Fast-growing Hepatoma Research Articles

Mitochondrial biogenesis in the liver during development and oncogenesis.

The analysis of the expression of oxidative phosphorylation genes in the liver during development reveals the existence of two biological programs involved in the biogenesis of mitochondria. Differentiation is a short-term program of biogenesis that is controlled at post-transcriptional levels of gene expression and is responsible for the rapid changes in the bioenergetic phenotype of mitochondria. In contrast, proliferation is a long-term program controlled both at the transcriptional and post-transcriptional levels of gene expression and is responsible for the increase in mitochondrial mass in the hepatocyte. Recently, a specific subcellular structure involved in the localization and control of the translation of the mRNA encoding the beta-catalytic subunit of the H(+)-ATP synthase (beta-mRNA) has been identified. It is suggested that this structure plays a prominent role in the control of mitochondrial biogenesis at post-transcriptional levels. The fetal liver has many phenotypic manifestations in common with highly glycolytic tumor cells. In addition, both have a low mitochondrial content despite a paradoxical increase in the cellular representation of oxidative phosphorylation transcripts. Based on the paradigm provided by the fetal liver we hypothesize that the aberrant mitochondrial phenotype of fast-growing hepatomas represents a reversion to a fetal program of expression of oxidative phosphorylation genes by the activation, or increased expression, of an inhibitor of beta-mRNA translation.

DNA polymerase α from normal rat liver is different than DNA polymerases α from Morris hepatoma strains

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.

Expression of different members of heat shock protein 70 gene family in liver and hepatomas.

The levels of expression of some genes of the HSP 70 family have been assessed in rat liver and in a series of transplantable hepatomas with different growth rates, subjected to heat shock in vivo. For this purpose, the mRNAs for the constitutive cognate HSC 73, the heat-inducible HSP 70 and the glucose-regulated GRP 78 have been analyzed by: (i) translation in reticulocyte lysates; (ii) hybrid-selected translation, and (iii) Northern blot analysis. In comparison with the liver, the fast-growing 3924A hepatoma has an increased constitutive amount of HSC 73 mRNA and a lower induction of HSP 70 mRNA after heat shock. The behavior of the 9618A slow-growing hepatoma is more similar to that of the liver, indicating that the changes detected in the fast-growing hepatoma are correlated to the high growth rate of the tumor rather than to carcinogenesis. This conclusion is reinforced by the results obtained with Yoshida AH-130 cells, growing at two different rates imposed by the environment in which they develop. When the Yoshida hepatoma grows rapidly in the peritoneal cavity, constitutive expression of HSC 73 mRNA is high and the inducibility of HSP 70 mRNA is poor: the opposite occurs when the tumor grows slowly in the subcutaneous compartment. The amount of GRP 78 mRNA increases progressively from the liver to the fast-growing hepatoma. The level of HSC 73 mRNA seems to correlate with the methylation state of the corresponding gene.

Superoxide dismutase depletion and lipid peroxidation in rat liver microsomal membranes: correlation with liver carcinogenesis

The depletion of superoxide dismutase in the liver of rats held on a copper-deficient diet for 8 weeks induces two profound modifications in microsomal membrane characteristics. These membranes show: (1) a low degree of peroxidation induced in vitro by both endogenous (NADPH and tert-butylhydroperoxide) and exogenous sources (xanthine/xanthine oxidase) of oxygen radicals as revealed by malondialdehyde and diene-conjugate production; (2) a strong decrease of polyunsaturated and an increase of monounsaturated fatty acid content. These alterations are similar to those found in microsomal membranes from fast-growing hepatomas which exhibit a pronounced saturation of fatty acid pattern and lack superoxide dismutase. These observations support the hypothesis that during hepatocarcinogenesis the loss of superoxide dismutase causes an oxidative stress that increases cellular membrane lipid peroxidation, as a consequence of which the cell responds by synthesizing more saturated fatty acids that permanently modify cell membrane structure and properties.

Constitutive and induced synthesis of heat shock proteins in transplantable hepatomas.

The synthesis of heat shock proteins (HSP) was studied in rat liver and in a series of transplantable Morris hepatomas with different growth rates, subjected to heat shock in vivo and in vitro. Different from the liver, hepatomas synthesized HSP constitutively, i.e., also before exposure to heat. This constitutive synthesis was low and limited to one HSP in the slowest-growing tumor, more marked and involving other HSP in the intermediate- and fast-growing hepatomas. In tumor that synthesized HSP constitutively, the induction of HSP in response to heat was proportionately reduced. These patterns of reaction were essentially similar in vivo ad in vitro. The amount of HSP 68 was well correlated to the levels of its mRNA in liver and in all hepatomas, whereas the increase in HSP 89 was accompanied by a corresponding increase in the related mRNA in liver and in slow-growing hepatoma, not in the other tumors, thus suggesting a different mechanism of control of HSP 89 synthesis in the more malignant hepatomas.

