with a correlation coefficient of 0.932. This value approaches the ideal maximal value of 2 for the ratio of lactate production:glucose consumption. In glycolysis 1 mol of glucose is converted into 2 mol of lactate; therefore, a ratio between lactate production and glucose consumption of nearly 2 indicates that all lactate produced must be derived from glucose. A slope of 1.7 means that 85% of the glucose consumed was converted to lactate. Therefore, in MCF-7 cells 37 nmol of glucose consumed were converted to lactate, and 7 nmol were used for synthetic processes (calculated with data from Table III). The intercept of the regression line reflects the lactate production without glucose consumption (Fig. 2 and Table I, Table II). This lactate can derive from glutamine and/or pyruvate consumption (Table I, Table II). In MCF-7 cells with 5 mM glucose 91 nmol of lactate/(h·;dish) were derived from sources other than glucose (Table I). If cultured in a medium with 5 mM glucose, there was a significant correlation between glutamine consumption and lactate production (Table I) but not between pyruvate consumption and lactate production (data not shown). Therefore, when no glucose was consumed by MCF-7 cells, the mass of lactate derived from glutamine. Furthermore, glutamine consumption increased with glucose consumption when the cells were cultured in 5 mM glucose (slope = 0.08; intercept = 6.2 nmol/(h·;dish); r = 0.777; n = 33). A reduction of the glucose concentration to 0.5 mM led to an inhibition of cell proliferation (Fig. 1A) and to a reduction of the total glucose conversion (Table III), but the strong linkage between glucose consumption and lactate production was not influenced at low glucose concentrations (Fig. 2A and Table I). The slope of the regression line was 1.6 with a regression coefficient of 0.642. The intercept of the regression line dropped from 91 nmol/(h·;dish) in medium with 5 mM glucose to 25 nmol/(h·;dish) in medi" @default.
• W2051219431 created "2016-06-24" @default.
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• W2051219431 date "1997-02-01" @default.
• W2051219431 modified "2023-10-17" @default.
• W2051219431 title "Effect of Extracellular AMP on Cell Proliferation and Metabolism of Breast Cancer Cell Lines with High and Low Glycolytic Rates" @default.
• W2051219431 cites W1482902326 @default.
• W2051219431 cites W1483212110 @default.
• W2051219431 cites W1484073306 @default.
• W2051219431 cites W1488748351 @default.
• W2051219431 cites W1500038515 @default.
• W2051219431 cites W1503075767 @default.
• W2051219431 cites W1503540946 @default.
• W2051219431 cites W1504666203 @default.
• W2051219431 cites W1507021851 @default.
• W2051219431 cites W1524643819 @default.
• W2051219431 cites W1531051436 @default.
• W2051219431 cites W1533084749 @default.
• W2051219431 cites W1534578439 @default.
• W2051219431 cites W1542594322 @default.
• W2051219431 cites W1548610182 @default.
• W2051219431 cites W1553739607 @default.
• W2051219431 cites W1559768539 @default.
• W2051219431 cites W1562919645 @default.
• W2051219431 cites W1566297179 @default.
• W2051219431 cites W1581773394 @default.
• W2051219431 cites W1590988749 @default.
• W2051219431 cites W1599545708 @default.
• W2051219431 cites W1607009526 @default.
• W2051219431 cites W1607470331 @default.
• W2051219431 cites W170239507 @default.
• W2051219431 cites W1745111908 @default.
• W2051219431 cites W1754310888 @default.
• W2051219431 cites W1828734010 @default.
• W2051219431 cites W1864900316 @default.
• W2051219431 cites W1969608892 @default.
• W2051219431 cites W1972440163 @default.
• W2051219431 cites W1972753440 @default.
• W2051219431 cites W1973365347 @default.
• W2051219431 cites W1974378085 @default.
• W2051219431 cites W1976598277 @default.
• W2051219431 cites W1978212802 @default.
• W2051219431 cites W1978533301 @default.
• W2051219431 cites W1978550183 @default.
• W2051219431 cites W1980963932 @default.
• W2051219431 cites W1986157611 @default.
• W2051219431 cites W1993119123 @default.
• W2051219431 cites W1995154934 @default.
• W2051219431 cites W1996500617 @default.
• W2051219431 cites W2003044258 @default.
• W2051219431 cites W2013729223 @default.
• W2051219431 cites W2019079338 @default.
• W2051219431 cites W2019344539 @default.
• W2051219431 cites W2022776160 @default.
• W2051219431 cites W2024611202 @default.
• W2051219431 cites W2025590706 @default.
• W2051219431 cites W2025716683 @default.
• W2051219431 cites W2026950763 @default.
• W2051219431 cites W2029001889 @default.
• W2051219431 cites W2037247859 @default.
• W2051219431 cites W2039485076 @default.
• W2051219431 cites W2042868752 @default.
• W2051219431 cites W2042926264 @default.
• W2051219431 cites W2043864472 @default.
• W2051219431 cites W2045454875 @default.
• W2051219431 cites W2048806994 @default.
• W2051219431 cites W2050843429 @default.
• W2051219431 cites W2058086823 @default.
• W2051219431 cites W2058312102 @default.
• W2051219431 cites W2069998681 @default.
• W2051219431 cites W2075636558 @default.
• W2051219431 cites W2091424804 @default.
• W2051219431 cites W2092173702 @default.
• W2051219431 cites W2127042719 @default.
• W2051219431 cites W2134207436 @default.
• W2051219431 cites W2140192657 @default.
• W2051219431 cites W2141172423 @default.
• W2051219431 cites W2158108870 @default.
• W2051219431 cites W2160694063 @default.
• W2051219431 cites W2328172021 @default.
• W2051219431 cites W2441513897 @default.
• W2051219431 cites W268181665 @default.
• W2051219431 cites W4243645905 @default.
• W2051219431 cites W4243903519 @default.
• W2051219431 cites W4317649971 @default.
• W2051219431 doi "https://doi.org/10.1074/jbc.272.8.4941" @default.
• W2051219431 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9030554" @default.
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