Physico-chemical and kinetic properties of hexokinase isoenzymes from human normal and tumor tissues]

Individual hexokinase isoenzymes (isoHK) are isolated from normal and malignant human stomach mucosa. IsoHK from tumour tissue are found to have KM for glucose 10 times as low as isoHK from normal tissue. Molecular weights of individual isoHK from normal and tumour tissues are similar (at the range of 112,000-125,000). The treatment of protein preparation with 8M urea in the presence of 1% sodium docecyl sulphate resulted in the appearance of a single band with molecular weight of 58,000-60,000 for all the isoHK under polyacrylamide gel electrophoresis. Intensive bands with molecular weight of 60,000 and 96,000 and a number of minor bands were observed under polyacrylamide gel disc elect-ophoresis in the absence of urea. 2-Mercaptoethanol did not affect the results of disc electrophoresis. It is concluded that the molecule of human hexokinase consists of two subunits with molecular weight of 60,000.     

Hexokinase isoenzymes in diabetes

Hexokinase is present in the tissues in four isoenzymic forms. Cerebral tissue contains predominantly Type I hexokinase which is believed to be insulin-insensitive. In cerebral tissue about 60 to 70% of the hexokinase is bound to the particulate fraction. The changes in the distribution of hexokinase Type I and Type II together with the bound and free hexokinase have been studied in control, diabetic and diabetic animals treated with insulin. The results indicate that the presence of insulin is essential for the normal binding of the hexokinase to the particulate fraction. In heart tissue, Type II hexokinase bound to the pellet shows a significant decrease in diabetes, which is reversed on insulin administration.     

Hexokinase isozymes in human neoplastic and fetal tissues: the existence of hexokinase II in malignant tumors and in placenta.

The distribution of electrophoretic fractions of hexokinase (HK E.C. 2.7.1.1) in extracts of 53 human primary tumors, homologous normal tissues and fetal organs was investigated. It was found that the fraction HK II is present in every malignant tumor and in placenta tissue.     

Determination of the Deoxyglucose and Glucose Phosphorylation Ratio and the Lumped Constant in Rat Brain and a Transplantable Rat Glioma

Mitochondrially bound hexokinase (ATP-D-hexose-6-phosphotransferase; EC 2.7.1.1) was dissociatively extracted from normal rat brains and intracerebral and subcutaneous implants of the 36B-10 glioma. At least 70% of the total hexokinase enzyme activity in normal and glioma tissue was associated with the mitochondrial fraction. Purification of the crude tissue extracts by ion-exchange and affinity chromatography followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a successive purification of the enzyme to homogeneity with a molecular size of 98 kilodaltons. Enzyme kinetics with glucose or 2-deoxyglucose (2-DG) as the substrate were measured spectrophotometrically by coupling the appropriate reactions to either NADPH or NAD+ formation. The Km of hexokinase with glucose as the substrate in the intracerebral glioma (0.138 mM) and subcutaneous glioma (0.183 mM) tissues was 2.1-2.7-fold higher than that observed in normal brain tissue (0.067 mM) (p less than 0.001). No significant differences were observed in the Km for hexokinase with 2-DG as the substrate in the glioma and normal brain tissue. The phosphorylation ratio for normal brain was 0.320 and was increased in the intracerebral glioma to 0.694 and in the subcutaneous glioma to 0.519. The ratios of deoxyglucose and glucose volumes of distribution in normal brain and intracerebral glioma tissues were 1.70 and 1.85, respectively. The lumped constants calculated directly from the phosphorylation ratios and the volumes of distribution of deoxyglucose and glucose were 0.517 in normal brain and 1.168 in intracerebral glioma. Our results indicate the lumped constant is increased 2.26-fold in intracerebral glioma compared with normal brain.     

Topography of genetic loci in tissue samples: towards new diagnostic tool using interphase FISH and high-resolution image analysis techniques.

