Properties of the genome in experimental hepatomas: variations in the composition of chromatin

The chromosomal proteins of two rapidly growing and poorly differentiated Morris hepatomas were compared with those of liver from normal and tumor bearing animals. While the total quantity of histone associated with DNA in all tumor and liver chromatin preparations studied were similar, tumor chromatin contained an increased quantity of nonhistone chromosomal proteins. Variations in specific classes of histones and nonhistone chromosomal proteins associated with the genome of the two tumors, host liver and liver of tumor bearing animals were observed.     

Properties of the inactive ribosomal components in rat liver and hepatoma

1. The relative concentrations of the inactive ribosomal components were compared in normal and regenerating rat liver and in two transplantable rat hepatomas (hepatomas 7800 and 5123D). 2. The size of the ribosomal-subunit pools in normal liver was not significantly affected by partial hepatectomy or neoplasia although, as shown previously, significant changes do occur in the monomer pool. 3. Further, the subunit pools in both liver and hepatoma were not significantly influenced by several treatments that caused dramatic changes in the size of the ribosomal monomer (and dimer) pools. 4. The high concentration of inactive monomers and dimers in the hepatomas appears to arise from limitations at the translational level, since they can be incorporated into pre-existing polyribosomes under the influence of cycloheximide.     

Nuclear topoisomerase I and DNase activities in rat diethylnitrosamine-induced hepatoma, in regenerating and fetal liver.

DNase activity in the presence of Ca2+ + Mg2+, Mg2+ alone, Mn2+ alone, or EDTA, and topoisomerase I activity were measured in nuclear extracts of diethylnitrosamine (DEN)-induced hepatomas, regenerating, fetal, and normal rat livers. In hepatoma tissue, the Ca/Mg-dependent DNase activity was lower than in normal tissue and nearly the same as in fetal liver. In the poorly differentiated hepatomas, Mn-dependent DNase activity was higher than in both moderately and well differentiated ones and than in normal liver tissue. The activity of topoisomerase I in hepatomas and in regenerating liver was lower than in normal liver tissue.     

Increased fraction of acid-soluble proteins in 0.35 M NaCl extracts of nuclei from rat liver tumors

1. The fraction of proteins extracted from nuclei with 0.35 M NaCl and soluble in 2% trichloroacetic acid was examined in five Morris hepatomas and rat liver. 2. This fraction was a much greater percentage of the total 0.35 M NaCl soluble proteins in the tumors than in normal or regenerating liver. 3. In part, this difference was due to proteins with molecular weights greater than high mobility group proteins. 4. The conditions for precipitation of high mobility group proteins 1 and 2 with trichloroacetic acid were found to differ in hepatoma and liver fractions.     

Gel-based proteomics of liver cancer progression in rat

A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control "sham-operated" liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.     

Hepatoma-associated nuclear matrix nonhistone antigens

Polyclonal antibodies generated against a group of high molecular weight nonhistone proteins from Morris hepatoma 7777 were used in immunological studies of hepatoma-associated nonhistone proteins in rat and hamster. We revealed the presence of cross-reactive antigens in rat Morris hepatomas 7777 and 8994, and in hamster Kirkman-Robbins hepatoma, but not in normal rat or hamster livers. These specific nonhistone proteins were found to be preferentially localized in the nuclear matrix of rat Morris hepatoma 7777 as well as hamster Kirkman-Robbins hepatoma.     

Localization of antigens in the chromatin of normal rat liver cells, not detectable in hepatoma chromatin

Antibodies against rat liver chromatin interact with homologous chromatin as well as with chromatin of Zajdela ascite hepatoma and solid hepatoma 27, but not with the nuclear matrix isolated from these hepatomas. Rat liver chromatin regions hypersensitive to DNAase I and endogenous Mg2+-dependent nuclease are enriched with immunogenic nonhistone proteins. Using antiliver IgG pretreated with chromatin of Zajdela ascite hepatoma and solid hepatoma 27, it was shown that liver chromatin antigens that are not detectable in hepatoma cells are localized in hypersensitive to nucleases chromatin regions buy not in actively transcribed ones.     

