Determining and understanding the control of glycolysis in fast-growth tumor cells. Flux control by an over-expressed but strongly product-inhibited hexokinase.
Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V(max)) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphofructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V(max) values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.
Marín-Hernández
Alvaro
A
Instituto Nacional de Cardiología, Departamento de Bioquímica, Juan Badiano no. 1, Colonia Sección XVI, Tlalpan, México, Mexico.
Rodríguez-Enríquez
Sara
S
Vital-González
Paola A
PA
Flores-Rodríguez
Fanny L
FL
Macías-Silva
Marina
M
Sosa-Garrocho
Marcela
M
Moreno-Sánchez
Rafael
R
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation; D005260:Female; D005947:Glucose / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000737:chemistry* / Q000378:metabolism; D006019:Glycolysis; D006367:HeLa Cells; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D006593:Hexokinase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D019344:Lactic Acid; D019298:Glucose-6-Phosphate; D006593:Hexokinase; D010732:Phosphofructokinase-1; D005947:Glucose