Cell Hexokinase Research Articles

The Complex Role of the Triphenylmethyl Motif in Anticancer Compounds

Compounds incorporating the triphenylmethyl motif constitute an emerging family of potent anticancer agents. Although several small molecules containing this pharmacophore have now been identified, the mechanism of cell death induction for some of these compounds is unknown. In an effort to define their mechanism of action, and to distinguish subtypes within the group of compounds containing the triphenylmethyl moiety, we have created novel triphenylmethyl-containing small molecules and have evaluated them in a battery of biological assays. Here we show that several phosphonate and phosphonochloridates possessing the triphenylmethyl motif potently induce death of multiple cancer cell lines in culture. Further assays evaluating the ability to cause cell cycle arrest, inhibit tubulin polymerization, dissociate mitochondrial-bound hexokinase in cancer cells, and inhibit calcium-dependent potassium ion channels indicate that triphenylmethyl-containing compounds can be placed into at least four distinct categories, each with a different mechanism of action.

Selective depletion of the Type I, Type II, and Type III isozymes of hexokinase in mammalian cells using small interfering RNAs

Constructs based on the pSUPER vector [Science 296 (2002) 550] and encoding small interfering RNAs specific for the Type I, Type II, or Type III isozymes of mammalian (rat) hexokinase were prepared. Transfection of Chinese hamster ovary and HeLa cells with these vectors resulted in selective depletion of the respective isozymes. A Zeocin marker was incorporated into the modified pSUPER vector, permitting isolation of stably transfected cell lines selectively depleted of the respective isozyme.

Selective depletion of the Type I, Type II, and Type III isozymes of hexokinase in mammalian cells using small interfering RNAs*1

Constructs based on the pSUPER vector [Science 296 (2002) 550] and encoding small interfering RNAs specific for the Type I, Type II, or Type III isozymes of mammalian (rat) hexokinase were prepared. Transfection of Chinese hamster ovary and HeLa cells with these vectors resulted in selective depletion of the respective isozymes. A Zeocin marker was incorporated into the modified pSUPER vector, permitting isolation of stably transfected cell lines selectively depleted of the respective isozyme.

Proinflammatory interleukin-1 cytokines increase mesangial cell hexokinase activity and hexokinase II isoform abundance.

Mesangial cell hexokinase (HK) activity is increased by a diverse array of factors that share both an association with pathological conditions and a common requirement for classic MAPK pathway activation. To better understand the relationship between glucose (Glc) metabolism and injury and to indirectly test the hypothesis that these changes constitute a general adaptive response to insult, we have sought to identify and characterize injury-associated factors that couple to mesangial cell HK regulation. Proinflammatory interleukin-1 (IL-1) cytokines activate the MAPK pathway and have known salutary effects in this cell type. We therefore examined their ability to influence mesangial cell HK activity, Glc utilization, MAPK pathway activation, and individual HK isoform abundance. IL-1beta increased HK activity in both a time- and concentration-dependent manner: activity increased maximally by approximately 50% between 12 and 24 h with an apparent EC(50) of 3 pM. IL-1alpha mimicked, but did not augment, the effects of IL-1beta. Specific IL-1 receptor antagonism and selective MAPK/ERK kinase or upstream Ras inhibition prevented these increases, whereas PKC inhibition did not. Changes in HK activity were associated with both increased Glc metabolism and selective increases in HKII isoform abundance. We conclude that IL-1 cytokines can regulate cellular Glc phosphorylating capacity via an IL-1 receptor-, Ras-, and classic MAPK pathway-mediated increase in HKII abundance. These findings suggest a novel, previously undescribed mechanism whereby metabolism may be coupled to inflammation and injury.

LPA is a novel lipid regulator of mesangial cell hexokinase activity and HKII isoform expression.

