Morphological and functional heterogeneity of rat ascitic hepatoma Zajdela cells

This work is devoted to the study of molecular and cellular mechanisms of disdifferentiation during neoplastic transformation of cells by investigating the malignant tumor cell heterogeneity. We have revealed two cell fractions of hepatoma Zajdela which differ in patterns of growth in primary culture. The cells of one fraction were attached to the culture plastic and grew in a monolayer (S-fraction), whereas cells of another fraction floated in the culture medium (F-fraction). Using method of lifetime supervision of primary culture cells (1-2 passages) at the limit of the resolving power of DIC-microscopy it has been revealed, that both fractions contain cells of several types. Some of them were specific for one of the fractions, and others were found in both fractions, but their frequencies differed. It has been shown by the same method, that long separate cultivation of these fractions in vitro (more than 50 passage) change both cellular structure and the initial ratio of different types of cells in both fractions. According to DNA flow cytometry, the cells of both fractions were hypotetraploid and had insignificant differences in DNA contents. After adaptation to in vitro conditions, S-fraction cells raised their proliferative activity in comparison with the F-fraction cells, and after long cultivation showed 2.3 times higher DNA content. Greater amount of cell surface laminin, a hepatocellular carcinoma marker, was observed on F-fraction cells than on S-fraction cells. Interfractional distinctions were confirmed also by immunologic assessment of hepatoma cells resistance to natural killer lyses: the sensitivity of S-fraction cells in primary culture was 2.4 times higher than F-fraction cells sensitivity, and, after long cultivation, F-fraction cells became practically resistant to cytotoxic action of natural killers. Based on the data obtained, the most probable paths of cell disdifferentiation during hepatoma Zajdela formation and during long cultivation of this tumor cells in vitro are discussed.     

Interaction between ricin and Zajdela ascitic hepatoma cells

The binding to and toxicity of ricin on Zajdela hepatoma ascites cells were studied. The kinetic analysis of [125I]-ricin binding to hepatoma cells indicated that maximal specific binding was reached within 30 min. at 4 degrees C and 60 min. at 25 degrees C and that toxin binding to hepatoma cells was saturable. When the binding data were plotted according to the method of Scatchard, curvilinear graphs were obtained suggesting that hepatoma cells have both high and low affinity receptors for ricin. The number of high and low affinity receptors was identical at 4 and 25 degrees C, i.e., 8 x 10(5) and 1.2 x 10(7) sites per cell respectively. However, the capacity of hepatoma cells to bind ricin is stronger at 4 degrees C than at 25 degrees C. The toxic activity of ricin was totally abolished in the presence of lactose suggesting that ricin binding to cells occurs through binding sites containing galactosyl residues.     

Biosynthesis of high molecular weight polylactosamine-type glycopeptides in rat Zajdela hepatoma ascites cells

The first steps of the biosynthetic pathway of high molecular weight polylactosamine-type glycopeptides from rat Zajdela hepatoma cells were studied by pulse-chase experiments, biochemical analysis and by inhibition of N-glycosylation. It is clear that this process involves firstly the transfer of a lipid-linked high-mannose oligosaccharide precursor to a protein moiety in a similar way to that of N-linked glycopeptides of a more common size range according to the classical 'cycle of dolichol'. In the presence of enzymes which are inhibitors of the processing of high-mannose oligosaccharide chains, this class of oligosaccharides was considerably increased, whereas polylactosamine chains and lower complex N-linked glycopeptides were concomitantly decreased in the same kinetics and the same ratio. As expected in the presence of N-methyldeoxynojirimycin, which is an alpha-glucosidase inhibitor, high-mannose oligosaccharides remained glycosylated and are mostly of the Glc1-3Man9GlcNAc type. In the presence of swainsonine, which is an alpha-mannosidase (EC 3.2.1.24) inhibitor, these chains were devoid of glucose residues. In addition, some chains displayed hybrid structures. It appears, therefore, that the first steps of the biosynthesis of polylactosamine-type and N-linked oligosaccharides of a more common size range proceed similarly and that differences between their biosynthetic pathways occur during the elongation phase, which leads to their final respective structures. Glycopeptides prepared from the cell surface by mild trypsin treatment as well as from entire cells, previously treated or not by processing inhibitors, display the same gel filtration patterns indicating that modifications in protein glycosylation do not prevent glycoprotein insertion into the cell membrane.     

