Study on ATP-generating system and related hexokinase activity in mitochondria isolated from undifferentiated or differentiated HT29 adenocarcinoma cells.
The functional properties of mitochondria bound hexokinase are compared in two subpopulations of the HT29 human colon cancer cell-line: (1) the HT29 Glc+ cells, cultured in the presence of glucose, which are poorly differentiated and highly glycolytic and (2) the HT29 Glc- cells, adapted to grow in a glucose-free medium, which are 'enterocyte-like' differentiated and less glycolytic when given glucose (Zweibaum et al. (1985) J. Cell Physiol. 122, 21-28). The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase are found to be twice as high in Glc+ cells when compared to Glc- cells. Besides, the respiration rate is decreased in Glc+ cells compared to Glc- cells. These results correlate with the higher glycolytic rate in Glc+ cells. In many tissues, it has been shown that the binding of hexokinase to the mitochondrial outer membrane allows a preferential utilization of the ATP generated by oxidative phosphorylation which, in turn, is activated by immediate restitution of ADP. In highly glycolytic cancer cells, although a large fraction of hexokinase is bound to the mitochondria, the existence of such a channeling of nucleotides is still poorly documented. The rates of glucose phosphorylation by bound hexokinase were investigated in mitochondria isolated from both Glc+ and Glc- cells either with exogenous ATP or with ATP generated by mitochondria supplied with ADP and succinate (endogenous ATP). Diadenosine pentaphosphate (Ado2P5), oligomycin and carboxyatractyloside (CAT) were used in combination or separately as metabolic inhibitors of adenylate kinase, ATP synthase and ATP/ADP translocator, respectively. Exogenous ATP appears to be 6.5-times more efficient than endogenous ATP in supporting hexokinase activity in the mitochondria from Glc+ cells and only 1.8-times cells. The rate of oxidative phosphorylation being higher in mitochondria from Glc- cells, hexokinase activity is higher in this model when ATP is generated by respiration. Furthermore, in Glc+ mitochondria, the adenylate kinase reaction appears to be an important source of endogenous ATP for bound hexokinase, while, in Glc- mitochondria, hexokinase activity is almost totally dependent on the ATP generated by oxidative phosphorylation. This result might be explained by our previous finding that mitochondria from Glc+ cells lack contact sites between outer and inner membrane, whereas numerous contacts were observed in mitochondria from Glc- cells (Denis-Pouxviel et al. (1987) Biochim. Biophys. Acta 902, 335-348).
Gauthier
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T
INSERM U 317, Institut de Physiologie, Université Paul Sabatier, Toulouse, France.
Denis-Pouxviel
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C
Paris
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H
Murat
J C
JC
D000230:Adenocarcinoma; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D003110:Colonic Neoplasms; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D010732:Phosphofructokinase-1 / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000378:metabolism*
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D006593:Hexokinase; D010732:Phosphofructokinase-1; D011770:Pyruvate Kinase; D005947:Glucose