Elevation of liver-galactosylhydroxylysyl glucosyltransferase activity in human primary hepatocellular carcinoma

The activity of L-GGT (EC 2.4.1.66), an enzyme catalyzing the intracellular biosynthesis of collagen, was determined in human primary hepatic cancer, acute viral hepatitis and cirrhotic liver tissues and compared to the mean level of enzyme activity in normal human liver tissues. The mean levels of L-GGT activity in primary hepatocellular carcinoma (PHC), acute viral hepatitis and cirrhotic tissues were 7.78, 2.69 and 2.16 times the mean level of enzyme activity in normal human liver tissues. The mean level of L-GGT activity in PHC was 3.61 times the mean level of L-GGT activity in cirrhosis and 2.90 times the mean value of liver enzyme activity in acute viral hepatitis. The findings in this study provide a basis for the highly elevated serum values of this intracellular enzyme in patients with primary hepatic cancer and the data indicate that L-GGT activity may be increased in primary liver cancer to compensate for an increased rate of collagen synthesis.     

High concentration of neutral endopeptidase (enkephalinase E.C. 3.4.24.11) in a malignant tumor: rat hepatoma 3924A

The activity of the membrane-bound neutral endopeptidase 24.11 was low in the normal liver (21 +/- 3 pmol/h/mg protein, mean +/- SE) but it increased 56-fold in rapidly-growing rat hepatoma 3924A. The identity of the enzyme in the tumor was established by immunoprecipitation and by using a specific inhibitor of neutral endopeptidase. The endopeptidase concentration in the differentiating and regenerating liver was lower than in normal tissue, 39 and 8% of the corresponding control. The activity of a plasma membrane marker enzyme carboxypeptidase M in the normal liver was 1.0 +/- 0.2 nmol/h/mg protein, it increased about 2-fold in the rapidly-growing hepatoma and in the differentiating liver, but was unchanged in regenerating liver. The function of the strikingly increased neutral endopeptidase activity in the rapidly growing hepatoma may relate to activation of autocrine or exocellular growth factors or to inactivation of cell proliferation-inhibitory factors. Such a biochemical change should confer selective advantages to the cancer cells.     

Mouse liver cytoplasmic malate dehydrogenase: rapid isolation, characterization and immunochemical comparison with hepatoma enzyme.

Cytoplasmic NAD-dependent malate dehydrogenase is decreased in activity in three transplantable mouse hepatomas compared to the activity of this enzyme in liver tissue. This enzyme is composed of several molecular forms of similar size which differ slightly in charge; the total activity and the discernible number of forms of the enzyme are decreased in both hepatoma and fetal liver. Mixing experiments suggest the absence of a significant quantity of unbound inhibitor of enzyme activity in the tumor or an activator in the liver. The liver cytoplasmic enzyme was purified to homogeneity by a relatively rapid method using Blue Sepharose affinity chromatography, which results in a good yield and high specific activity of the enzyme. Cytoplasmic and mitochondrial enzymes bind and elute differentially from this affinity resin. Molecular weight, kinetic constants and amino acid composition of the cytoplasmic enzyme were determined. Monospecific antiserum to the cytoplasmic enzyme has been produced in a goat and used to demonstrate a lack of immunological cross-reactivity between the mitochondrial and cytoplasmic enzyme. The tumor and liver cytoplasmic enzymes possess similar, if not identical, immunological determinants. Immunotitration experiments have been used to demonstrate that liver and hepatoma enzyme are identical in specific activity. Thus, the reduction in level of cytoplasmic enzyme in hepatoma is due to a decrease in the number of molecules per tissue mass.     

The activity of polynucleotide phosphorylase in polyribosomes of regenerating liver of adult rats, liver of newborn rats and in some reinoculated tumours]

Specific activity and level of polynucleotide phosphorylase (PNPase) in polyribosomes of regenerating liver of adult rats, liver of newborn rats and in malignant tumours of rat (sarcoma M-1 and hepatoma 27) were studied. 24 hours after partial hepatectomy the specific activity and level of PNPase in regenerating liver decreased 3--4 times in the fraction of polyribosomes, bound to the endoplasmic reticulum membranes, and remained at a constantly low level in the fraction of free polyribosomes. The PNPase activity also showed a sharp decrease in the fraction of membrane-bound polyribosomes from newborn rats liver and could not be detected either in free or in bound polyribosomes from sarcoma M-1 or hepatoma 27. The PNPase activity in the fraction of bound polyribosomes increased with a decrease in the rate of liver growth (regenerating liver and newborn rats liver), and reached the level normal for adult animals. Possible mechanisms of regulation of the PNPase activity in animal tissue were studied. It was found that a 2-fold administration of cyclic 3,5'-AMP to intact animals (5 mg per 100 g of body weight) with an interval of 8 hours, corresponding to the interval between two peaks of the increase in cyclic 3,5'-AMP concentration following partial hepatectomy, diminished the PNPase specific activity in polyribosomes by 30%. A factor, presumably of protein origin, which induced a release of PNPase from polyribosomes of normal rat liver but did not affect the activity of the liberated enzyme, was detected in the cell sap of sarcoma M-1 and hepatoma 27.     