Using single and dual colour fluorescence in situ hybridisation (FISH) combined with image analysis techniques the topographic characteristics of genes and centromeres in nuclei of human colon tissue cells were investigated. The distributions of distances from the centre-of-nucleus to genes (centromeres) and from genes to genes (centromeres to centromeres) were studied in normal colon tissue cells found in the neighbourhood of tumour samples, in tumour cell line HT-29 and in promyelocytic HL-60 cell line for comparison. Our results show that the topography of genetic loci determined in 3D-fixed cell tissue corresponds to that obtained for 2D-fixed cells separated from the tissue. The distributions of the centre-of-nucleus to gene (centromere) distances and gene to gene (centromere to centromere) distances and their average values are different for various genetic loci but similar for normal colon tissue cells, HT-29 colon tumour cell line and HL-60 promyelocytic cell line. It suggests that the arrangement of genetic loci in cell nucleus is conserved in different types of human cells. The investigations of trisomic loci in HT-29 cells revealed that the location of the third genetic element is not different from the location of two homologues in diploid cells. We have shown that the topographic parameters used in our experiments for different genetic elements are not tissue or tumour specific. In order to validate high-resolution cytometry for oncology, further investigations should include more precise parameters reflecting the state of chromatin in the neighbourhood of critical oncogenes or tumour suppresser genes.     

Role of hexokinase in the regulation of erythrocyte hexose monophosphate pathway under oxidative stress

Human erythrocytes overloaded with homogeneous human hexokinase (up to 15-times the activity of normal RBC) show almost unmodified rates of glucose metabolized in the HMP, however hexokinase-loaded RBC are able to metabolize 1.5 fold more glucose than controls through the HMP when an oxidizing agent like methylene blue (5 to 100 microM) is present. Similarly, RBC loaded with inactivating anti-hexokinase IgG (12 +/- 3% residual hexokinase activity) show HMP rates unchanged under resting conditions, but only 12% of the HMP rate found in normal controls under oxidative stress. These data provide clear evidence that the HMP rate under conditions of oxidative stress is controlled by hexokinase activity and suggest that RBC from patients with hexokinase deficiency are not able to increase the HMP rate under oxidative stress like erythrocytes from individuals with G6PD deficiency.     

Human hexokinase type I microheterogeneity is due to different amino-terminal sequences

Human placenta hexokinase type I was previously shown to be present in two subtypes with similar isoelectric points but different molecular masses of 112 and 103 kDa, respectively. In order to exclude that these subtypes arise by artifact(s) occurring during the protein purification, we have developed a single-step immunoaffinity chromatography for the isolation of microgram quantities of hexokinase. The results obtained confirmed the presence of both hexokinase subtypes in human placenta. By Northern blot analysis a single mRNA species that hybridized with a hexokinase-I cDNA was found to be present in human placenta. Furthermore, in vitro translation of placenta mRNA in a rabbit reticulocyte lysate followed by hexokinase immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that only one hexokinase with apparent molecular mass of about 112 kDa is expressed in this tissue and suggests a post-translational modification as a probable cause of hexokinase I microheterogeneity. To further investigate this point we have purified the high and low Mr hexokinase and determined their NH2-terminal sequences. The results obtained show that when compared with the amino acid sequence deduced from a cDNA the high Mr hexokinase starts at amino acid 11 while the low Mr hexokinase starts at amino acid 103. Since the first 10 amino acids are involved in the binding of hexokinase to mitochondrial porin these data provide an explanation both for the inability of these hexokinases to bind to mitochondria and for their differences in Mr.     

Levels, phosphorylation status and cellular localization of translational factor eIF2 in gastrointestinal carcinomas

The level of expression and the phosphorylation status of the subunit of initiation factor 2 (eIF2) protein have been determined by comparing samples from human stomach, colon and sigma-rectum carcinomas with normal tissue from the same patients. The unphosphorylated and phosphorylated levels of cytoplasmic eIF2, as well as the percentage of phosphorylated factor over the total, were significantly higher in stomach, colon and sigma-rectum tumours compared with normal tissue. The expression of this factor was also studied by using immunocytochemical methods, where redistribution towards the nucleus in tumour cells as compared with normal tissue was observed. Our results support a likely implication of eIF2 in gastrointestinal cancer.     

