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.     

Nucleic acids from subcellular fractions of N-nitrosodiethylamine-induced hepatoma in mice.

During eight successive isologous passages of hepatoma induced in male C3HA mice by N-nitrosodiethylamine, no common features of tumor progression were observed, although both the mitotic pattern and ploidy differed from generation to generation. These additional cytologic criteria allowed the biochemical examination of material least changed due to tumor progression. Tumor nDNA's were characterized by greater actinomycin D (AD)- and acridine orange (AO)-binding abilities than were normal nDNA's; this could have resulted from a higher proportion of double-stranded regions in tumor DNA. Isolated tumor deoxyribonucleoprotein had both lower template activity in an RNA polymerase system and fewer AD- and AO-binding sites, when compared with the activity and sites from normal mouse liver. RNA-DNA hybridization data with the above-mentioned findings showed that in hepatoma, part of the nuclear genome was repressed. Also, RNA "new classes" appeared and a certain proportion of nuclear genes controlling mitochondrial protein biosynthesis were derepressed in tumor mitochondria. The hybridization of mitochondrial RNA (mtRNA) and DNA revealed new classes of pulse-labeled RNA's in in vitro-incubated liver mitochondria that were absent from intact cell organelles; the hybridization properties of in vivo- and in vitro-formed hepatoma mtRNA's were similar. Competition and hybridization experiments demonstrated that in tumor mitochondria in vivo, some new classes of RNA existed. Hepatoma mitochondrial mRNA had a higher metabolic stability than did normal mRNA.     

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.     

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.     

Superoxide dismutase activity of normal murine liver, regenerating liver, and H6 hepatoma.

By means of both direct assay and gel electrophoresis, normal A/J mouse liver was shown to possess both Cu-Zn and Mn superoxide dismutase (SD) activity. H6 hepatoma cells contained Cu-Zn SD activity, but no Mn SD activity was detectable. Isolated mitochondria from normal liver contained both forms of the enzyme, but isolated mitochondria from H6 hepatoma cells contained no SD activity. To ascertain whether this loss of Mn SD activity was characteristic of these tumor cells or was simply a property of rapidly dividing cells, SD activity was measured in regenerating liver. Mn SD activity was present in the regenerating liver at all times after surgery. Hence loss of the Mn SD activity seemed to be a characteristic of some tumor cells but not of corresponding rapidly dividing normal cells.     

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.     

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.     

Proton-cation translocation in tumor cell mitochondria

The capacity of mitochondria isolated from tumor cells to conserve the transmembrane electrochemical proton gradient set up by respiration has been studied. In a K+ medium, mitochondria from Ehrlich ascites tumor cells exhibit a capacity to conserve aerobic delta microH comparable to that displayed by normal rat liver mitochondria. Mitochondria from Morris hepatoma 3924A show a decreased capacity to store delta microH+, which is principally due to lowering of delta pH. In a Na+ medium, both species of tumor mitochondria show a significant decrease of aerobic delta pH, while delta psi is the same, with respect to rat liver mitochondria. Experiments on passive swelling show that mitochondria from ascites tumor cells have an enhanced permeability to chloride salts of monovalent cations and increased activity of the Na+ (K+)-H+ exchange system of the mitochondrial membrane with respect to normal mitochondria. The enhanced activity of this system in ascites cells is also shown by the characteristics of respiration-linked proton translocation in submitochondrial particles and subsequent anaerobic proton diffusion. It is concluded that the decreased capacity of mitochondria from tumor cells to conserve aerobic delta pH is due to enhanced cyclic flow of Na+ across the membrane.     

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.     

Co-expression of macrophage colony-stimulating factor with its receptor in human hepatoma cells and its potential roles

Recently,theanti-cytokinetherapyandantireceptortherapyisbeingidentifiedasanefficacioustreatmentinthefieldofbiologicaltherapyagainstcancerandrelateddiseases.[llPathogenesisofhepatomaisquitecomplicateinwhichmulti-factors,multi-genesandmulti-processesareinvolved.Activationofcertainoncogenes,inactivationofsometumorsuppressergenes,orabnormalexpressionofcancerrelatedgenesmightcontributetothedevelopmentofthetumor.TheN-ras,c-myc,p53,cfmsandIGF-2/IGF-ZRarerecognizedasoncogenesorcancerrelatedgenesof…     

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.     