The prototypical extracellular phospholipid mediator, lysophosphatidic acid (LPA), exhibits growth factor-like properties and represents an important survival factor in serum. This potent mesangial cell mitogen is increased in conditions associated with glomerular injury. It is also a known activator of the classic mitogen-activated protein kinase (MAPK) pathway, which plays an important role in the regulation of mesangial cell hexokinase (HK) activity. To better understand the mechanisms coupling metabolism to injury, we examined the ability of LPA to regulate HK activity and expression in cultured murine mesangial cells. LPA increased total HK activity in a concentration- and time-dependent manner, with maximal increases of >50% observed within 12 h of exposure to LPA concentrations > or =25 microM (apparent ED(50) 2 microM). These effects were associated with increased extracellular signal-regulated kinase (ERK) activity and were prevented by the pharmacological inhibition of either MAPK/ERK kinase or protein kinase C (PKC). Increased HK activity was also associated with increased glucose (Glc) utilization and lactate accumulation, as well as selectively increased HKII isoform abundance. The ability of exogenous LPA to increase HK activity was both Ca2+ independent and pertussis toxin insensitive and was mimicked by LPA-generating phospholipase A2. We conclude that LPA constitutes a novel lipid regulator of mesangial cell HK activity and Glc metabolism. This regulation requires sequential activation of both Ca2+-independent PKC and the classic MAPK pathway and culminates in increased HKII abundance. These previously unrecognized metabolic consequences of LPA stimulation have both physiological and pathophysiological implications. They also suggest a novel mechanism whereby metabolism may be coupled to cellular injury via extracellular lipid mediators.

Gleevec (STI571) Influences Metabolic Enzyme Activities and Glucose Carbon Flow toward Nucleic Acid and Fatty Acid Synthesis in Myeloid Tumor Cells

Chronic myeloid leukemia cells contain a constitutively active Bcr-Abl tyrosine kinase, the target protein of Gleevec (STI571) phenylaminopyrimidine class protein kinase inhibitor. Here we provide evidence for metabolic phenotypic changes in cultured K562 human myeloid blast cells after treatment with increasing doses of STI571 using [1,2-13C2]glucose as the single tracer and biological mass spectrometry. In response to 0.68 and 6.8 microm STI571, proliferation of Bcr-Abl-positive K562 cells showed a 57% and 74% decrease, respectively, whereas glucose label incorporation into RNA decreased by 13.4% and 30.1%, respectively, through direct glucose oxidation, as indicated by the decrease in the m1/Sigma(m)n ratio in RNA. Based on the in vitro proliferation data, the IC50 of STI571 in K562 cultures is 0.56 microm. The decrease in 13C label incorporation into RNA ribose was accompanied by a significant fall in hexokinase and glucose-6-phosphate 1-dehydrogenase activities. The activity of transketolase, the enzyme responsible for nonoxidative ribose synthesis in the pentose cycle, was less affected, and there was a relative increase in glucose carbon incorporation into RNA through nonoxidative synthesis as indicated by the increase in the m2/Sigma(m)n ratio in RNA. The restricted use of glucose carbons for de novo nucleic acid and fatty acid synthesis by altering metabolic enzyme activities and pathway carbon flux of the pentose cycle constitutes the underlying mechanism by which STI571 inhibits leukemia cell glucose substrate utilization and growth. The administration of specific hexokinase/glucose-6-phosphate 1-dehydrogenase inhibitor anti-metabolite substrates or competitive enzyme inhibitor compounds, alone or in combination, should be explored for the treatment of STI571-resistant advanced leukemias as well as that of Bcr-Abl-negative human malignancies.

Identification and characterization of hexokinase isozyme predominantly expressed in malignant tumor cells

Tumor cells show a higher glycolytic rate than normal cells. Of glycolytic enzymes, the activity of hexokinase, known as a rate limiting enzyme in glycolysis, is amazingly high in malignant tumor cells. In mammals, four isozymes of hexokinase are expressed but the question which isozyme is responsible for the high hexokinase activity observed in tumor cells was not yet clearly answered. By Northern blot analysis, we found that the type II isozyme, which is only slightly expressed in normal heart, muscle and adipose tissue, was remarkably expressed in malignant tumor cells. We next tried to understand how the expression of type II hexokinase gene is regulated in tumor cells. For this purpose, we first isolated the type II hexokinase gene and characterized its structural features. We further investigated the regulatory mechanisms of the expression of type II hexokinase in tumor cells. Results indicate the potential involvement of a serum responsive factor in the regulation of the expression of type II hexokinase in tumor cells. In addition to the remarkable expression, binding of the type II hexokinase to mitochondria is another characteristic of tumor cells, however, the physiological meaning of hexokinase binding to mitochondria was not yet fully understood. Our results clearly showed that the mitochondria-bound hexokinase utilize mitochondrially generated ATP more preferentially under normal conditions. However, when the rate of extramitochondrial ATP generating system (glycolysis) exceed that of mitochondrial ATP generating system (oxidative phosphorylation), the mitochondria-bound hexokinase utilize extramitochondrial ATP. This result indicates that the hexokinase binding enables a cross talk between oxidative phosphorylation and glycolysis.