Correlation of membrane-bound and free ribosomes in normal rat liver, Zajdela hepatoma rat liver and ascite cells proper]

The content of membrane-bound ribosomes in normal rat liver cells is 3 times as high as compared to that of free ribosomes. (K=membrane-bound ribosome RNAs divided by free ribosome RNAs=3, the opposite effect being observed in case of ascites hepatoma cells. A considerable increase in the free ribosome fraction in the liver of hepatoma-bearing rats occurs by the sixth day due to a decrease in the content of hepatoma-bearing rats occurs by the sixth day due to a decrease in the content of membrane-bound ribosomes (K=0.6). Similar, but less-pronounced changes were observed in liver cells of control animals after 48-hour starvation (K=0.9), simulating the condition occurring during the last days of tumour animals' life. Thus, changes in the rativ of membrane-bound to free ribosomes in liver during the ascites tumour growth are probably specifics and are not only due to anorexia in Zajdela hepatoma animals.     

Membrane antigens of Zajdela ascitic hepatoma]

The membrane antigens of Zajdela ascitic hepatoma cells were investigated. Living cells were studied by immunofluorescence method, and solublized membrane preparations by the precipitation reacting in agar gel. Testing of the tumor cells with organospecific anti-kidney serum caused a specific fluorescence of tumor cells surface. This can be due to incorporation into the antigenic structure of the Zajdela hepatoma cell membranes of at least one organospecific antigen. Treatment of the tumor cells with organospecific anti-liver serum led to specific fluorescence of tumor cells surface. In solubilizates of the tumor cells one of the three organospecific antigens peculiar for the normal liver cells, was detected.     

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.     

Interaction of laminin with the plasma membrane components of ascitic Zajdela hepatoma cells

The laminin affinity chromatography was used for isolating laminin-binding proteins from the plasma membrane of Zajdela hepatoma cells synthesizing laminin. These were components with mol. weights about 80, 67, 60, 55, 52, 48 and 43 kDa. The isolation of laminin integrin receptors from plasma membranes of Zajdela hepatoma cells in the presence of MnCl2 detected only a protein with mol. weight about 80 kDa in EDTA-elution conditions. This protein was identified by mass spectrometry method as the 78 kDa glucose-regulated protein precursor (GRP78). It belongs to the family of 70 kDa heat shock proteins, recently GRP78 was reported to be localized on the surface of different cell types, including hepatocytes.     

Fractional composition of cytoplasmic RNP-particles in the cells of Zajdela ascitic hepatoma and the liver of a tumor-bearing animal]

The relative content of poly(A)-RNA in the cytoplasm was greater in the cells of Zajdela hepatoma and the liver of tumour-bearing rats than in the normal hepatocytes of rats. Besides, the tumour cells (and to a lesser extent the hepatocytes of the tumour-bearing animal) were characterized by the changes in the ratio of the polyribosomes and monoribosomes usual for normal hepatocytes (and correspondingly between the m-RNP-particles and the informosomes) in favour of the latter, this pointing to definite changes in their protein-synthesizing apparatus. As judged by some of the parameters, the cells of the tumour-bearing by some of the parameters, the cells of the tumour-bearing animal occupied an intermediate position between the normal and tumour cells.     

Revelation and identification of laminin in the structure of plasma membrane of ascitic Zajdela hepatoma cells

Cell dysdifferentiation during neoplastic transformation is a crucial problem of cell biology and oncology. Antigenic diversion of cancer cells is a typical characteristic of dysdifferentiation. It involves the appearance of antigens which are unusual for normal tissue of this type. Components organospecific for membrane proteins of normal kidney were previously found among plasma membrane proteins of hepatocellular rat tumors, rat hepatocytes after carcinogen treatment, and regenerating liver, respectively. In the present work we showed that a protein with mol. weight about 200 kDa reacting with laminin-1 immunoserum is the basic component of plasma membranes of the rat Zajdela hepatoma cells, which is responsive for organospecific anti-kidney immunoserum in Western blot. A mass-spectrometer analysis of trypsin proteolysis fragments was carried out in SDS-PAGE slices containing the investigated component. The analysis showed the presence of beta1, beta2 and alpha4 laminin chains peptides. The component with mol. weight about 180 kDa, found in the Western blot with laminin-1 immunoserum, was also subjected to the mass spectrometer analysis. As a result, a gamma1 laminin chain was found. An increased amount of laminin was revealed in the ascitic liquid and sera of rat with developed Zajdela hepatoma, in comparison with sera of normal rats. In addition, we found the appearance of laminin on the hepatocyte surface on the 4th day after hepatocarcinogen injection (N-diethylnitrosamine, DENA). Thus, for the first time tumor associated antigens were revealed and identified in the structure of plasma membranes of Zajdela hepatoma cells, being specific to rat kidneys. Our results allow to conclude that in the process of carcinogenesis in rat liver laminin synthesis occurs, which is also characteristic of the rat hepatoma Zajdela cells.     