Mitochondrial and microsomal phospholipids of Morris hepatoma 7777.

The phospholipids of both mitochondrial and microsomal membranes from normal liver, host liver, and Morris hepatoma 7777 were isolated, separated, and quantitated. The total as well as the individual fatty acid concentrations and compositions were determined. The total phosphlipids isolated from tumor mitochondria were idly altered, compared with mitochondria from other normal or host liver. The polyenoic acids were decreased, and there was a concomitant increase in the monoenes. When the respiratory control was determined, the tumor mitochondria exhibited a significant decrease in this parameter. The tumor microsomal membrane fraction, on the other hand, contained about 50% less phospholipid than the controls. The fatty acid patterns of the total as well as the individual phospholipids were quite similar to those observed in the mitochondria. The species of phosphatidylcholine from both membrane fractions were separated by argentation chromatography of the intact molecules, and, as predicted by the fatty acid compositions, the major species of the tumor was the monoenoic/dienoic fraction. The acyl coenzyme A:1-acyl glycerophosphorylcholine acyltransferases, which aid in controlling the fatty acid composition of phospholipids, were measured. The very marked increase in activity of these enzymes toward polyenoic as well as monoenic fatty acids suggested that the polyenoic acids were not available for use in the resynthesis of the phosphatidylcholines in the tumor.     

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%).     

Differences in total mitochondrial proteins and proteins synthesized by mitochondria from rat liver and Morris hepatomas 9618A, 5123C, and 5123tc.

Mitochondria were isolated from a slow-growing (9618A) and two intermediate-to-fast-growing (5123C, 5123tc) Morris hepatomas and host livers. The mitochondrial proteins were solubilized and fractionated on sodium dodecyl sulfate:polyacrylamide slab gels. One Coomassie blue-stained band was absent or reduced in amount in all tumors relative to host livers. In addition, a major mitochondrial enzyme present in normal liver, carbamyl phosphate synthetase, was missing or greatly reduced in the slow-growing, highly differentiated hepatoma 9618A, a tumor that is considered to be similar to normal liver in many biochemical and morphological respects. Incubation of mitochondria with [35S]methionine and a suitable amino acid incorporation system resulted in labeling of specific mitochondrial proteins. Autoradiography of the slab gels disclosed four prominently labeled fractions and a number of minor fractions. Preparations from hepatoma 5123tc demonstrated two labeled bands that were absent or greatly reduced in host liver. Host liver preparations displayed a minor band that was absent or greatly reduced in hepatoma 5123C. However, no single change in labeling pattern was common to all three tumors, suggesting the absence of a causal relationship between carcinogenesis and mutations in mitochondrial DNA.     

Lectin-binding proteins in nuclear preparations from rat liver and malignant tumors

Lectin binding [concanavalin A, biotinylated ricinus communis agglutinin, and biotinylated succinylated wheat germ agglutinin (B-SWGA)] was used to detect the glycosylated proteins associated with a residual protein fraction [insoluble in 4% sodium dodecyl sulfate and termed the nuclear residual fraction (NRF)] or with nuclear matrix preparations from normal rat liver, azo dye (3'-MeDAB)-induced rat hepatoma, and Walker 256 transplantable carcinosarcoma. One- and two-dimensional gel electrophoresis were used with lectins, polyclonal antisera, and monoclonal antibody binding to characterize some of the glycoconjugates. Two polypeptide bands with approximate molecular weights of 95,000 and 55,000, shown previously to be present only in the induced tumor cells and the Walker 256 tumor, were reactive with lectins. In addition, a Mr 62,000 protein reacted only with B-SWGA in the nuclear matrix fractions from normal rat liver and the induced hepatoma. A polypeptide band (approximate molecular weight, 213,000) in the Walker 256 NRF reacted with concanavalin A and biotinylated ricinus communis agglutinin. One polypeptide band (approximate molecular weight, 182,000) reacted with concanavalin A in all three tissues, with biotinylated ricinus communis agglutinin and B-SWGA in the Walker NRF, and with B-SWGA in the hepatoma NRF. Another polypeptide band (approximate molecular weight, 138,000), reactive with all three lectins, was present in all three tissues. Our findings are consistent with previous reports of lectin binding proteins in the eukaryotic cell nucleus and indicate that certain glycoproteins isolated in nuclear preparations are found specifically in 3'-MeDAB-induced hepatoma and Walker 256 transplantable carcinosarcoma.     