Regulation of mesangial cell hexokinase activity by PKC and the classic MAPK pathway.

Phorbol esters increase glucose (Glc) uptake and utilization in a variety of cell types, and, in some cells, these changes have been attributed to increased Glc phosphorylation and better functional coupling of hexokinases (HKs) to facilitative Glc transporters. Phorbol esters are potent mesangial cell mitogens, but their effects on HK-catalyzed Glc phosphorylation and metabolism are unknown. When examined in murine mesangial cells, active, but not inactive, phorbol esters increased HK activity in a time- and dose-dependent manner. Maximal induction of HK activity at 12-24 h was accompanied by parallel increases in both Glc utilization and lactate production and was blocked by the specific MEK1/2 inhibitor PD-98059 (IC(50) approximately 3 microM). This effect involved early activation of protein kinase C (PKC), MEK1/2, and ERK1/2, and the prolonged time course of subsequent HK induction was attributable, in part, to requirements for ongoing gene transcription and de novo protein synthesis. Mesangial cell HK activity thus exhibits novel regulatory behavior involving both PKC and classic MAPK pathway activation, suggesting specific mechanisms whereby PKC activation may influence Glc metabolism.

Hexokinase Inactivation Induced by Ascorbic Acid/Fe(II) in Rabbit Erythrocytes Is Independent of Glutathione-Reductive Processes and Appears to Be Mediated by Dehydroascorbic Acid

Recent studies performed in our laboratory demonstrated that rabbit red blood cell hexokinase was remarkably inhibited by the cocktail ascorbic acid/Fe(II) (Stocchiet al.,1994,Arch. Biochem. Biophys.311, 160–167) and that the formation of dehydroascorbic acid was a key event in this process (Fioraniet al.,1996,Arch. Biochem. Biophys.334, 357–361). The present study was undertaken to determine the final hexokinase-inactivating species using cell-free extract as a model. Our results demonstrate superimposable kinetics of hexokinase decay promoted by either ascorbic acid/Fe(II) or dehydroascorbic acid in erythrocyte lysates in which the reduced glutathione (GSH) levels were variously manipulated. In particular, neither removal nor addition of this tripeptide was able to significantly alter the rate or extent of hexokinase inhibition. Thus, GSH-reductive processes are dispensable events in the process of hexokinase inhibition promoted by ascorbic acid/Fe(II) in red blood cells. As a consequence, dehydroascorbic acid appears to be the species which directly inhibits hexokinase. This inference is further supported by the observation that addition of dehydroascorbic acid to the purified enzyme leads to a remarkable inhibition in its activity.

Glucose metabolism and insulin release in mouse beta HC9 cells, as model for wild-type pancreatic beta-cells.

Glucose metabolism and its relationship with glucose-induced insulin release were studied in beta HC9 and beta TC3 cells to identify and characterize key factors controlling the intermediary metabolism of glucose and glucose-induced insulin release. The beta HC9 cell line, derived from pancreatic islets with beta-cell hyperplasia, is characterized by a normal concentration-dependency curve for glucose-stimulated insulin release, whereas the beta TC3 cell line, derived from pancreatic beta-cell tumors, shows a marked leftward shift of this curve. Maximum velocity and the Michaelis-Menten constant of glucose uptake in beta HC9 and beta TC3 cells were similar, even though GLUT-2 expression in these two cell lines differed. In both cell lines, the kinetic characteristics of glucose usage, glucose oxidation, and glucose-induced oxygen consumption were similar to those of glucose phosphorylation, indicating that the kinetics of glucose metabolism from the glucose phosphorylation step in the cytosol to the mitochondrial process of oxidative phosphorylation are determined by the glucose-phosphorylating enzyme, that is, by glucokinase in beta HC9 cells and by hexokinase in beta TC3 cells. Thus beta HC9 cells provide an opportunity for the quantitative analysis of glucose metabolism, the associated generation of coupling factors, and other essential beta-cell functions involved in glucose sensing and insulin secretion.

