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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The efflux of adenine nucleotides was studied in mitochondria isolated from normal rat liver, host livers, and the tumors from four Morris hepatoma lines of varying growth rates. [3H]Adenosine diphosphate (ADP) or [3H]adenosine triphosphate (ATP) was preloaded to the energized mitochondria, and the initial rates of exchange with unlabeled extramitochondrial nucleotides were measured with the carboxyatractyloside stop method. Results indicate that the Vmax values of ATP efflux in mitochondria from fast and intermediately growing tumors (hepatoma cell lines 7777, 7800, and 5123D) are significantly smaller than that of host or normal liver mitochondria, while in slow growing tumor (line 16) the Vmax is not different. On the other hand, for ADP efflux, the opposite (namely, higher in tumor than in host) is observed in the mitochondria of fast growing tumors. Preincubation with the divalent cation ionophore A23187 and calcium chelator ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid increases the efflux of both ATP and ADP (to a lesser extent) in these tumor mitochondria, indicating that the extraordinarily high concentrations of calcium form complexes with adenine nucleotides (particularly ATP) and thus lower the effective concentrations of free nucleotides for translocation. Together with previously published results (R. L. Barbour and S. H. P. Chan, Cancer Res., 43: 1511-1517, 1983) on lower nucleotide uptake rates in these tumor mitochondria, we propose that the lower ATP efflux and higher ADP efflux rates may cause a futile cycle of ADP transport across the mitochondrial membrane which may contribute to high rates of aerobic glycolysis (by stimulating key glycolytic enzymes such as hexokinase and phosphofructokinase) observed in these fast and intermediately growing tumors.
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PMID:Efflux of adenine nucleotides in mitochondria from rat tumor cells of varying growth rates. 646 6

The distribution of hexokinase between bound and soluble forms was studied by digitonin fractionation of Zajdela hepatoma ascites cells maintained under various metabolic conditions. Addition of glucose to Zajdela cells respiring on endogenous substrates induces an immediate inhibition of respiration by 50-60% ( Crabtree effect), and a production of acid due to glycolysis. Acid production decreases abruptly after 60s to 50% of the initial rate. The ATP/ADP ratio is not altered by the addition of glucose or by different rates of glycolysis. The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone decreases the ATP/ADP ratio by 10-fold in cells respiring on endogenous substrate, but has little effect on cells oxidizing glucose. Rapid fractionation of the cells under these various metabolic conditions revealed no change in the distribution of hexokinase. Approx. 75% of hexokinase is bound in all cases, in contrast with lactate dehydrogenase, 95% of which was in the soluble form. Longer-term incubations (to 20 min) revealed only slight (10-15%) increases in soluble hexokinase in cells incubated with glucose. Various metabolic inhibitors had little additional affect on the subcellular distribution of hexokinase. Thus a rapid release of hexokinase from mitochondrial membrane is not a mechanism by which glycolysis is regulated in rapidly growing Zajdela hepatoma.
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PMID:Altered metabolic states do not change the intracellular distribution of hexokinase in Zajdela hepatoma ascites cells. 672 51

The highly glucolytic hepatoma cell line H-91 is characterized by a high hexokinase activity to rat liver; 50% of this activity is associated with the mitochondrial fraction [Bustamante, E., & Pederson, P.L. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3735--3739]. Treatment of mitochondria from this cell line with adenosine 5'=triphosphate (ATP) or glucose 6-phosphate solubilizes bound hexokinase activity. Solubilization of the enzyme by ATP results in a six- to sevenfold purification. Free ATP, unchelated by Mg ions, induces the release of the enzyme from the membrane, whereas the MgATP complex is ineffective. Ethylenediaminetetraacetic acid (EDTA) fails to release mitochondrial hexokinase indicating that the enzyme is not attached to the membrane by divalent cations. Energization of mitochondria is not required for ATP to induce solubilization of bound hexokinase. This is evidenced by (a) the ability of the nonhydrolyzable ATP analogue adenylyl imidodiphosphate to solubilize the enzyme, (b) the inability of uncouplers and inhibitors of oxidative phosphorylation to either solubilize or prevent the release of mitochondrial hexokinase, and (c) the inability of atractyloside to solubilize or prevent the release of bound hexokinase. The bound and the ATP-solubilized forms of mitochondrial hexokinase from H-91 hepatoma cells are kinetically different. When membrane bound, the enzyme has a significantly higher apparent affinity (Km = 0.25 mM) for its substrate MgATP than when solubilized (Km = 1.2 mM). Free ATP acts as a competitive inhibitor of mitochondrial hexokinase. Both the membrane-bound and the solubilized forms of mitochondrial hexokinase have about the same apparent affinity for glucose (Km = 56 and 83 microM, respectively). The experiments reported here provide the first description of the properties and the nature of binding of mitochondrial hexokinase from a tumor cell line growing in tissue culture.
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PMID:Mitochondrial hexokinase of rat hepatoma cells in culture: solubilization and kinetic properties. 677 59