Effect of alloxan-diabetes on multiple forms of hexokinase in adipose tissue and lung

Comparison has been made of the effect of alloxan-diabetes on the multiple forms of hexokinase (EC 2.7.1.1) in adipose tissue and lung. Types I and II hexokinase were distinguished in adipose tissue by their different stabilities to heat treatment, which made it possible to determine the activity of each form spectrophotometrically; additional confirmatory evidence was obtained from starch-gel electrophoresis. Type II hexokinase was markedly depressed in adipose tissue from alloxan-diabetic rats. Lung contained types I, II and III hexokinase, type I predominating. There was no significant change in the pattern of these multiple forms of hexokinase in lung from alloxan-diabetic rats. These results are discussed in relation to current ideas that the insulin-sensitivity of a tissue may be correlated with the content of type II hexokinase.     

Hexokinase receptors: Preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude mitochondrial fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude mitochondrial fraction, a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to non-mitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.     

Effect of streptozotocin-induced diabetes and insulin treatment on the synthesis of hexokinase II in the skeletal muscle of the rat

The relative rate of synthesis of hexokinase II in the skeletal muscle of the normal, streptozotocin-diabetic, and diabetic insulin-treated rat was determined by the rate of incorporation of [3H]leucine into hexokinase II and the total cytosolic proteins to determine if the rate of hexokinase II synthesis was altered relative to that of the average protein. This relative rate of synthesis of hexokinase II is approximately 1.9 times higher in the normal than in the diabetic rat. The administration of insulin to the diabetic animal increases the rate of hexokinase synthesis to approximately normal levels. An enzyme-linked immunosorbent assay procedure was developed to determine the amount of hexokinase II protein in the skeletal muscle extracts, and immunoprecipitation was utilized to determine the hexokinase II activity. The specific activity of hexokinase II was determined from these analyses. The specific activity of hexokinase II was the same in the skeletal muscle extracts from normal, streptozotocin-diabetic, and diabetic insulin-treated rats. These results suggest that the decrease in muscle hexokinase activity is not caused by the loss of an activator of the enzyme nor by the increased formation of a hexokinase inhibitor in streptozotocin-induced diabetes; rather the decrease in hexokinase II activity reported in diabetic rats relative to normal animals is a result of decreased synthesis coupled to increased degradation in the diabetic relative to the normal animal.     

Autoradiographic study of human scirrhous gastric carcinoma during cultivation in diffusion chambers]

The growth of the epithelial and connective tissue cells was noted in cultivation of human schirrous carcinoma of the stomach in diffuse chambers. In difference to the initial tissue of the tumour in vitro in which no incorporation of thymidine-H3 into the connective tissue cells was noted, these cells proved to be labeled under conditions of growth in the chambers. One hour after the administration of thymidine-H3 the percentage of cells with labeled nuclei averaged 25.1% this considerably exceeding the value of the label index determined under conditions of incubation in vitro of the initial tumour tissue (6.6%). Under conditions of cultivation there was revealed a rapidly proliferating subpopulation of cells with the mitotic cycle duration of 14.8 hours.     

Adrenal androgen concentrations in breast tumours and in normal breast tissue. The relationship to oestradiol metabolism

Concentrations of ADIOL, DHA and DHAS were measured in human breast tumours and normal tissue from the same breast and related to 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity in these tissues. ADIOL and DHA were significantly higher in tumour tissue compared to normal tissue from the same breast (paired t-test: P less than 0.05 and P less than 0.01 respectively) whereas the difference between concentrations of DHAS in normal tissue and tumour tissue was not significant. There was a positive correlation between ADIOL and DHA in both tissues (P less than 0.001) but for DHAS the relationship was only significant in normal tissue (ADIOL:DHAS, P less than 0.001; DHA:DHAS, P less than 0.002). An increase in 17 beta-HSD activity was associated with an increase in DHAS concentrations in both normal and tumour tissue (P less than 0.01 and P less than 0.001 respectively) and with an increase in DHA concentrations in normal tissue (P less than 0.05). These results might be explained by an impairment in the balance between sulphatase and sulphotransferase activity in breast tumours.     

