Characterization of nucleolar antigens of normal rat liver, regenerating rat liver, and Novikoff ascites hepatoma cells following in vitro translation of polyadenylic acid-containing messenger RNA

Nucleolar antigens of normal rat liver, regenerating liver, and Novikoff ascites hepatoma cells were transplanted in vitro from polyadenylic acid-containing messenger RNAs isolated from the respective tissues and immunoprecipitated with specific antinucleolar antibodies and Protein A. By two-dimensional gel electrophoresis of the translation products, five antigens were detected in normal rat liver. The antigens detected in 18-hr regenerating rat liver were the same as those of normal rat liver when immunoprecipitated with the anti-liver nucleolar antibodies. In the Novikoff tumor, 11 antigens were detected with anti-Novikoff nucleolar antibodies. Of these, two were not found in either normal or regenerating liver. Four major antigens were detectable in both Novikoff hepatoma and regenerating liver messenger RNA translation products with anti-Novikoff nucleolar antibodies. Two antigens were found in normal and regenerating liver that were not found in Novikoff hepatoma. In agreement with previous results, these immunoprecipitation analyses provide further evidence that some nucleolar antigens are present in Novikoff hepatoma that are not found in either normal or regenerating rat liver.     

Retinyl palmitate, retinyl phosphate, and dolichyl phosphate of postnuclear membrane fraction from hepatoma, host liver, and regenerating liver: marginal vitamin A status of hepatoma tissue

The retinyl palmitate content of the postnuclear membrane fraction from 10 Morris hepatomas, their host rat livers, one acetylaminofluorene-induced rat liver hepatoma, and the host liver and of regenerating rat liver was measured by reverse-phase high-pressure liquid chromatography of the chloroform:methanol extracts. Membranes from the hepatoma tissue contained less than detectable levels of retinyl acyl esters, whereas membranes from host liver tissue and regenerating liver contained levels of retinyl palmitate within normal ranges. The amount of cellular retinol-binding protein was also decreased considerably in cytosols from 9618 and 7777 hepatomas. The ratio of endogenous retinyl phosphate to the polyisoprenoid dolichyl phosphate available for mannosylation in an assay containing postnuclear membranes and guanosine dephospho[14C] mannose was decreased by a factor of 3 to 10 in hepatoma tissue. Such change in ratio was not attributable to specific changes in retinyl phosphate mannose-synthesizing activity, but it appeared to be related to the vitamin A deficiency condition of the membrane from tumors. As for membranes from vitamin A-deficient liver tissue, postnuclear membranes from rat cystic hepatocarcinoma, Morris 7777, 3924A1-1, and 5123D-1-2 transplantable rat hepatomas and guinea pig line 10 hepatoma all synthesized a mannolipid with intermediate hydrophobic properties between retinyl phosphate mannose and dolichyl phosphate mannose and not normally found in liver tissue. These alterations in patterns of lipid intermediates may be responsible for altered glycosylation of glycoproteins in neoplastic cells. In conclusion, the present investigation establishes that hepatoma cell membrane is in a status of vitamin A and of retinyl phosphate depletion, while dolichyl phosphate contents appear similar to host liver membrane.     