gamma-Glutamyltranspeptidase and dipeptidyl peptidase IV activity in the serum of normal and hepatoma-bearing chickens and in the plasma membranes from liver and hepatoma Mc-29.

Investigations on the activity of gamma-glutamyltranspeptidase (GGT) and dipeptidyl peptidase IV (DPP IV) in the serum of healthy chickens and those bearing hepatoma Mc-29, and in liver and hepatoma plasma membranes were carried out. There was no difference in the serum enzyme activities of control and tumor-bearing chickens but the activity of GGT was twice higher and that of DPP IV 20 times lower in hepatoma plasma membranes than in chicken liver plasma membranes. Using thin-layer analytical isoelectric focusing in agarose gels it was established that the pI range of GGT from host serum and hepatoma plasma membranes was shifted to more acidic values. This could be interpreted as a specific feature for this enzyme considered as a tumor marker.     

Molecular cloning and expression of Galbeta1,3GalNAc alpha2, 3-sialyltransferase from human fetal liver.

Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding Galbeta1, 3GalNAc alpha2,3-sialyltransferase (SIATFL) has been isolated from human fetal liver. Expression analysis of the gene has been performed with various carcinoma cell lines, fetal tissues, fetal and adult liver and both hepatoma and the surrounding tissue from the same liver. The SIATFL gene was expressed poorly in fetal liver and in adult liver, slightly in hepatoma and highly in the surrounding tissue of hepatoma. The cDNA encoding the putative active domain was expressed in COS-1, Escherichia coli, and Pichia pastoris. The recombinant protein expressed in COS-1 could catalyse the transfer of NeuAc from CMP-NeuAc to asialo-fetuin. No enzyme activity was detected with a 32-kDa protein in E. coli and both 32-kDa and 41-kDa proteins in P. pastoris. These results suggested that correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity.     

Isolation of gamma-glutamyl transpeptidase from human primary hepatoma and comparison of its kinetic and catalytic properties with the enzyme from normal adult and fetal liver

gamma-Glutamyl transpeptidase (gamma-GT) from human primary hepatoma was solubilised and purified 290-fold with 25% recovery. The kinetic and catalytic properties were compared with those purified from human fetal and normal adult liver. Hepatoma gamma-GT did not differ from the fetal and adult liver gamma-GT in its pH optima for transpeptidation and auto-transfer reaction, heat stability, Km for the two substrates and inhibition by L-serine + borate. Enzyme from the three sources behaved in a similar manner towards various cations, sulphhydryl reagents, amino acid dipeptides. Adult liver enzyme showed a 4 time higher Ki value for anthglutin than hepatoma and fetal liver. The hepatoma gamma-GT could not be differentiated from that of adult and fetal liver by concanavalin-A Sepharose 4B column chromatography. The tissue concentration of gamma-GT was 3 to 13 times higher in hepatoma and fetal liver than in adult liver.     

Increased concentration of CTP synthetase in hepatoma 3924A: immunological evidence

CTP synthetase (UTP:L-glutamine ligase, EC 6.3.4.2) was purified 370-fold from rapidly growing rat hepatoma 3924A. A major band was demonstrated by acrylamide gel electrophoresis which corresponded to this enzymic activity. It was estimated that the enzyme was 90% pure. Antibodies were produced in rabbit using this purified hepatoma enzyme. The specificity of the anti-serum was proved by the absence of the reaction between control serum and CTP synthetase. The amount of anti-serum required to inactivate completely the cytosolic CTP synthetase of hepatoma 3924A was 11-fold of that required for normal liver which is in good agreement with the 11-fold increase in CTP synthetase activity in this hepatoma. These results demonstrate that the liver and hepatoma 3924A CTP synthetases were immunologically similar or identical and that the markedly increased enzymic activity in hepatoma 3924A reflected an increase in the enzyme protein amount. These studies provide further evidence that in the neoplastic transformation a reprogramming of gene expression takes place which is manifested in the emergence of increased concentrations of CTP synthetase which should provide selective advantages to cancer cells by increasing the capacity for this rate-limiting step in $\text{de novo}$ CTP biosynthesis.     