Endocrine cells in gastric carcinoma and adjacent mucosa. An immunohistochemical and ultrastructural study

Endocrine cells are often found in human gastric carcinoma and may be recognized by the immunoreactivity of their chromogranin A, peptides and biogenic amines content. Anti-chromogranin A was used to investigate the morphology of endocrine cells using light and electron microscope immunohistochemical techniques. The hormone content of endocrine cells was examined in both tumour tissue and tumour-adjacent mucosa. It was found that the endocrine cells in tumour tissue were malignant, often had amphocrine differentiation and did not resemble a normal cell type. The hormone content of endocrine cells in tumour tissue seldom corresponded to the hormonal content of endocrine cells in tumour-adjacent mucosa. In intestinal-type carcinoma and in some parts of diffuse-type gastric carcinomas, endocrine cell hyperplasia and an alteration of the differentiation in the tumour-adjacent mucosa were discovered. The distribution of endocrine cells in the tumour tissue was different in both types of gastric carcinoma. The results reported here suggest that endocrine cell differentiation of malignant endocrine cells in human gastric carcinoma develops in a different way from that of endocrine cells in tumour-adjacent mucosa, and as a result, diverse hormonal products may appear in tumour tissue.     

Glucose catabolism in brain. Intracellular localization of hexokinase

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.     

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour cells.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

Simultaneous preparation from human placenta of several enzymes of glucose metabolism

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.     

Fast quantification of immunohistochemistry tissue microarrays in lung carcinoma

Tissue microarrays (TMAs) are an effective tool for high-throughput molecular analysis of tissues to help identify new diagnostic and prognostic markers and targets in human cancers. We have developed a fully automated method for rapid, continuous and quantitative analysis of TMAs based on immunohistochemistry. The method deals with complex and varying tissue architectures, segments tumour cells from normal cells, conducts cell compartmentalisation, identifies nuclei and cytoplasm and produces three different continuous measurements of marker expression levels within tumour cell nuclei, tumour cell cytoplasm and total tumour cell protein expression. We have demonstrated this method using three independent protein markers (BAK, BAX and a novel biomarker, named KS) over 7 TMAs, involving 2 BAK stained TMAs with 229 tumour tissue cores, 2 BAX stained TMAs with 229 tumour tissue cores and 3 KS stained TMAs with 373 tumour cores of lung carcinomas. We validated the automated method, showing that the automated scoring is significantly correlated with the pathologist-based scoring.     

Glucose metabolism in the mucosa of the small intestine. The effect of glucose on hexokinase activity

1. The effect of three dietary components on hexokinase activity in the mucosa of rat small intestine was studied in vivo. Glucose, amino acids or an emulsion of monoglyceride with long-chain fatty acids were given by stomach tube to previously starved rats, and hexokinase activity was determined in the particle-free supernatant of mucosal homogenates. The formation of lactate from glucose and glucose 6-phosphate respectively was also measured. 2. When the three dietary components were given in isocaloric amounts, only glucose brought about an increase in hexokinase activity. 3. Intravenous injection of a similar amount of glucose to that given orally did not alter hexokinase activity. 4. An increase in the hexokinase activity of the particle-free supernatant prepared from mucosal homogenates was also observed after sacs of the small intestine of starved rats had been incubated in vitro in a medium containing glucose. Hexokinase activity increased to the values observed in corresponding preparations from fed rats, and this increase was strictly glucose-dependent.