鼠肝细胞癌变中DNA甲基化作用的研究

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.     

A comparative study of plasma membrane Mg2+-ATPase activities in normal,regenerating and malignant cells

Plasma membranes have been prepared from rat normal liver cells, regenerating liver cells and Yoshida ascites hepatoma 66 cells after intact cells were first bound to polylysine-coated polyacrylamide beads, and the membrane-associated Mg²⁺-ATPase activity was assayed directly on beads with membrane attached. With plasma membranes from normal liver cells, K_m for ATP and V were found to be higher than those in regenerating liver cells and hepatoma cells. Vanadate caused a different sensitivity of the activity, without an effect in normal liver cells and with an inhibition in regenerating liver cells and hepatoma cells. The activity in normal and regenerating liver cells decreased with increasing temperature above 24–30°C, while the activity in hepatoma cells continued to increase linearly to 37°C. Unlike the enzyme in normal and regenerating liver cells, the hepatoma enzyme was shown to have a higher phase transition temperature and lower activation energies. In all three kinds of cells the activity was increased by the dephosphorylation of plasma membranes and unaffected by the phosphorylation. By means of histochemical Mg²⁺-ATPase staining applied on polyacrylamide gels, at least three major bands which show the enzymic activity were visible in normal and regenerating liver and a single band was detected in hepatoma cells.     

Sialyl transferase activities of rat ascites hepatoma cells and rat liver

Sialyl transferase activities of the homogenate of rat ascites hepatome cells were compared with normal rat liver homogenate. The former had only 20% of the activity of the latter when an exogenous acceptor was added in the reaction mixture.Toward endogenous receptors, the activity of the hepatoma cell homogenate was 50% of that of the normal cell homogenate. A stimulation of the activity toward endogenous acceptors was observed when the homogenate of rat ascites hepatoma cells and that of rat liver were mixed. This stimulatory effect seems to be the consequence of utilization of acceptors from ascites hepatoma cells by the sialyl transferases of the rat liver.     

序列固相凝集素亲和层析法研究大鼠诱发肝癌中碱性磷酸酶ALP的糖链变化

 凝集素是一种能同复合糖中不同糖基序列特异性相结合的糖结合蛋白质。本文在研究二乙基亚硝胺诱发肝癌中碱性磷酸酶(ALP)活力和同工酶的基础上,利用四种固相凝集素柱组成的序列亲和层析,配合外切糖苷酶β-N-酰氨基葡萄糖苷酶,研究了诱发肝癌ALP糖链中“Bisecting”GlcNAc残基的变化。研究结果表明:(1)大鼠诱发肝癌ALP的活性较正常肝脏明显增高,其性质和正常肝脏ALP基本相同,两者均属于组织非特异性ALP,提示大鼠肝癌发生过程中没有同工酶谱的变化。(2)大鼠诱发肝癌ALP糖链的“Bisecting”GlcNAc残基含量较正常鼠肝明显增加。     

Increased content of calmodulin in morris hepatoma 5123 t.c.(h)

The content of calmodulin in the 100,000 × g supernatant and particulate fractions in Morris hepatoma 5123 t.c.(h), assayed by its ability to activate the Ca²⁺-activatable cAMP phosphodiesterase, was significantly higher (about 44%) than that in normal or host liver. Only one peak of calmodulin activity was detected when 100,000 × g supernates (350 mg protein) from sonicated homogenates of normal liver and hepatoma were fractionated on a DEAE cellulose column, eluting at a sodium acetate concentration of 0.65 M. The total calmodulin activity eluted from the hepatoma supernatant was 240% higher than that from nornal liver. The increased content of calmodulin in the hepatoma may contribute to the state of abnormal cell proliferation in this tissue.     

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.     

Induction of gamma-glutamyl transpeptidase by hexachlorocyclohexane

Hexachlorocyclohexane (BHC) induced gamma-Glutamyl transpeptidase in rat liver. The enzyme was partially purified from normal BHC fed and fetal liver and also from hepatoma. The gel filtration and electrophoretic properties of the BHC-induced enzyme was compared against that of the other three. Chemical induced hepatoma showed an additional peak of activity in Sephadex G-200 filtration. The other enzymes could be cleaved by papain to give a fraction which cochromatographed with the additional peak of hepatoma enzyme. BHC-induced enzyme and normal enzyme had similar electrophoretic mobility but differed from that of hepatoma and fetal liver enzyme which showed a slightly slower movement.     