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: II. Measurement of enzyme activities utilizing microsomal glycoproteins as exogenous acceptors

Microsomal sialyltransferase was assayed in chicken liver and hepatoma Mc-29 utilizing liver and hepatoma microsomal glycoprotein fractions, treated with Triton X-100, as exogenous acceptors. In a homologous assay system containing enzyme and acceptor from one and the same tissue no quantitative dependence of enzyme activity was revealed with increasing amount of the acceptor. In mixed experiments in which liver enzyme activity was tested towards hepatoma acceptor glycoproteins, a gradual drop in sialyltransferase activity occurred with increasing quantities of the acceptor. This effect seems to be a consequence of the presence of some inhibitor in the microsomal fractions from the hepatoma cells.     

大鼠肝癌模型中Piwil4、Mael和Ddx4基因mRNA和蛋白表达水平及其与肿瘤检测的关系

PIWI和piRNA的表达水平与肿瘤类型密切相关。Piwil2 mRNA在肝癌中的表达量比肿瘤周围的肝脏组织的表达量高。PIWI/piRNA通路基因Piwil4、Mael和Ddx4为候选癌基因,在多种癌组织中表达,而在肝癌组织中的研究尚无报道。为探讨Piwil4、Mael和Ddx4基因在肝癌组织中表达水平变化的影响,建立了大鼠肝癌模型,并提取血清用ELISA方法检测肿瘤标记物,采用实时荧光定量PCR、蛋白质免疫印迹方法和免疫组织化学方法检测正常肝组织与模型组肝组织中Piwil4、Mael和Ddx4基因mRNA转录和蛋白表达水平。结果表明,大鼠肝癌动物模型血清中肿瘤标记物含量明显升高。Piwil4、Mael和Ddx4基因mRNA及蛋白在肝癌模型组织中高表达。研究表明,肝癌模型组织中Piwil4、Mael和Ddx4基因表达水平有望作为肝癌检测的一种分子标志物。     

Sodium selenite modulates tumour marker indices in N-nitrosodiethylamine-initiated and phenobarbital-promoted rat liver carcinogenesis

The effect of sodium selenite (Se) was investigated against two-stage rat liver carcinogenesis initiated by a single intraperitoneal injection of N-nitrosodiethylamine (DEN, 200 mg kg(-1) i.p.) followed by promotion with phenobarbital (PB, 0.05%) in a basal diet. Se (4 p.p.m.) was administered per os daily throughout the entire experiment, before the initiation, or during the promotion stage. The plasma, liver (hepatoma and surrounding tissue) and kidney tissue were investigated biochemically for lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and 5'-nucleotidase. These enzyme activities were increased (p < 0.001) in plasma of hepatoma-bearing rats compared with normal control rats. The elevation of these enzyme activities in plasma was indicative of the persistent deteriorating effect of DEN in cancer-bearing animals. Aminotransferase levels were decreased in hepatoma and surrounding liver tissue, whereas lactate dehydrogenase, alkaline phosphatase and 5'-nucleotidase were increased in the cancer condition. These enzyme activities were reversed to near normal control values in animals treated with Se. It is apparent that the beneficial effect of Se is primarily exerted on the initiation phase and secondarily during the promotion stage of DEN-initiated rat liver carcinogenesis. The analysis of marker enzyme activities taken together with our previous findings clearly indicates the antitumour efficacy of sodium selenite on DEN-induced hepatoma animals.     

Nuclear poly(A) polymerase from rat liver and a hepatoma. Comparison of properties, molecular weights and amino acid compositions.

Poly(A) polymerase was extracted from isolated nuclei of rat liver and a rapidly growing solid tumor (Morris hepatoma 3924A). The enzyme from each tissue was purified by successive chromatography on DEAE-Sephadex, phosphoecllulose, hydroxyapatite and QAE-Sephadex. Purified enzyme from both liver and tumor was essentially homogeneous as judged by polyacrylamide gel electrophoresis. Under nondenaturing conditions, enzyme activity corresponded to visible protein and, upon denaturation, a single polypeptide was detected. The enzymes had absolute requirements for Mn2+ as the divalent ion, ATP as the substrate and an oligonucleotide or polynucleotide as the primer. Both enzymes were inhibited by sodium pyrophosphate, N-ethylmaleimide, Rose Bengal, cordycepin 5'-triphosphate and several rifamycin derivatives. The reactions were unaffected by potassium phosphate, alpha-amanitin and pancreatic ribonuclease. However, the liver and hepatoma enzymes differed from each other with respect to apparent Km, primer saturation levels and sensitivity to pH changes. The most striking differences between the enzymes were in their calculated molecular weights (liver, 48000; hepatoma, 60000) and amino acid compositions. Finally, the level of the hepatoma enzyme relative to that of the liver enzyme was at least 1.5-fold higher when expressed per mg DNA.     