Negative correlation of L-glutamine concentration with proliferation rate in rat hepatomas

The concentration of L-glutamine was determined in freeze-clamped samples of normal liver of adult male fed rats (5.7-6.1 mumol/g) and in transplantable hepatomas of vastly different proliferative rates. The L-glutamine concentration in the slowly growing hepatomas was in the range of the normal liver and it decreased in relation to the increase of hepatoma growth rate, in the most rapidly growing tumors amounting to 12% of that of normal liver. In 24-hour regenerating liver, the glutamine content was slightly reduced (by 17%). In normal rat organs of high cell renewal, such as testis, intestinal mucosa, spleen, and thymus, the L-glutamine concentration was 18 to 46% of that of normal rat liver. The L-glutamine content was similar in rat brain and liver, but it was 1.6-fold higher in the heart, and low in the blood. Glutamine synthetase (EC 6.3. 1.3) activity in normal adult liver of ACI/N strain rats was 1,000 nmol per hr per mg protein; the activity increased in the very slowly growing hepatoma 20, but decreased markedly in all the other hepatomas. Thus, glutamine synthetase activity was essentially transformation-linked. The negative correlation of glutamine content with growth rate in transplanted hepatomas appears to be more closely linked with the activities of enzymes that utilize glutamine. The low L-glutamine concentration in the rapidly growing hepatomas provides a potential marker for anti-glutamine chemotherapy selectively targeted against the glutamine-utilizing enzymes.     

Chromatin protein methylation in proliferating liver and hepatoma cells.

Chromatin protein methylation in proliferating liver cells and hepatomas was examined in vivo and in vitro. Methylation in vivo was estimated using 2-14 C-L-methionine and 3H-methyl-L-methionine. 3H/14C ratios were calculated for crude histone and nonhistone chromatin protein fractions and compared to those calculated for serum albumin. From this, determination of a methylation index was calculated using serum albumin as a standard. Results of this study indicate that both histones and nonhistone chromatin proteins are methylated to a greater extent in proliferating liver cells and in hepatomas than in control preparations. These observations are generally supported by our in vitro methylation studies. The results are discussed in terms of chromatin protein methylation and its possible relationship to DNA replication.     

Fractionation of rat-liver-chromatin nonhistone proteins into two groups with different metabolic rates.

In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40 000-60 000 remained in the alkali-resistant group. L-[14C]Leucine pulse-labelling experiments showed that the specific radioactivity of the alkali-labile nonhistone proteins was 2-3 times higher than that of the alkali-resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver. In the course of a 21-day chase the specific radioactivity of the alkali-labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali-resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali-resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase. A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali-resistant and is conserved in chromatin like histones.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

Isolation and characterization of the plasma membranes from rat ascites hepatomas and from normal rat livers, including newborn, regenerating, and adult livers.

Plasma membranes (PM) were isolated from island-forming types of rat ascites hepatoma (AH 130, AH 602, and AH 7974) and from their free-cell sublines (AH 130FN and AH 7974F), and were characterized in terms of electron-microscopic morphology, marker enzyme activities, and lipid contents. The results were compared with those of the PM isolated in a similar way from newborn, regenerating, and adult livers. The marker enzyme activities, such as Na+, K+-insensitive Mg2+-ATPase [EC 3.6.1.3] (Mg2+-ATPase) and 5'-nucleotidase [EC 3.1.3.5], as well as the phospholipid composition of the PM isolated from hepatomas by Wallach's nitrogen gas cavitation method were similar to those obtained with the PM isolated by a modification of Emmelot's method, although the former method gave a much lower yield in terms of protein than the latter. Based on the modified Emmelot method, sufficiently pure PM preparations could be obtained from the hepatomas in the form of large membrane sheets without any contamination by other identifiable components, as determined with an electron microscope, and with high specific activities of the marker enzymes, such as Na+, K+-sensitive ATPase [EC 3.6.1.3] (Na+, K+ -ATPase), Mg2+ -ATPase, and 5'-nucleotidase. As for the characteristics of the hepatoma PM, lower specific activity of 5'-nucleotidase and higher fatty aldehyde molar percentages in total phospholipids were noted in all the PM from the hepatomas in comparison with normal liver PM of various origins. The PM from the hepatomas showed an increased amount of cholesterol (mumole per mg protein), whereas actively growing newborn and regenerating livers gave rather lower amounts in comparison with that of normal adult liver.     