大鼠肝癌细胞中失活的肿瘤相关基因的克隆和筛选

本文描述了运用减式杂交技术筛选只在大鼠正常肝细胞中特异性表达,而在肝癌细胞中失活的肿瘤相关基因的实验。实验中用肝癌细胞mRNA杂交筛减正常肝细胞cDNA得到探针A,用正常肝细胞mRNA杂交筛减肝癌细胞cDNA得到探针B。文章描述了用这两种探针对2500个重组子点杂交筛选得到的与探针A杂交强的cDNA克隆中的6个:TR1-6,并主要分析了其中两个在正常肝细胞中特异性表达的情况。实验中还建立了cDNA库,以用于体外转录mRNA及今后的全长cDNA筛选。     

Differential response of the NADH oxidase of plasma membranes of rat liver and hepatoma and HeLa cells to thiol reagents

NADH oxidase activity of plasma membranes from rat hepatoma and HeLa cells responded to thiol reagents in a manner different from that of plasma membranes of liver. Specifically, the NADH oxidase activity of plasma membranes of HeLa cells was inhibited by submicromolar concentrations of the thiol reagentsp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), or 5,5-dithiobis-(2-nitrophenylbenzoic acid) (DTNB), whereas that of the rat liver plasma membranes was unaffected or stimulated over a wide range of concentrations extending into the millimolar range. With some hepatoma preparations, the NADH oxidase activity of hepatoma plasma membranes was stimulated rather than inhibited by PCMB, whereas with all preparations of hepatoma plasma membranes, NEM and DTNB stimulated the activity. In contrast, NADH oxidase activity of rat liver plasma membrane was largely unaffected over the same range of PCMB concentrations that either stimulated or inhibited with rat hepatoma or HeLa cell plasma membranes. Dithiothreitol and glutathione stimulated NADH oxidase activity of plasma membranes of rat liver and hepatoma but inhibited that of HeLa plasma membranes. The findings demonstrate a difference between the NADH oxidase activity of normal rat liver plasma membranes of rat hepatoma and HeLa cell plasma membranes in addition to the differential response to growth factors and hormones reported previously (Brunoet al., 1992). Results are consistent with a structural modification of a NADH oxidase activity involving thiol groups present in plasma membranes of rat hepatoma and HeLa cells but absent or inaccessible with plasma membranes of rat liver.     

A simple procedure for the purification of calmodulin bound to membranes; calmodulin bound to the particulate fraction of AH-66 hepatoma ascites cells

We have demonstrated the identity of calmodulin tightly bound to the particulate fractions of AH-66 hepatoma cells and normal liver with authentic soluble calmodulin and have compared the particulate calmodulin content of AH-66 cells with that of normal liver. Calmodulin bound to the particulate fractions of the hepatoma and normal liver cells was purified to electrophoretic homogeneity by a simple procedure which involves solubilization of the particulate fractions with LIS, extraction of the solubilized solution with 37.5% phenol, gel filtration, and affinity chromatography on Fluphenazine-Sepharose. There were no detectable differences between the particulate calmodulin thus purified and authentic soluble calmodulin of rat brain. The particulate calmodulin in the hepatoma and normal liver cells was assayed based on its ability to activate calmodulin-deficient phosphodiesterase after partial purification of calmodulin from the particulate fractions by solubilization with LIS and extraction with phenol as described above. In addition, the particulate calmodulin content in the hepatoma and normal liver cells was also measured after solubilization of the particulate fractions with Lubrol PX. The results obtained by these two different procedures indicate that calmodulin content in the particulate fraction as well as in the soluble fraction of the hepatoma is significantly higher than that in the corresponding fractions of normal liver.     

Evidence for inherent differences in the system A carrier from normal and transformed liver tissue. Differential inactivation and substrate protection in membrane vesicles and reconstituted proteoliposomes