Superoxide dismutase and superoxide radical in Morris hepatomas

Total superoxide dismutase (SOD) and manganese superoxide dismutase (Mn SOD) specific activities were measured in tissue homogenates and in isolated mitochondria from normal rat liver and three Morris hepatomas of different growth rates. Total SOD and Mn SOD specific activities were decreased in all tumor homogenates when compared to normal liver; the lowest activity was associated with the fastest growing tumor. These results are consistent with the hypothesis that total Mn SOD specific activity is decreased in all tumors. The Mn SOD specific activity was similar to the total SOD specific activity of isolated mitochondria, indicating that mitochondrial SOD is almost entirely manganese containing. This activity was decreased in the fast- and medium-growth-rate hepatomas but was slightly increased in the tumor with the slowest growth rate when compared to liver. The normal or higher than normal mitochondrial Mn SOD specific activity indicates that decreased mitochondrial SOD specific activity is not a characteristic of all tumors. Superoxide radical (O2-.) formation was measured in submitochondrial particles obtained by sonication of isolated mitochondria and subsequent washings to remove the SOD. The difficulty encountered in reducing the SOD activity suggests that at least part of the mitochondrial SOD might be associated with the mitochondrial membrane. In liver submitochondrial particles, O2-. was formed only when succinate and antimycin A were used together, as substrate and inhibitor of the electron transport chain, respectively. In the hepatomas studied for O2-. production (slow- and fast-growth rates), the formation of the radical was detected in the presence of succinate even when no inhibitor was present. Antimycin A stimulated the production of O2-. in normal rat liver and slow-growth-rate tumor, but not in fast-growth-rate tumor submitochondrial particles. Reduced nicotinamide adenine dinucleotide did not lead to the production of O2-. by normal liver or hepatoma submitochondrial particles. Mitochondrial membrane damage was seen in micrographs of the medium- and fast-growing hepatomas. This could be a consequence of low mitochondrial SOD concomitant with a flux of superoxide, if the radical is produced in vivo by these mitochondria.     

COMPARATIVE STUDY OF PHOSPHOTYROSYL PROTEIN PHOSPHATASE OF MICE NORMAL LIVER, REGENERATING LIVER, AND MICE H22A HEPATOMA ASCITES CELL

In regenerating liver of mice, marked increase of the activity of phosphotyrosyl protein phosphatase (PTPP) in cytosol was observed. The PTPP activity varied with time and reached the highest level between 24 to 48 hours after partial hepatectomy. In H22a cells the PTPP activity found in every subcellular fraction was lower than that of the normal liver. The PTPP activity was mostly concentrated in lysosomes of normal liver, but mainly distributed in nucleus, cytosol and microsome of regenerating liver. In H22a cells PTPP activity seemed distribute evenly. Five similar major PTPP peaks (I-V) were obtained on DEAE cellulose chromatography of cytosols from all three of liver cells studied. However, two additional PTPP peaks, a and b, were also obtained from cytosol of liver.     

Characteristics of mitochondria isolated by rate zonal centrifugation from normal liver and Novikoff hepatomas

Mitochondria were isolated from whole homogenates of normal liver and Novikoff hepatomas using reorienting rate zonal centrifugation on sucrose gradients. The activities of several mitochondrial-specific enzymes and ultrastructure were compared in the two tissues. Our results indicate that cytochrome oxidase, lipoamide dehydrogenase, malate dehydrogenase, and succinate dehydrogenase activities are all higher in liver homogenates than in Novikoff hepatoma homogenates. Mitochondrial hexokinase, however, is much greater in the hepatoma than in liver. The activity of these enzymes in isolated mitochondria displayed a much different pattern. Both cytochrome oxidase and succinate dehydrogenase activities were higher in hepatoma mitochondria than in liver mitochondria. Lipoamide dehydrogenase and malate dehydrogenase, conversely, were higher in liver mitochondria. Hexokinase was found to be virtually absent in liver mitochondria but plentiful in hepatoma mitochondria. Ultrastructural studies have shown that the hepatoma mitochondria are much smaller in size, which results in a decreased rate of migration into the gradient. These studies have also shown that normal liver consists of predominantly "condensed" forms of mitochondria, whereas hepatoma contained a majority of "twisted" species. Experiments using 1% bovine serum albumin in the homogenization procedures and in the gradient have confirmed earlier observations that bovine serum albumin is essential for optimal isolation of neoplastic mitochondria.     

Nuclear protein phosphokinases in normal and neoplastic tissues.