Glucose metabolism in cancer cells: immunogold localization of hexokinase to the outer mitochondrial membrane

Hexokinase (ATP: D-hexose 6-phophotransferase EC 2.7.1.1) is the first enzyme of the glycolytic pathway which commits glucose to catabolism by catalyzing the phosphorylation of glucose with ATP. Previous studies have shown diat hexokinase activity is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. A large fraction (50-80%) of this enzyme activity is bound to the mitochondrial fraction (1,2) where it has preferred access to ATP (3). In contrast,the hexokinase activity of normal tissues is quite low, with one exception being brain which is a glucose-utilizing tissue (4). Biochemical evidence involving rigorous subfractionation studies have revealed striking differences between the subcellular distribution of hexokinase in normal and tumor cells [See review by Aroraet al(4)].In the present report, we have utilized immunogold labeling techniques to evaluate die subcellular localization of hexokinase in highly glycolytic AS-30D hepatoma cells and in the tissue of its origin, i.e., rat liver.

Inactivation of Rabbit Red Blood Cell Hexokinase Activity Promoted in Vitro by an Oxygen-Radical-Generating System

Rabbit red blood cell hexokinase (EC 2.7.1.1) has been shown to be inactivated in vitro by incubating intact erythrocytes in the presence of an oxygen-radical-generating system represented by ascorbate and iron. It was interesting to note that among the glycolytic enzymes, only hexokinase was found to be susceptible to the action of oxygen radicals, suggesting that the loss of activity of this enzyme may be one of the first signals of cellular damage in rabbit red blood cells. This statement is supported by the fact that, under the experimental conditions used, we did not observe any significant plasma membrane lipid peroxidation nor intracellular proteolysis. Furthermore, mature erythrocytes are unable to synthesize hexokinase as well as other proteins de novo; therefore, the inactivation of this enzyme, which is both the first and one of the rate-limiting enzymes of the glycolytic pathway, could play an important role in determining metabolic impairment of red blood cells, with possible physiological implications. We also investigated the effect of various radical scavengers and antioxidants (glucose, vitamin E, dithiothreitol, flavonoids) which are able to influence the inactivation of hexokinase activity to different extents. Finally, under the experimental conditions used (90 min of incubation at 37°C), we did not observe any difference in the hemolysis of rabbit red blood cells incubated in the presence or absence of ascorbate and iron (hemolysis was about 1% after 90 min of incubation), suggesting that the system used was able to furnish information about the cellular damage produced by oxygen radicals without provoking cell lysis.

Reflection confocal imaging of type I and type III isozymes of hexokinase in PC12 cells

Reflection confocal microscopy was used to determine the intracellular distribution of Type I and III isozymes of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in PC12 cells; detection was by staining with diaminobenzidine as a substrate for horseradish peroxidase-conjugated antimouse immunoglobulins bound to isozyme-specific monoclonal antibodies. With both isozymes, detection of the staining pattern was significantly enhanced by reflection confocal imaging compared with viewing with transmitted brightfield optics. For Type I, prominent staining of cytoplasmic organelles having a distribution consistent with that of mitochondria was noted. For Type III, intense staining at the nuclear periphery was observed. A distinct punctate pattern along the nuclear surface implied a nonuniform distribution of the Type III hexokinase and may represent preferential association with nuclear pore structures. A study of technical factors involved in optimizing the reflection image was conducted. We demonstrate that both the choice of objective and the thickness of the mounting medium are critical to successful imaging, and we describe a simple test for assessing the suitability of objectives in any system.

Expression of yeast hexokinase in pancreatic beta cells of transgenic mice reduces blood glucose, enhances insulin secretion, and decreases diabetes.