Adenine nucleotide translocase activity and sensitivity to inhibitors in hepatomas. Comparison of the ADP/ATP carrier in mitochondria and in a purified reconstituted liposome system.

Adenine nucleotide uptake was found to be lower in mitochondria from hepatoma 7777, 7800, and 9618A than in the host livers. Moreover, in the fast-growing hepatoma 7777 the sensitivity of the adenine nucleotide translocase to inhibition by carboxyatractylate and bongkrekic acid was considerably decreased. Purification of the ADP/ATP carrier from hepatoma 7777 mitochondria and its reconstitution into an artificial liposome system reversed the abnormal kinetics in that the adenine nucleotide uptake and response to inhibitors were identical in proteoliposome preparations from host liver and tumor mitochondria. Analysis of the lipids of the hepatoma inner mitochondrial membrane indicated considerable differences from normal in the levels of phospholipids and cholesterol. Most striking was the increase in cholesterol and sphingomyelin of the hepatoma 7777 inner membrane. An artificial liposome system containing cholesterol in addition to the standard phospholipids could produce alterations in kinetics of the purified ADP/ATP carrier from heart mitochondria similar to those seen in the hepatoma 7777. In general, these results support the suggestion that alterations in the lipid environment of the inner mitochondrial membrane rather than intrinsic changes in the carrier protein itself produce the aberrant observations of adenine nucleotide translocase activity in hepatoma mitochondria.

Ultrastructural study of somatotroph cells from mice bearing a fast growing transplanted hepatoma, in different periods of the tumor development

The presence of a transplanted, fast-growing hepatoma (SS1-K) produces conspicuous ultrastructural changes in pituitary STH cells of C3H-S male mice. These changes are suggestive of an increased secretion of growth hormone only during the first stages of the tumor development. The hepatoma influence does not seem to be clearly related to the illumination regimen or time of killing.

Cell localization of aldolase fetal isozymes in rat regenerating liver: Differences with hepatoma

Using the indirect immunoperoxidase technique with optical and electron microscopy, we have localized both fetal aldolases A and C from regenerating rat liver in Kupffer and endothelial cells (by contrast with the localization of aldolase B in hepatocytes only). These results have been confirmed by biochemical methods. The localization of the 3 aldolases differs widely from that observed in fast-growing hepatoma and suggests that control mechanisms of gene regulation are different in cancer and in liver regeneration.

A morphologic and morphometric study of the mitochondria in several hepatoma cell lines and in isolated hepatocytes

Some characteristics of the mitochondria of hepatocytes and of three hepatoma cell lines have been compared. By means of stereologic analysis of electron micrographs of cross-sections through cells the volume of mitochondria per unit volume of cell cytoplasm and the surface areas of the mitochondrial envelope and cristae membranes have been measured. The relative mitochondrial volume in the cytoplasm decreases with increasing growth rate but the surface area of outer and cristae membranes per unit volume of mitochondria is not altered. The internal organization of hepatoma mitochondria, however, differs distinctly from that of normal liver mitochondria as evident from electron micrographs; the hepatoma cells contain mitochondria in which parallel cristae appear to cross the whole mitochondrial profile unlike the irregular, short cristae seen in normal liver mitochondria. Furthermore, in the fast-growing hepatoma cells the mitochondrial matrix appears less dense than in the hepatocyte. Hepatoma cells contain less organized rough endoplasmic reticulum than normal liver cells and the spatial relationship of the mitochondria to the rough cisternae, seen in the hepatocyte, is absent in the fast-growing hepatoma cell lines. It is concluded that hepatoma cells have fewer mitochondria than normal liver cells, but that the organelles have a normal content of inner membranes.

Properties of guanylate cyclase in adult rat liver and several Morris hepatomas

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) activity was examined in preparations from normal rat liver and a series of Morris hepatomas. Homogenate guanylate cyclase activities were 3.2, 1.6 and 1.2 nmol cyclic GMP formed per min/g tissue in normal liver and in two fast-growing hepatomas, 3924A and 9618A2, respectively. Enzymatic activity was found in all particulate fractions and in the supernatant fraction of all tissues examined. Liver had a particulate:soluble guanylate cyclase ratio of 0.24. Five hepatomas displayed particulate:soluble enzymatic ratios greater than one. Particulate and soluble guanylate cyclase from hepatoma 3924A and hepatoma 9618A2 were examined in greater detail and compared to normal liver. Triton X-100 stimulated particulate guanylate cyclase 11-, 5-, and 2-fold in preparations from liver and hepatomas 3924A and 9618A2, respectively. Liver and hepatoma particulate and soluble guanylate cyclase preferred Mn 2+ as the sole cation. While liver preparations could utilize Mg 2+ when Mn 2+ was present in low concentrations, the tumor enzymes did not utilize Mg 2+ in the presence of Mn 2+. Calcium inhibited all particulate and stimulated all soluble preparations. ATP inhibited all preparations of guanylate cyclase. Sodium azide stimulated the particulate and soluble enzyme from liver 5- and 6-fold, respectively. Sodium azide did not alter guanylate cyclase activity from tumor preparations.