Effects of formamidoxime on macromolecular synthesis in regenerating liver and hepatomas

Formamidoxime caused an inhibition of [³H]thymidine incorporation into DNA in regenerating liver and transplanted hepatomas of different growth rates when administered by i.p. injection to rats. A dose level of formamidoxime (500 mg/kg body weight) which caused at least a 75% inhibition of DNA synthesis in these tissues had little or no effect on the incorporation of [³H]orotate into total RNA. After administration of formamidoxime there was no significant effect on amino acid nitrogen concentration in the tissues. The incorporation of ³H-labeled amino acids into acid-soluble material, cytoplasmic proteins and acid-insoluble nuclear proteins were either unaffected or showed only small changes after treatment of rats with the drug. In regenerating rat liver and Morris hepatomas 7787 and 7777, formamidoxime caused an inhibition of incorporation of ³H-labeled amino acids into both lysine-rich and arginine-rich histones. In the host livers of rats bearing the transplanted hepatomas, histone synthesis was less affected. The data indicated that formamidoxime causes inhibitory effects which are similar in nature and extent to those previously shown for the structurally related compound, hydroxyurea, in the regenerating rat liver and demonstrated that these effects can also be observed in liver tumors.     

The effect of preparations of non-histone chromosomal proteins on the expression of hetero-organic membrane antigens of kidney origin in a primary rat hepatocyte culture

The expression of membrane hetero-organic antigens of kidney origin, associated with the rat hepatomas in primary culture of intact adult rat hepatocytes, was investigated by means of the indirect immunofluorescence method using a specific immune serum. These antigens were observed on the membrane of some hepatocytes after their contact with nonhistone chromosomal proteins (NHCP), which were obtained from the kidney of intact rats from cells of hepatoma 27 and Zajdela hepatoma, or from the carcinogenic liver after a single diethylnitrosamine injection. Negative results were obtained after the incubation of hepatocytes in the medium lacking some of NHCP, or in that with NHCP obtained from the liver of intact rats.     

Transforming growth factor-beta (TGF-beta) isoforms in rat liver regeneration: messenger RNA expression and activation of latent TGF-beta.

Expression of transforming growth factor-beta s (TGF-beta s) 1-3 was studied in normal liver and during liver regeneration after partial hepatectomy in the rat to determine whether each of these isoforms might be involved in hepatocyte growth in vivo. Expression of the mRNAs for all three TGF-beta isoforms increases in the regenerating liver. In addition, the levels of expression of the mRNAs for several extracellular matrix proteins, including fibronectin, vitronectin, laminin, and collagen, also increase in the regenerating liver. Immunohistochemical staining analysis shows a similar distribution of all three TGF-beta s in normal and regenerating liver; however, in both tissues, the level of expression of TGF-beta 1 is 8- to 10-fold higher than that of TGF-beta 2 as determined by sandwich enzyme-linked immunosorbent assay. Expression of all three TGF-beta mRNAs is restricted to liver nonparenchymal cells. Although hepatocytes from normal and regenerating livers do not synthesize TGF-beta, they are sensitive to inhibition of growth by all three TGF-beta isoforms. Hepatocytes from regenerating livers are capable of activating latent TGF-beta 1 complexes in vitro, whereas normal hepatocytes are not. The different TGF-beta isoforms may function in an inhibitory paracrine mechanism that is activated during liver regeneration and may also regulate the synthesis of extracellular matrix components in the regenerating liver.     

Gangliosides of hepatoma 27, normal and regenerating rat liver.