Protein phosphokinases were isolated from the nuclei of normal and fetal liver and neoplastic tissues. Chromatography on phosphocellulose columns resolved the normal and fetal liver kinases into five reproducible fractions. Each of the fractions differed in optimal divalent cation and substrate requirements. Hepatic proliferation was accompanied by quantitative changes in the kinase activity profiles (with endogenous phosphoprotein as natural substrate). An additional phosphoprotein kinase activity stimulated by Mn2+ was found in the nuclei of malignant cells. This tumor-specific kinase could not be detected either in tumor cytoplasm or in fetal or regenerating liver nuclei. Mn2+-dependent phosphoprotein kinase from Novikoff hepatoma phosphorylated only one major protein band detectable by polyacrylamide gel electrophoresis. This substrate could not be detected in chromatin of normal tissues.     

Comparison of nuclear nonhistone phosphoproteins of rat liver and Novikoff hepatoma.

As part of a continuing comparison of nuclear proteins of tumors and other tissues, 32P-labeled nuclear proteins were extracted successively with 0.15 and 0.35 m NaC1 from the nuclei of normal, regenerating, and thioacetamide-treated rat liver as well as Novikoff hepatoma 3 hr after injection of 32Pi into rats. Separation of proteins of these fractions with aqueous phenol was carried out before two-dimensional electrophoresis on polyacrylamide gels. By autoradiography many common spots were found, but four 32P-labeled protein spots, CU', C13p, C21p, and CMp, were found in the Novikoff hepatoma and not in the various liver samples studied. Two spots, B6 and B10, were found in the liver patterns and not in the tumor. Sot B33 was very dense in regenerating liver but was only a faint spot in thioacetamide-treated liver. The greater density of Spots CU', C13p, C21p, and CMp in the tumor patterns is consistent with the increased density reported earlier for spots of the C-region of a variety of tumors.     

Membrane lipids of hepatic tissue. II. Phospholipids from subcellular fractions of liver and hepatoma 7288CTC

Phospholipids from homogenate, nuclei, mitochondria, endoplasmic reticulum (ER), plasma membrane (PM), and cytosol of liver and hepatoma 7288CTC (from inbred male BUF rats) were analyzed for their concentrations, fatty acid compositions of individual lipid classes, and levels of octadecenoate positional isomers. The phospholipid concentrations of hepatoma mitochondria and ER were less than 60% of liver values. Sphingomyelin concentrations were elevated dramatically in hepatoma nuclei, mitochondria, and PM. Hepatoma nuclei and mitochondria contained only 25% or less the concentrations of phosphatidylethanolamine (PE) as those of liver, whereas ER, PM, and cytosol fractions of hepatoma contained equal or greater concentration of PE than did the corresponding liver fractions. The fatty acid profiles of the individual lipid classes were somewhat characteristic of liver organelles but not of hepatoma. Lipid classes thought to be located preferentially on the outer bilayer of liver PM contained lower percentages of polyunsaturated fatty acids than did hepatoma PM. For hepatoma generally the lipid classes tended to exhibit a more uniform fatty acid profile among organelles, the polyunsaturated fatty acid percentages were decreased, and the octadecenoate percentages were increased. Octadecenoates isolated from individual lipid classes of organelles contained high levels of cis-vaccenate, in addition to oleate, and some class and organelle specificity was observed in liver. In contrast, hepatoma octadecenoates exhibited little class or organelle specificity, and much higher oleate concentrations were found in PM phosphatidylcholine, PM PE, and ER PE in hepatomas than in liver.     

Glutaminase and glutamine synthetase activities in human cirrhotic liver and hepatocellular carcinoma.