Increased ornithine decarboxylase activity in murine sarcoma virus infected cells

POLYAMINE biosynthesis is one of the earliest events occurring during cellular proliferation1,2, and the rate at which it proceeds3 is limited by ornithine decarboxylase (ODC), an enzyme which catalyses the formation of putrescine from ornithine. ODC levels are low, but stable, in resting cells, and a large increase in activity occurs rapidly after the onset of growth4,5. Elevated ODC levels occur in certain tumour cells, including hepatoma, neuroblastoma and SV40-transformed cells3–7. Fast growing hepatomas have higher enzyme levels than slow growing ones6. During Rous sarcoma virus (RSV) transformation, elevation of ODC levels precedes morphological change8.

The dedifferentiated pattern of enzymes in livers of tumor-bearing rats.

Summary Seven days after the s.c. implantation of a neoplasm, there are significant decreases of ornithine aminotransferase and glucokinase, as well as an increase of hexokinase in livers of adult rats. The concentration of 10 additional enzymes changes in the course of the following week, while the levels of nine other enzymes and the liver size remain normal. Growth of tumors in suckling rats inhibits the appearance of liver enzymes that normally emerge at this stage of differentiation. An implanted tumor inhibits the developmental formation in liver of ornithine aminotransferase, glucokinase, glutamine synthetase, and malate-NADP dehydrogenase, but it does not prevent glucocorticoids from inducing a premature rise of ornithine aminotransferase. The enzymes that increase in host liver upon tumor transplantation are among those that are relatively high in fast-growing hepatomas and in fetal liver. Those that decrease are those that are low or absent in hepatomas and fetal liver. Thus, by gain or partial loss of these enzyme activities, the quantitative pattern of enzymes in host liver diverges from normal adult liver toward that of immature liver and the well-differentiated hepatomas.

Formes moléculaires fœtales d'enzymes dans l'hépatome

Summary The resurgence of fetal type aldolases in hepatoma offers a striking example of the fetal pattern of enzymes in some cancerous tissues. In slow growing hepatomas (transplantable or induced by 3'MeDAB) we have observed an increase of aldolase A (muscle type) which is relatively abundant in fetal liver but present only in small amounts in normal adult liver. This increase is proved by the elevation of the aldolase activity ratio, by the action of specific antiserum, and by electrophoresis. Electrophoresis on starch gel shows that aldolase A is hybridized with the normal liver type aldolase (aldolase B). This hybridization indicates a synthesis of the two aldolases by the same type of cells. In fast-growing hepatomas (ascitic or solid) aldolase B is absent or nearly absent ; aldolase A is present in large amounts. Moreover, the presence of a third type of aldolase, aldolase C (brain type), which is completely absent from normal adult rat liver, has been demonstrated in fetal and in cancerous liver. This was proved by (a) the aldolase activity ratio (about 30.0 instead of 1.0), (b) electrophoresis which shows in fetal liver and in hepatoma anodic isozymes similar to brain aldolase isozymes ; (c) the action of specific antiserum anti aldolase C. The degree of inhibition by each specific antiserum corresponds roughly to that expected from the electrophoretic pattern. Fetal liver contains the three types A, B and C ; fast-growing hepatomas contain aldolases A and C and very little aldolase B. Aldolase C from fast-growing hepatomas was partly purified. Similar observations were made in human primary liver cancer. A fetal liver pattern has also been found for some other enzymes in hepatomas by several authors ; mainly hexokinases, lactic dehydrogenases, glutaminases and branched chain amino acid transaminases ; it can be compared with the resurgence of fetal antigens in cancer tissues and sera. And although the placental type of alkaline phosphatase found in cancer by Fishman was not found in fetus, it is noteworthy that placenta is an embryonic tissue. S. Weinhouse has shown that the liver-type isozyme of several enzymes is replaced by non-hepatic types in fast-growing hepatomas. This « fetal patternis not specific for carcinogenesis : for example « fetal-typeof several muscle enzymes is present in atrophied and dystrophic muscle. In conclusion, it seems likely that in several pathological states the biosynthesis of the adult form of enzymes is preferentially « repressedwhile the fetal, or less differentiated molecular form would be derepressed.

Resurgence of two Fetal-type of aldolases (A and C) in some fast-growing hepatomas

We have previously demonstrated the resurgence of aldolase “A” (Muscle type) in hepatomas. In this communication we shown that in some solid transplantable hepatomas, this aldolase “A” is hybridized with a third type of aldolase present also in fetal-liver. The kinetic and electrophoretic properties of this third type of aldolase are identical to those found in aldolase “C” (Brain type).