The highly malignant rat hepatoma 27 was found to have increased amounts of lipid-bound sialic acid as compared with normal liver whereas in regenerating liver the lipid-bound sialic acid level was reduced. In contrast to the liver the hepatoma contained higher amounts of disialogangliosides and no trisialogangliosides, which are abundant in the liver. The main disialoganglioside of the hepatoma had no analogue among the liver gangliosides and was identified as Gal-GalNAc(AcNeu-AcNeu)-Glc-Cer (GD1b), which in other tissues is known to be a precursor of trisialogangliosides. These findings may be explained by a reduced activity of glycosyltransferases in the hepatoma and apparently do not simply reflect differences in growth rate since the ganglioside pattern of regenerating rat liver was not altered significantly in comparison with the liver. Liver and hepatoma microsomes were found to be enriched in gangliosides as compared with whole cells, liver mitochondria were slightly poorer, while the ganglioside level of hepatoma mitochondria was much higher than that of the hepatoma cells. It thus appears that the existing image of the plasma membranes as the only sites of high ganglioside concentration may not hold true for weakly differentiated hepatomas of high malignancy.     

Neoplastic transformation-linked alterations in adenylosuccinate synthetase activity.

Adenylosuccinate synthetase has been measured in rats in normal, differentiating, and regenerating liver, transplantable hepatomas of different growth rates, kidney cortex, and a transplantable kidney tumor. The activity was increased 1.6 to 3.7-fold in all the tumors. The activity showed no correlation with the degree of histological or biochemical differentiation of the tumors, nor with their growth rate. Adenylosuccinate synthetase activity in regenerating liver was unchanged and in neonatal liver it was much lower than in adult liver. It is concluded that the ubiquitous increase in the tumors of liver and kidney was linked with the neoplastic transformation.     

Altered activation state of hydroxymethylglutaryl-coenzyme A reductase in liver tumors

Extensive studies have demonstrated that the normal inhibition of cholesterol synthesis by cholesterol feeding is decreased in all hepatomas studied in vivo. This loss of the normal feedback regulation of cholesterol synthesis has been shown to be due to the failure of cholesterol ingestion to inhibit the activity of hydroxymethylglutaryl (HMG)-CoA reductase. The basis for this absence of feedback control of cholesterogenesis is unknown. Studies to date have not demonstrated structural or kinetic differences between the HMG-CoA reductase of normal liver and hepatoma. The present study, however, demonstrates significant differences in the activation state of HMG-CoA reductase from normal liver and hepatoma. In normal liver only approximately 10-20% of the microsomal HMG-CoA reductase is in the dephosphorylated, active form while 80-90% is in the phosphorylated, inactive state. In contrast, in three different Morris hepatomas in vivo, from 53 to 73% of the HMG-CoA reductase is in the active state. That the increased activation state in hepatomas is a property of tumor tissue and is not solely due to rapid growth is demonstrated by the fact that in both fetal and regenerating liver an enhanced activation state of HMG-CoA reductase is not observed. Additionally, preincubation with magnesium and ATP results in the inhibition of HMG-CoA reductase both in tumor and in liver. Presumably, this decrease in HMG-CoA reductase activity is due to the phosphorylation of the enzyme. Similarly, the preincubation of tumor and liver microsomes with phosphatase results in an increase in HMG-CoA reductase activity presumably by the dephosphorylation of the enzyme to its active form. The relationship between the altered activation state of HMG-CoA reductase in hepatomas and the reduction in the feedback regulation of this enzyme in liver tumors remains to be explored.     

The histochemical demonstration of gamma-glutamyl transpeptidase activity in different populations of rat liver during azo dye carcinogenesis

To better assess the reliability of gamma-glutamyl transpeptidase (gamma-GTase) as a marker of preneoplastic liver lesions and hepatomas, the gamma-GTase activity of different cell populations was examined in liver sections from rats fed 4-dimethylaminoazobenzene. The results indicated that the biliary ductular cells in trabeculae of cirrhotic livers may exhibit appreciable gamma-GTase activity in addition to that shown by islands of regenerating parenchyma. At later stages of azo dye carcinogenesis, the epithelial cells of bile duct cysts and cholangiomas, as well as those of hepatomas, gave positive reactions for gamma-GTase. Thus biochemical data on liver gamma-GTase in different models of hepatocarcinogenesis cannot be translated directly in terms of alterations in a particular cell type unless such interpretation is justified by parallel histochemical investigations.