Plasma membrane vesicles isolated from intact rat liver (normal hepatocyte) or cultured rat H4 hepatoma cells retain Na+-dependent uptake of 2-aminoisobutyric acid mediated by System A. The carrier was inactivated in normal liver membrane vesicles by either N-ethylmaleimide (NEM) or p-chloromercuribenzene sulfonate (PCMBS). The concentrations required to produce half-maximal inhibition were approximately 370 and 110 microM for NEM and PCMBS, respectively. In contrast, transport of System A in H4 hepatoma membrane vesicles was sensitive to PCMBS (K 1/2 = 180 microM), yet totally unaffected by NEM at concentrations up to 5 mM. Substrate-dependent protection from PCMBS activation was observed for the System A activity in H4 hepatoma membranes, but not in vesicles from normal hepatocytes. Subsequent inactivation of the substrate-protected carrier by sulfhydryl-specific reagents, added following the removal of the protective amino acid, suggests that one or more cysteine residues become less reactive in the presence of System A substrates. Treatment of solubilized membrane proteins with NEM prior to reconstitution into artificial proteoliposomes showed that the selective inactivation by NEM of the carrier in normal liver membranes is not dependent on the lipid environment or on the integrity of the plasma membrane. The results support the hypothesis that there are inherent differences in the System A carriers that are present in normal and transformed liver tissue.     

Oxidative phosphorylation properties of mitochondria isolated from transplanted hepatoma

Mitochondria were isolated from Morris hepatomas with rapid (types 3683, 7777, and 3924A) and intermediate (types 5123D and 7800) growth rates, using proteolytic digestion of minced tumor tissue to release the particles. Mitochondria isolated by the same procedure from rat liver were employed as controls. All the hepatoma mitochondria were capable of coupled respiration with normal phosphorylation yields (ADP/O) and respiratory control ratios ranging from 2 to considerably more than 10. Particles from hepatomas 7777 and 7800 exhibited properties closest to liver mitochondria, while those from hepatomas 3683 and 3924A showed the greatest difference. All the hepatoma mitochondria were capable of oxidizing succinate, 3-hydroxybutyrate and monoamines. However, the oxidation rates of the latter two substrates by mitochondria from hepatomas 3683 and 3924A were only a fraction of the control rates. These differences appeared to be due, at least in part, to the structural instability of the isolated hepatoma mitochondria. In contrast to the reports of others, all hepatoma mitochondria exhibited considerable stimulation of ATPase activity by uncouplers. Maximal stimulation of ATPase activity by representatives of three classes of uncouplers was in all instances comparable to the values obtained for rat liver mitochondria.     

Oxidative phosphorylation properties of mitochondria isolated from transplanted hepatoma.

Mitochondria were isolated from Morris hepatomas with rapid (types 3683, 7777, and 3924A) and intermediate (types 5123D and 7800) growth rates, using proteolytic digestion of minced tumor tissue to release the particles. Mitochondria isolated by the same procedure from rat liver were employed as controls. All the hepatoma mitochondria were capable of coupled respiration with normal phosphorylation yields (ADP/O) and respiratory control ratios ranging from 2 to considerably more than 10. Particles from hepatomas 7777 and 7800 exhibited properties closest to liver mitochondria, while those from hepatomas 3683 and 3924A showed the greatest difference. All the hepatoma mitochondria were capable of oxidizing succinate, 3-hydroxybutyrate and monoamines. However, the oxidation rates of the latter two substrates by mitochondria from hepatomas 3683 and 3924A were only a fraction of the control rates. These differences appeared to be due, at least in part, to the structural instability of the isolated hepatoma mitochondria. In contrast to the reports of others, all hepatoma mitochondria exhibited considerable stimulation of ATPase activity by uncouplers. Maximal stimulation of ATPase activity by representatives of three classes of uncouplers was in all instances comparable to the values obtained for rat liver mitochondria.     

Gangliosides of plasma membranes from normal rat liver and Morris hepatoma.

Plasma membranes were isolated from normal rat liver and Morris hepatoma 5123tc by discontinuous sucrose gradient centrifugation. There was an average two and one-halffold enrichment of gangliosides in plasma membranes from normal liver and hepatoma as compared with their respective whole cells. The amount of total gangliosides in plasma membranes from hepatoma was eight times greater than that found in normal liver. This increase resulted from a fivefold increase in hematosides, an eightfold increase in monosialogangliosides and a twenty-twofold increase in disialogangliosides. Trisialogangliosides were present in normal liver but not in hepatoma.     

Reversible phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase in Morris hepatomas

The reversible phosphorylation of microsomal 3-hydroxy-3-methylglutaryl CoA reductase in host liver and hepatoma 5123C has been investigated. The percentage of the total enzyme activity in vivo was similar in the normal liver, host liver and hepatoma 5123C. The inclusion of 30 mM EDTA and 10 mM mevalonic acid in assays of 3-hydroxy-3-methylglutaryl CoA reductase inactivation in vitro eliminated artifacts generated by the presence of mevalonate kinase. Inactivation of 3-hydroxy-3-methylglutaryl CoA reductase from normal liver, host liver and hepatoma occurred at a similar rate with similar half-times. We conclude that phosphorylation/dephosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase occurs in hepatomas and that the lack of dietary cholesterol feedback inhibition in the hepatomas is not a result of a defect in this particular aspect of the reversible phosphorylation system.     