Glutamine synthetase and glutaminase activities in human cirrhotic liver tissues and hepatocellular carcinomas were determined for comparison with normal liver tissues. In hepatocellular carcinoma, glutamine synthetase activity was approximately one-third of that in normal liver, whereas no detectable change in the enzyme activity was observed in cirrhotic liver. Phosphate-dependent and phosphate-independent glutaminase activities were increased approximately 20-fold and 6-fold, respectively, both in the carcinoma and cirrhotic liver compared with those from normal liver, Oxypolarographic tests showed that the rate of glutamine oxidation in the tumor and cirrhotic liver mitochondria was about 5-fold higher than that in the liver mitochondria. The rate of glutamate oxidation in the liver mitochondria was comparable to that in the cirrhotic liver and tumor mitochondria. Glutamine oxidation was inhibited by prior incubation of the mitochondria with 6-diazo-5-oxo-L-norleucine, which inhibited mitochondrial glutaminase. These results indicate that the product of glutamine hydrolysis, glutamate, is catabolized in the tumor and cirrhotic liver mitochondria to supply ATP. In the liver and cirrhotic liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via a transamination pathway in the tumor mitochondria.     

Spermine-induced variations in the adenosine 5'-diphosphate ribosylation patterns of nuclear proteins from rat liver and hepatoma.

Rat liver and hepatoma nuclei were incubated in vitro with [3H]nicotinamide adenine dinucleotide to allow synthesis of a polymer of adenosine diphosphoribose subunits joined in an 1',2' ribose-ribose linkage. The addition of 1 mM spermine altered the adenosine 5'-diphosphate (ADP) ribosylation patterns of nuclear proteins in hepatoma, host liver, and regenerating liver. Spermine-treated nuclei showed a greater incorporation of ADP-ribose into H1 histones and nonhistone nuclear proteins with isoelectric points between pH 3.0 and 6.0 when separated on polyacrylamide gels. Conversely, a large reduction in ADP ribosylation was seen in core histones (H2A, H2B, and H3) from the same nuclei. The proportion of ADP-ribose incorporated into histones was reduced in the nuclei from proliferating cells relative to their respective control livers. These results imply that polyamines, which are higher in concentration in rapidly dividing cells, may elicit a regulatory function by causing the preferential ADP ribosylation of H1 histones, as well as the more acidic of the nuclear proteins.     

Physicochemical characterization of Novikoff hepatoma mitochondrial DNA.

Mitochondrial DNA's (mtDNA) isolated from rat liver and the Novikoff hepatoma grown as both solid tumors and cells in monolayer culture were examined by a variety of physicochemical techniques. Buoyant densities in analytical CsCl equilibrium gradients and thermal denaturation profiles revealed no significant differences in base composition among the mtDNA's isolated from liver, tumor, and hepatoma cells. Sedimentation in neurtral and alkaline CsCl showed no differences in mtDNA size. However, tumor and hepatoma cell mtDNA's were slightly smaller and more heterogeneous in size than liver mtDNA when molecular contour lengths were measured in the electron microscope. Based on chemical determinations, neoplastic mitochondria contained four to five times more DNA per mitochondrion than liver. Also, electron microscopy showed the proportion of mtDNA in complex forms (catenated dimers and oligomers) to be much higher in tumor (18%) and hepatoma cells (15%) than liver (4%).     

Elevated activity and increased mannose-6-phosphate in the carbohydrate moiety of cathepsin D from human hepatoma

A significant elevation of cathepsin D activity was observed in six human hepatoma tissues as compared to 12 normal human livers. In isoelectric focusing experiments, cathepsin D purified from normal liver exhibited three different forms, with isoelectric points of 5.6, 6.1, and 6.7, while cathepsin D purified from hepatoma contained another five to six more acidic forms in addition to the forms observed in normal liver cathepsin D. When the tumor enzyme was treated with endo-beta-N-acetylglucosaminidase H followed by isoelectric focusing, the acidic components disappeared and were converted to forms identical to those of the normal liver cathepsin D. Determination of the mannose-6-phosphate content showed that hepatoma cathepsin D contains twice as much mannose-6-phosphate as normal liver cathepsin D. Peptide mapping and amino acid analysis showed that the protein moiety of cathepsin D from hepatoma is almost identical with that from normal liver. These findings indicate that the appearance of acidic variants in hepatoma cathepsin D is mainly due to changes in the oligosaccharide chains of the enzyme, which are closely associated with the increase of mannose-6-phosphate in the tumor enzyme.     