Glutathione and GSH-dependent enzymes in patients with liver cirrhosis and hepatocellular carcinoma

We investigated glutathione level, activities of selenium independent GSH peroxidase, selenium dependent GSH peroxidase, GSH S-transferase, GSH reductase and the rate of lipid peroxidation expressed as the level of malondialdehyde in liver tissues obtained from patients diagnosed with cirrhosis or hepatocellular carcinoma. GSH level was found to be lower in malignant tissues compared to adjacent normal tissues and it was higher in cancer than in cirrhotic tissue. Non-Se-GSH-Px activity was lower in cancer tissue compared with adjacent normal liver or cirrhotic tissue, while Se-GSH-Px activity in cancer was found to be similar to its activity in cirrhotic tissue and lower compared to control tissue. An increase in GST activity was observed in cirrhotic tissue compared with cancer tissue, whereas the GST activity in cancer was lower than in adjacent normal tissue. The activity of GSH-R was similar in cirrhotic and cancer tissues, but higher in cancer tissue compared to control liver tissue. An increased level of MDA was found in cancer tissue in comparison with control tissue, besides its level was higher in cancer tissue than in cirrhotic tissue. Our results show that the antioxidant system of cirrhosis and hepatocellular carcinoma is severely impaired. This is associated with changes of glutathione level and activities of GSH-dependent enzymes in liver tissue. GSH and enzymes cooperating with it are important factors in the process of liver diseases development.     

Increased plasma α (1 → 3)-l-fucosyltransferase activities in patients with hepatocellular carcinoma

Alpha(1 3)-l-fucosyltransferase (1,3FT) activity was determined in plasma of patients with chronic liver diseases, namely, chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The plasma 1,3FT activity was significantly higher (p<0.01) in chronic liver diseases than that in normal controls. The enzyme activity in plasma of patients with HCC was also significantly higher than that in LC (p<0.05) or that in CH (p<0.01). However, no significant difference was observed in the enzyme activity between LC and CH. Plasma 1,3FT activity in patients with HCC was not significantly changed before and after transcatheter arterial embolization. In addition, the enzyme activity in the homogenate of the cirrhotic liver tissue was higher than that in the preparation of the hepatoma tissue in the same patient. These results suggest that the increased plasma 1,3FT activity in patients with HCC reflects mainly the enzyme activity of cirrhotic liver tissue, not that of hepatoma tissue. The significance of the elevated levels of plasma 1,3FT and its decreased hepatoma tissue activity in patients with HCC, compared with that in LC, remains to be clarified.     

Variations in some molecular events during the early phases of the Reuber H35 cell cycle. IV-regulation of tyrosine aminotransferase

Tyrosine aminotransferase activity increased during conversion of serum depleted quiescent Reuber H35 rat hepatoma cells into the proliferative state. Increased activity coincides with the actual increase of cells into S phase. The rate of tyrosine aminotransferase synthesis along the cell cycle was studied. The rate of enzyme synthesis fluctuated through the cell cycle but could not explain the increase of specific activity. Apparently enzyme activity is predominantly regulated by a post-translational event. Intracellular levels of cyclic AMP and cyclic GMP were measured at various times of G1 and S phases. In the early part of the cell cycle tyrosine aminotransferase decreased while intracellular levels of cyclic AMP increased. At later stages cyclic AMP rises concurrently with increased rates of enzyme synthesis. Induction of tyrosine aminotransferase by N6,O2'-dibutyryladenosine 3', 5'-monophosphate (Bt2cAMP) was studied. Inducibility by Bt2cAMP fluctuated through the cell cycle. Alternation of positive and negative control of tyrosine aminotransferase synthesis was observed. In early serum induced cells, Bt2cAMP increased enzyme activity without any increased rate of enzyme synthesis, on the contrary, a decreased rate of synthesis was observed. The data support the view that alternation of positive and negative control of tyrosine aminotransferase synthesis and temporary post-translational control of enzyme activity determine the enzyme level during the transition of quiescent hepatoma cells into proliferation.     

Increased content of natural ATPase inhibitor in tumor mitochondria

The ATPase activity of Zajdela hepatoma and Yoshida sarcoma submitochondrial particles was several times lower than the enzyme activity in rat heart and rat liver submitochondrial particles. The content of F1-ATPase in the tumor mitochondria was found not to be very different from that in mitochondria of rat liver. Immunochemical determination of the amount of the natural ATPase inhibitor revealed that the tumor mitochondria contain 2-3-times more ATPase inhibitor than control mitochondria. It is concluded that the low ATPase activity of the tumor mitochondria results from the inhibition of the enzyme activity by the natural ATPase inhibitor.     