A detailed investigation concerned with localizing hexokinase in the Novikoff ascites tumor is presented. At least 50% of the total hexokinase activity was shown by differential and density gradient centrifugation techniques to be associated with tumor mitochondria. None of this activity was latent. Fractionation of isolated tumor mitochondria with digitonin revealed an outer membrane location for this enzyme. Treatment of tumor mitochondria with glucose 6-phosphate released about 80 to 85% of the hexokinase activity without disrupting the intermembrane compartment. This suggests that at least this proportion of the activity is bound to the outer surface of the outer membrane. Successive treatments did not remove the remaining hexokinase activity. At 30 degrees C, an incubation time of about 10 min with glucose 6-phosphate was required to achieve maximal release. No solubilization occurred at 0-4 degrees C. The isozymes derived from Novikoff mitochondria were identified by anion exchange chromatography as types I and III. Glucokinase activity was not detectable. Evidence is also presented which indicates that the hexokinase obtained from Novikoff mitochondria binds to the outer membrane of rat liver mitochondria. In contrast, the low endogenous hexokinase activity present in isolated liver mitochondria was found not to fractionate with outer membrane markers, but rather with contaminating microsomal membrane markers. Results described here provide the first direct evidence for the submitochondrial location of hexokinase in a tumor. They reveal an outer membrane location and an involvement of two hexokinase isozymes. Because these findings are characteristic of the hepatoma and not observed in control liver preparations, it is suggested that they may be very relevant to the general property of rapidly growing tumors to catabolize large amounts of glucose.
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PMID:Intracellular localization and properties of particulate hexokinase in the Novikoff ascites tumor. Evidence for an outer mitochondrial membrane location. 688 6

The effect of extracellular pH on hexose uptake by cultured Novikoff hepatoma cells has been examined with respect to four distinct parameters: i) the transport of 3-O-methyl-D-glucose, ii) the uptake of 2-deoxy-D-glucose by intact cells, iii) the phosphorylation of 2-deoxy-D-glucose in vitro by cell-free preparations, and iv) the intracellular pH as measured by 5,5'-dimethyl[2-14C]oxazolidine-2,4-dione. Transport per se was not affected by pH in the range of 6 to 8, while uptake of 2-deoxy-D-glucose increased with pH 2- to 3-fold over this range. The pH sensitivity of uptake can be explained on the basis of a change in hexokinase activity effective in situ. The pH dependence of hexokinase observed in vitro, however, was insufficient to account for that apparent in situ.
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PMID:Hexose transport and phosphorylation by Novikoff rat hepatoma cells as function of extracellular pH. 745 78

Mouse sperm contain a major phosphotyrosine-containing protein of M(r) 95,000 (nonreducing conditions) which has been implicated as a sperm membrane receptor for the egg zona pellucida glycoprotein, ZP3 (Leyton, L., and Saling, P. (1989) Cell 57, 1123-1130; Leyton, L., LeGuen, P., Bunch, D., and Saling, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11692-11695). This protein was purified and subjected to limited tryptic digestion and subsequent amino acid analysis. Three sequenced peptides revealed 100% amino acid identity to a mouse hepatoma hexokinase (Arora, K. K., Fanciulli, M., and Pederson, P. L. (1990) J. Biol. Chem. 265, 6481-6488). The purified protein, which migrated at M(r) 116,000 under reducing conditions (p95/116), reacted with an antiserum to the purified rat brain hexokinase, type 1, and comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the purified rat brain enzyme under both nonreducing and reducing conditions. Unlike p95/116, the rat brain enzyme was not a phosphotyrosine-containing protein. The p95/116 protein could be immunoprecipitated with the hexokinase antiserum or an O-phosphotyrosine antibody. Limited tryptic digestion of the purified p95/116 and the rat brain enzyme generated subsets of identical peptides which reacted with the hexokinase antiserum. However, p95/116 also contained phosphotyrosine-containing peptides that were not present in the rat brain hexokinase. When different mouse tissues were probed with the hexokinase antiserum all tissues, with the exception of liver, contained immunoreactive protein. In contrast, only sperm and testis possessed a phosphotyrosine-containing form of hexokinase. These data suggest that the germ cell component of the testis possesses a unique tyrosine-phosphorylated form of hexokinase.
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PMID:p95, the major phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with unique properties. 750 20