Hydroperoxide-stimulated release of calcium from rat liver and AS-30D hepatoma mitochondria

The presence of 50 microM t-butyl hydroperoxide induces the oxidation of intramitochondrial pyridine nucleotides and release of accumulated Ca2+ from rat liver but not AS-30D hepatoma mitochondria in the presence of succinate (plus rotenone) as a respiratory substrate. The effects of t-butyl hydroperoxide are mediated by the activities of glutathione peroxidase and reductase, which are less than 20 and 50% as active, respectively, in hepatoma than in normal liver mitochondria. However, the differences in the activities of these enzymes are not responsible for the insensitivity of succinate-energized tumor mitochondria to t-butyl hydroperoxide, since Ca2+ release and pyridine nucleotide oxidation can be elicited when ascorbate plus tetramethyl-p-phenylenediamine are used as alternative respiratory electron donors. In the presence of succinate alone, rat liver mitochondria generate malate exclusively, whereas AS-30D hepatoma mitochondria produce pyruvate and reduced nicotinamide adenine dinucleotide phosphate as well as malate due to the activity of a nicotinamide adenine dinucleotide phosphate-dependent malic enzyme which is not present in normal rat liver mitochondria. These results indicate that the maintenance of pyridine nucleotides in their reduced form by malic enzyme is responsible for the lack of t-butyl hydroperoxide-induced Ca2+ efflux by tumor mitochondria respiring on succinate. This altered pattern of mitochondrial metabolism may also influence the regulation of other reduced nicotinamide adenine dinucleotide phosphate-sensitive activities in addition to that of Ca2+ transport.     

Transport of pyruvate in mitochondria from different tumor cells

A comparative study of the transport of pyruvate in mitochondria isolated from normal rat liver and from three tumors has been carried out. The Km for net pyruvate uptake in mitochondria isolated from Ehrlich ascites tumor cells is practically equal to that measured in normal rat liver mitochondria while, on the other hand, it is higher in Morris hepatomas 44 and 3924A. The Vmax of pyruvate uptake is depressed in all three types of tumor mitochondria as compared to that in the rat liver mitochondria, with the depression being higher in Morris hepatoma 3924A mitochondria. The lower activity of pyruvate translocator in mitochondria isolated from tumor cells as compared to that in rat liver mitochondria is also shown by depression of the rate of pyruvate-supported oxygen uptake. The results document a decreased activity of the pyruvate translocator in tumor mitochondria which seems to be correlated with the growth rate of the tumor cells.     

Differences in the buoyant density of mitochondria from hepatoma, Krebs ascitic tumor II and normal liver]

Mitochondria isolated from normal liver, experimental hepatoma, and Krebs II ascites tumor tissues in mice were examined by isopycnic centrifugation in a sucrose density gradient (25--50%). Normal liver mitochondria were shown to be characterized by two visible zones, while those of tumors were distributed homogeneously. Such a distribution pattern was also confirmed by the investigation of marker enzyme activities (glucose-6-phosphatase and cytochromoxidase). A comparison of homologous tissues (hepatoma--liver) has evidenced that hepatoma mitochondria are "lighter" even than the "light" fraction of normal ones, that seems to evidence the disturbances in the biogenesis of these organelles. The results obtained are in a good agreement with the data reported by S. A. Neifakh on alterations in the structure of tumor mitochondrial membranes.     

Differences in nucleolar antigens of rat liver and Novikoff hepatoma ascites cells.