Characterization of a nonhepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach.

Ethanol oxidation by the soluble fraction of a rat hepatoma was compared to that of the liver. Ethanol oxidation by the hepatoma was NAD⁺-dependent and sensitive to pyrazole, suggesting the presence of alcohol dehydrogenase. At low concentrations of ethanol (10.8 mm) the alcohol dehydrogenase activities of hepatoma and liver supernatant fractions were comparable. When the concentration of ethanol was raised to 108 mm, the activity of the liver enzyme decreased, whereas the activity in hepatoma supernatant fractions was strikingly elevated. m-Nitrobenzaldehyde-reducing activity was also conspicuously higher in hepatoma supernatant fractions. By contrast the ability to metabolize steroids and cyclohexanone was less than that in supernatant fractions of the liver.Electrophoresis of the liver supernatant fractions on ionagar at pH 7.0 revealed only one component that oxidized ethanol. On the other hand, hepatoma supernatant fractions contained two components with alcohol dehydrogenase activity; one with the same electrophoretic mobility as the liver enzyme, the other showing a slower rate of migration. The latter component, which is absent in the liver, is referred to as hepatoma alcohol dehydrogenase. By electrophoresis on starch gels at pH 8.5, it could be demonstrated that the liver and hepatoma enzymes moved in opposite directions.The liver and hepatoma enzymes differ in electrophoretic mobility, susceptibility to heat treatment, pH activity optimum and some catalytic properties. The substrate specificity of the hepatoma enzyme is narrower than that of liver alcohol dehydrogenase; cyclohexanone or 3β-hydroxysteroids of A/B cis configuration and the corresponding 3-ketones are not substrates for the hepatoma enzyme. The overall substrate specificity characteristics are, however, similar to those of the liver enzyme in that the effectiveness of substrates increases with an increase in chain length and introduction of unsaturation or an aromatic group. Both liver and hepatoma alcohol dehydrogenase cross-react with antibody to horse liver alcohol dehydrogenase EE. The Michaelis constant for ethanol with the hepatoma enzyme is 223 mm, compared to 0.3 mm for liver alcohol dehydrogenase; at 1.0 m ethanol the hepatoma enzyme is not fully saturated with substrate. The Michaelis constant for 2-hexene-1-ol is 0.3 mm, indicating that the hepatoma enzyme is better suited for dehydrogenation of longer chain alcohols. Stomach alcohol dehydrogenase has kinetic properties comparable to those of the hepatoma enzyme, as well as similar electrophoretic mobility. The hepatoma enzyme can be detected in the serum of rats bearing hepatomas.     

Altered subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase in the Morris 7777 hepatoma.

The subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase is altered in the Morris 7777 hepatoma. Mitochondria in this poorly differentiated tumor are the principal sites of CDP-diacylglycerol synthesis, in contrast to normal rat liver where the endoplasmic reticulum is most active. This enzyme activity was increased 17-fold in the outer mitochondrial membrane, and a 22% increase was noted in the inner mitochondrial membrane of the 7777 hepatoma as compared with the corresponding fractions from normal rat liver. Increased mitochondrial CTP:phosphatidate cytidylyltransferase was present in six other Morris hepatomas, but it was not found in fetal rat liver mitochondria, suggesting that rapid growth alone is not responsible for the difference. Evidence is presented which indicates that mitochondrial lipid degradation is similar in normal liver and the 7777 hepatoma, in vitro. The increased activity of CTP: phosphatidate cytidylytransferase is thought to be responsible in part for the moderately increased diphosphatidylglycerol content of 7777 hepatoma mitochondria.     

Synthesis of lysyl oxidase in experimental hepatic fibrosis.

The synthesis of lysyl oxidase, which initiates the cross-linking of collagen and elastin, was investigated in carbon tetrachloride (CCl4) induced fibrotic liver of rat. Lysyl oxidase activity of the fibrotic liver was 4 times greater than that of normal liver. mRNAs from the livers of normal and CCl4-treated rats were prepared for in vitro protein synthesis and the products were analyzed by immunoprecipitation with a monoclonal antibody against lysyl oxidase. The mRNAs from the fibrotic liver gave more than 3 times higher level of messenger copies for lysyl oxidase than did mRNAs from normal liver. The molecular weight of the nascent lysyl oxidase was 48,000.