Enzymatic,biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTPγS. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5′-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.     

Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells

Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio-Gel P-6. Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo-beta-N-acetylglucosaminidase and tunicamycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype. The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O-glycosidically linked glycan chains, and did not result from a hypersialylation. N-glycosidically linked glycopeptides, high-mannose, and complex-type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high-mannose type oligosaccharides and an increase in the degree of branching of complex-type oligosaccharides. In addition, "bisected" glycopeptides were present only on hepatoma cells. The pattern of N-linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells. The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.     

Isolation and comparison of arylsulfatase A from rat liver and Morris hepatoma 7777

Rat liver and Morris hepatoma 7777 arylsulfatase A were isolated from the soluble lysosomal extract by a procedure involving blue-Sepharose affinity chromatography, DEAE-cellulose chromatography, hydrophobic chromatography on phenyl-Sepharose and preparative polyacrylamide gel electrophoresis. The preparation obtained by this method was apparently homogenous in disc electrophoresis and in immunoelectrophoresis. The comparative studies revealed that the properties of arylsulfatase A from rat liver and Morris hepatoma 7777 are very similar, considering molecular weight of the native monomer and its subunits, the ability to form tetramers, isoelectric point, Michaelis constant and the anomalous kinetics of the reaction. The twofold elevation of arylsulfatase B activity found in Morris hepatoma 7777 suggests that the enzyme may have certain functions in tumor growth.     

Tyrosine protein kinase in preneoplastic and neoplastic rat liver

When rats are subjected to chemical hepatocarcinogenesis according to the protocol of D. Solt and E. Farber ((1976) Nature (London) 263, 701-703), the liver exhibits elevated levels of tyrosine protein kinase activity as early as 3 weeks after the injection of diethylnitrosoamine. A more striking elevation in tyrosine protein kinase activity is noted in rat hepatomas induced by administration of chemical carcinogens, in particular that of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Tyrosine protein kinase solubilized from the particulate fraction of 3'-Me-DAB-induced hepatoma has a molecular weight identical to that of p60v-src, cross-reacts with p60v-src immunologically, phosphorylates the heavy chain of anti-p60v-src IgG, and probably belongs to a family of p60c-src. The tyrosine protein kinase from the particulate fraction of normal rat liver is indistinguishable from the hepatoma kinase in these properties; thus it apparently differs only in the level of activity. Whether the liver and hepatoma kinases differ merely quantitatively or whether they differ even qualitatively, however, remains to be elucidated.     

Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.

Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into phospholipid by a Ca(2+)-dependent base-exchange reaction. This reaction is virtually absent in normal liver mitochondria. The finding cannot be attributed to microsomal contamination of the sucrose gradient-purified 7777 hepatoma mitochondria. The reaction is also absent in the rapid-growth controls, fetal rat liver and regenerating rat liver. [(14)C]Serine incorporation into 7777 hepatoma mitochondrial phospholipid by base-exchange requires Ca(2+) and is inhibited by EDTA. Ca(2+) cannot be replaced by Mg(2+), Mn(2+), or Co(2+). The reaction is inhibited by a sulfhydryl reagent and by detergents and is abolished by heating to 70 degrees C for 10 min. Product analysis indicates that phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, are formed by 7777 hepatoma mitochondria, while phosphatidylserine is the sole product with microsomes. The conversion of phosphatidylserine into phosphatidylethanolamine in 7777 hepatoma mitochondria is inhibited by KCN. This study provides further evidence of abnormal mitochondrial biogenesis in the 7777 hepatoma. Our earlier study indicated a greatly increased mitochondrial activity of CTP:phosphatidate cytidylyltransferase in the 7777 hepatoma (Hostetler, Zenner, and Morris. 1978. J. Lipid Res. 19: 553-560). The presence in mitochondria of these two enzymes, which are primarily microsomal in normal liver, does not appear to be due to rapid growth alone, since their intracellular distribution was not altered in fetal or regenerating rat liver.-Hostetler, K. Y., B. D. Zenner, and H. P. Morris. Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.