One of the most characteristic phenotypes of rapidly growing cancer cells is their propensity to catabolize glucose at high rates. Type II hexokinase, which is expressed at high levels in such cells and bound to the outer mitochondrial membrane, has been implicated as a major player in this aberrant metabolism. Here we report the isolation and sequence of a 4.3-kilobase pair proximal promoter region of the Type II hexokinase gene from a rapidly growing, highly glycolytic hepatoma cell line (AS-30D). Analysis of the sequence enabled the identification of putative promoter elements, including a TATA box, a CAAT element, several Sp-1 sites, and response elements for glucose, insulin, cAMP, Ap-1, and a number of other factors. Transfection experiments with AS-30D cells showed that promoter activity was enhanced 3.4-, 3.3-, 2.4-, 2.1-, and 1.3-fold, respectively, by glucose, phorbol 12-myristate 13-acetate (a phorbol ester), insulin, cAMP, and glucagon. In transfected hepatocytes, these same agents produced little or no effect. The results emphasize normal versus tumor cell differences in the regulation of Type II hexokinase and indicate that transcription of the Type II tumor gene may occur independent of metabolic state, thus, providing the cancer cell with a selective advantage over its cell of origin.
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PMID:Glucose catabolism in cancer cells. Isolation, sequence, and activity of the promoter for type II hexokinase. 762 9

In a previous study, we found that the steady state transcript level of type II hexokinase was specifically and remarkably elevated in rat hepatoma AH130 cells. To determine the molecular mechanism of transcriptional control of the type II hexokinase gene, we examined the nucleotide sequence of its 5'-flanking region and analyzed putative transcription factor binding sites. We also examined the type II hexokinase promoter activity by the chloramphenicol acetyltransferase (CAT) assay.
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PMID:Nucleotide sequence of the 5'-flanking region of the rat type II hexokinase gene. 787 17

Phosphorylation of glucose by hexokinase is the key step in glucose and energy metabolism of the cell. In the Morris hepatoma 3924A, hexokinase II is the predominant hexokinase isoenzyme and occurs in the cytosol as well as bound to membranes. Hexokinase II was isolated by DEAE-cellulose chromatography from both the cytosolic and the mitochondria-enriched fractions and further resolved by hydrophobic-interaction chromatography on phenyl-Sepharose into two components designated hexokinase IIa and IIb. In both the soluble and the mitochondria-enriched fractions, type IIb was the predominant form, but the IIb/IIa ratio was higher in the particulate (6-8) as compared with the cytosolic fraction (1.5-2.0). Binding of the isolated forms of the enzyme to rat liver mitochondria resulted in a 2-10-fold activation of both subtypes. Biochemical characterization showed that both subtypes are closely related to the isoenzyme commonly referred to as hexokinase II, and that the microheterogeneity was not a consequence of contamination with hexokinase I or III. Both subtypes had a molecular mass of 110 kDa, they were inhibited by Pi at concentrations higher than 5 mM, and activated by the detergent CHAPS. The two subtypes differed in electrophoretic mobility (IIa > IIb), in Km values for glucose (IIa, 0.109 mM; IIb, 0.216 mM), in Ki values for glucose 6-phosphate (IIa, 25 microM; IIb, 0.106 mM), and in Ki values for glucose 1,6-biphosphate (IIa, 12.2 microM; IIb, 5.5 microM). An artificial proteolytic cleavage as cause of the hexokinase II microheterogeneity can be excluded, since both subtypes show the same molecular mass and the ability to bind to mitochondria and phenyl-Sepharose. In addition, the relative proportions of the two subtypes did not vary markedly between several enzyme preparations. Northern-blot analysis with a hexokinase II-specific cDNA probe revealed two distinct mRNA transcripts of 5.2 and 6.3 kb in length, which offers the possibility that hexokinase II microheterogeneity is due to differential RNA transcription and/or processing.
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PMID:Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A. 794 51

A pyranoid polyol, 1,5-anhydroglucitol (AG), generally occurs in the human body as a humoral component. The plasma AG concentration in healthy individuals is maintained at a constant level, but it is markedly decreased in diabetes mellitus. This is due to hyperglycemia-dependent abolishment of renal AG retention. Hence, the plasma AG concentration has been established as a clinical marker for duration of hyperglycemia and since 1991 it has been practically applied to diabetic care in Japan. However, the details of the metabolism of AG and its physiological significance generally remain to be studied. In this study, we confirmed AG synthesis in cultured cells of a rat hepatoma line, Reuber H-35, in which AG was found to be derived from glucose, with retention of all six carbon atoms in the pyranoid structure. The fraction of the total glucose consumed by the cells, which was converted to AG (conversion efficiency) was at most 5 x 10(-6). The conversion efficiency increased at higher glucose concentrations (mM orders) where the glucose consumption rate was saturated. Since the rate of the hexokinase reaction, one of the rate-limiting steps in glucose consumption, has been estimated to be saturated at microM orders of glucose concentration, this observation was interpreted as indicating that AG is synthesized through a pathway which does not share the hexokinase reaction with glucose utilization. The presence of precursors other than glucose was also indicated in the time-course study of AG synthesis. Further, the amount of AG synthesized daily in humans is significant in comparison with the amount obtained from the diet.
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PMID:Synthesis of 1,5-anhydro-D-glucitol from glucose in rat hepatoma cells. 818 42


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