Subcellular localization of acetoacetate coenzyme A transferase in rat hepatomas.

Succinyl coenzyme A:acetoacetate coenzyme transferase (EC 2.8.3.5), an initiator of ketone body usage and absent in normal liver, has been shown to be located in mitochondria from Morris hepatoma 7288ctc using differential and density gradient centrifugation. Furthermore, tumor mitochondrial subfractionation revealed that this transferase is associated with the matrix-soluble proteins. Comparison of the amounts of total transferase activity in several other hepatomas with the amounts found in the corresponding isolated mitochondria suggests that the results with the 7288ctc tumor pertain generally. The mitochondrial localization of coenzyme A transferase indicates the probable use of ketone bodies as energy sources for the hepatomas.     

Dosimetry and treatment planning for 90Y-labeled antiferritin in hepatoma

Radiation absorbed-dose estimates and treatment planning are reported for 11 patients with hepatoma who were administered 90Y-labeled polyclonal antiferritin IgG for therapy in a Phase 1-2 trial. Dosimetric studies included quantitation of the localization and clearance of 111In-labeled antiferritin IgG in tumor and normal tissues and computer-assisted tumor and normal liver volumetrics from X ray CT scans. For the group of patients studied, hepatoma volumes at the time of treatment ranged from 135 to 3442 cm3. Quantitative 111In antiferritin imaging prior to and following 600 or 900 cGy of external-beam irradiation of the primary tumor demonstrated that tumor uptake increased 1.1 to 5.8-fold (mean 2.8) following external beam. In contrast, changes in uptake of radiolabeled antiferritin in normal liver ranged from 0.35 to 2.1-fold (mean 0.93) after external irradiation. Administered activities of 90Y antiferritin ranged from 8 to 37 mCi and were dependent on tumor volume and tumor localization of radiolabeled antiferritin. Following external-beam irradiation, tumor dose rates achieved with 90Y antiferritin ranged from 10 to 20 cGy/hr and normal liver dose rates from 1.1 to 5.7 cGy/h. The corresponding absorbed dose in hepatomas ranged from 900 to 2150 cGy and in normal liver from 80 to 650 cGy. After external-beam irradiation, tumor and normal liver uptake of 90Y antiferritin was consistent with that of 131I antiferritin.     

Metabolism of nucleic acids in normal, regenerating and neoplastic tissues of the liver]

Under study was the kinetic growth of three hepatomas-22A, 60 and 46, characterized by a various degree of differentiation. There was found a correlation between the degree of differentiation and parameters of the hepatomas growth. For the hepatomas a correlation between the DNA content and rate of growth was observed, for hepatoma 46 a value of the DNA content proved to be near to that of normal liver. Moreover, in a regenerating liver the DNA content is the same as normal, that indicates the difference between normal and neoplastic actively proliferating tissues. The processes of DNA synthesis, studied by thymidine-C14 incorporation, showed a linear correlation with the rate of growth in regenerating, normal and neoplastic tissues. The ratio RNA/DNA in tumors is regularly decreased with respect to normal liver (a linear correlation between RNA/DNA and the maximum rate of growth). In a regenerating liver the ratio is high. Variations in the ratio RNA/DNA reflect changes in the functional activity and related disorders in the metabolism of nucleic acids in hepatomas. There was found a suppressed DNA decomposition in autolysis of homogenates from hepatoma tissues. It is essential to note that in a minimum deviated hepatoma some inhibition of the DNA decomposition is the only observed disorder in the nucleic acids metabolism. Spin-labelled DNA preparations of tissues under study were obtained. The presence of structural differences between separate DNA samples has been demonstrated.