Antisera to nucleoli of Novikoff hepatoma ascites and normal rat liver cells were produced in rabbits by injection of whole, isolated nucleoli. These antisera have been used to compare the nucleolar antigens that were partially fractionated by differential solubilization from nucleoli. Fourteen antigens were detected by these antisera; ten of these antigens were detected by both antisera. Ouchterlony double diffusion analysis of soluble extracts from normal rat liver and Novikoff hepatoma ascites nucleoli and fetal rat liver nuclei provided evidence for antigens found only in liver extracts, only in tumor extracts, or only in tumor and fetal extracts. Antisera preabsorbed to remove antibodies to common antigens of liver and tumor provided confirmatory evidence for one nucleolar antigen in liver that was not found in tumor or fetal rat liver, one antigen in tumor that was not found in adult or fetal rat liver, and three antigens in both tumor and fetal rat liver that were not found in adult rat liver. In addition, the antitumor nucleolar antiserum preabsorbed with liver nuclear extracts still produced positive nucleolar fluorescence in Novikoff hepatoma ascites cells but not in liver cells. Conversely, anti-liver nucleolar antiserum preabsorbed with tumor nucleolar extracts did not produce detectable tumor nucleolar fluorescence but did produce positive fluorescence in liver nucleoli.     

Soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA of Novikoff hepatoma cells.

The nature of soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA was studied in Novikoff hepatoma cells. The decreased activity in hepatoma preparations was due to loss of a high-molecular-weight heat-labile factor. Although this factor cochromatographed with arginase activity on Sephadex G-150, it does not appear to result from this activity as judged by the failure of arginine to prevent the inhibitory effect on [3H]thymidine incorporation. Both liver and hepatomas contained a heat-stable factor with inhibitory activity. Studies with ethanol-soluble material suggested that the action was not solely attributable to the presence of unlabeled thymidine, since the apparent molecular weight was too high and since the factor(s) inhibited [3H]leucine incorporation into protein in addition to inhibiting [3H]thymidine incorporation in DNA.     

Membrane lipids of hepatic tissue. I. Neutral lipids from subcellular fractions of liver and hepatoma 7288CTC

Enriched subcellular fractions of nuclei, mitochondria, endoplasmic reticulum (ER), plasma membrane, and cytosol were prepared from liver and hepatoma 7288CTC taken from male inbred BUF rats. Purity was established by marker enzyme activities and distribution of DNA, RNA, sialic acids, total phospholipids, and cholesterol. The subcellular fractions of hepatoma differed from those of liver: 5'-Nucleotidase activity was elevated in ER and mitochondria, cytosol RNA was increased, and cholesterol was elevated in all hepatoma subcellular fractions. Neutral lipid classes of hepatoma subcellular fractions differed quantitatively from those of liver: Hepatoma nuclei and mitochondria contained elevated levels of free fatty acids (FFA) and triglycerides (TG). Generally, the fatty acid profiles of FFA, TG, and sterol esters from hepatoma subcellular fractions were more uniform and showed less organelle specificity than did liver. Hepatoma FFA and TG contained lower percentages of palmitate and higher percentages of stearate in all subcellular fractions than did liver. The sterol esters from most hepatoma subcellular fractions compared to those from liver were characterized by much higher levels of long-chain fatty acids of 20 carbon atoms or longer. The oleate-to-vaccenate ratio in FFA of liver subcellular fractions exhibited some specificity, but not that of hepatoma subcellular fractions. The oleate-to-vaccenate ratio in the acyl chains of liver and hepatoma TG did not reveal organelle specificity.     

Comparison of thioredoxin reductases from Novikoff ascites hepatoma cells and normal liver of rats.

Adult rat liver contained variant forms of thioredoxin reductase with isoelectric points at pH 4.9 and at approximately pH 4.7 compared to pH 5.1 for the enzyme from Novikoff ascites hepatoma. Fetal and regenerating liver contained only the form with the isoelectric point at pH 4.9. All three enzymes precipitated with and were inhibited by a rabbit antibody to purified enzyme from Novikoff tumor.