It has been proposed that endogenous hexokinases of the pancreatic beta cell control the rate of glucose-stimulated insulin secretion and that genetic defects that reduce beta-cell hexokinase activity may lead to diabetes. To test these hypotheses, we have produced transgenic mice that have a 2-fold increase in hexokinase activity specific to the pancreatic beta cell. This increase was sufficient to significantly augment glucose-stimulated insulin secretion of isolated pancreatic islets, increase serum insulin levels in vivo, and lower the blood glucose levels of transgenic mice by 20-50% below control levels. Elevation of hexokinase activity also significantly reduced blood glucose levels of diabetic mice. These results confirm the role of beta-cell hexokinase activity in the regulation of insulin secretion and glucose homeostasis. They also provide strong support for the proposal that reductions in beta-cell hexokinase activity can produce diabetes.

L-Sorbose does not cause hemolysis in dog erythrocytes with inherited high Na, K-atpase activity

1. The hemolytic effect of l-sorbose on canine erythrocytes characterized by inherited high Na, K-ATPase activity and a high potassium concentration (HK RBCs) was compared with that on normal canine erythrocytes (LK RBCs). 2. Dogs having HK RBCs (HK dogs) revealed no clinical and hematological changes after administration of l-sorbose, whereas normal dogs (LK dogs) developed severe hemolytic anemia associated with hemoglobinuria and marked decreases of erythrocyte ATP concentrations. 3. In vitro, l-sorbose induced hemolysis in LK RBCs along with the depression of both ATP and lactate formation in these cells, but not in HK RBCs. The inhibition of glycolysis by l-sorbose in LK RBCs, however, was not observed when glucose-6-phosphate was used as a substrate instead of glucose. 4. These results suggest that the disparity of susceptibility to sorbose-induced hemolysis may be due to the difference in erythrocyte metabolism between HK and LK RBCs, especially the high activity of hexokinase in HK cells, which was 2-fold greater than that in LK RBCs.

Barbiturates suppress glucose utilization by inhibition of hexokinase in neuroblastoma cells

Exposure to the pentobarbital potently inhibited the 2-deoxy glucose uptake in cultured neuroblastoma cells. The inhibition was assumed to be due to saturation of the uptake in the early stage where the incorporation was linear in the nontreated cells. On the contrary, the incorporation of 3-O-methyl glucose, another glucose analog which is not phosphorylated by hexokinase, was not altered by the treatment with pentobarbital. These results suggest that the suppression of hexokinase is involved in the above-mentioned effect of pentobarbital.

Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support.

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.

Effect of B- and T-cell mitogens on the maximum activities of hexokinase, lactate dehydrogenase, citrate synthase and glutaminase in bone marrow cells and thymocytes of the rat during four hours of culture

1. 1. Cells from the bone marrow and cells from the thymus of the rat were incubated in the presence of glucose and glutamine and phytohaemagglutinin, concanavalin-A or lipopolysaccharide. Cells were harvested at times up to 4 hr, extracted and maximum activities of hexokinase, lactate dehydrogenase, citrate synthase or glutaminase measured. 2. 2. In bone marrow cells, there were little changes in enzyme activities except for an increase in the activity of citrate synthase which was prevented by concanavalin-A. This mitogen also caused a decrease in the activity of hexokinase. 3. 3. In contrast, in thymocytes, the activities of hexokinase and glutaminase were decreased in the control condition but addition of lipopolysaccharide, a B-cell mitogen prevented these decreases in activity and concanavalin-A maintained the activity of glutaminase. Concanavalin-A caused a decrease in hexokinase activity but a marked increase in that of glutaminase. 4. 4. It is suggested that changes in the maximum activities of hexokinase and glutaminase over this 4 hr period may represent the effect of removal of thymus-produced growth factors, whose effects can be replaced, at least in part, by two mitogens.

Synthesis and targeting of hexokinase to mitochondria in hepatoma cells

The synthesis and turnover of hexokinase has been measured in Zajdela hepatoma ascites cells labeled for short periods with [ 35S]methionine. Digitonin fractionation of the labeled cells into a soluble and a membrane fraction showed that only a small part of the newly labeled hexokinase is transferred to mitochondrial binding sites. The soluble enzyme disappears, however, with a half-life of less than 2 h. Glucose had no effect on the stability of the soluble enzyme in intact cells. Our experiments suggest that Zajdela cell hexokinase is synthesized in excess of binding sites and that the excess enzyme is not Stable.

Physiology of the mesangial cell.

Ce travail resume et discute de nombreuses investigations realisees dans le but d'etudier la structure, la physiologie et le role des cellules mesengiales