Adaptation of Zajdela ascitic hepatoma cells to monolayer growth: change in the cell ganglioside pattern

The Zajdela hepatoma is a transplantable ascitic tumor of the rat, characterized by a very simple ganglioside pattern, GM3 being the main compound. When these cells are adapted to monolayer culture, they undergo a maturation process and the total cellular ganglioside concentration increases progressively; GM2, GM1 and GD3 amounts rose and GD1a accumulated. These modifications in the ganglioside pattern complexity are not affected by the addition of ascitic fluid to the cultures, nor by growth in serum free, hormone-supplemented medium. They are totally reversible when the cultured hepatoma cells are reinjected into a rat and developed an ascitic tumour. Cell growth control and adhesion processes could be related to the maturation process of these hepatoma cells growing in monolayer, which may constitute a convenient model for further investigations on the regulation of membrane glycolipid composition by the external environment.     

Morphological and functional heterogeneity of Zajdela rat ascites hepatoma cells

This work studies the mechanisms of dysdifferentiation at cell neoplastic transformation based on the example of heterogeneity of the cell populations that form malignant tumors. Two natural fractions of Zajdela rat hepatoma cells are revealed that differ in the type of growth in the primary culture. Cells of one fraction are attached to substrate and are growing in monolayer (S-fraction), whereas cells of the other fraction are floating in the culture medium (F-fraction). Using the method of supravital observation of the primary culture cells (of 1–2 passages) at the limit of resolution of DIC microscopy, it has been established that both fractions contain cells of several types. Some of these cells are specific to one of the fractions and others are present in both fractions, but with different frequencies. Using the same method, it has been shown that, at the long-term separate cultivation of the fractions in vitro (more than 50 passages), both the cell composition and the initial ratio of cells of different types are changed in both of them. According to the data of flow DNA cytometry, cells of both fractions are hypotetraploid and have insignificant differences in the amount of DNA. After adaptation to conditions of cultivation in vitro, S-fraction cells have been found to have elevated proliferative activity compared to the cells of F-fractions; after long cultivation, the fractions already differ significantly (2.3 times) by this criterion. The content of the cell surface laminin, a marker of hepatocellular carcinomas, is higher on cells of the F-fraction than on those of the S-fraction. The interfraction differences are confirmed by immunologic estimations of the resistance of hepatoma cells to lyses of natural killer cells; cells of the S-fraction of the primary culture are 2.4 times more sensitive than cells of the F-fraction, while, after long-term cultivation, cells of the F-fraction become almost resistant to the cytotoxic action of natural killer cells. Based on the obtained data, the most probable pathways of the dysdifferentiation of rat hepatocytes upon the establishment of Zajdela hepatoma and at the long-term cultivation of cells of this tumor in vitro are discussed.     

Zajdela hepatoma cells cultured in vitro

The goal of this work was to study cellular mechanisms of tumor progression and metastasizing. As a result of explantation of cells of rat Zajdela ascitic hepatoma, we obtained two transplantable cell cultures—monolayer (ZH-ad) and suspension (ZH-fl)—that differ in levels of cell differentiation and tumorigenicity. By using tumor-specific immune serum, we revealed tumor-associated antigens, synthesis of which is reduced or inhibited in ZH-ad cells, in outer membranes of the ZH-fl cells. Intraperitoneal injection into rat of 0.5–12 × 10⁶ ZH-fl cells leads to development of an ascitic tumor and death of 100% of animals, whereas, in the case of administration of ZH-ad cells, to achieve a tumorigenic effect, the minimal dose needs to be elevated to 20 × 10⁶ cells. Clonogenic analysis of the ZH-fl cells revealed three types of the formed clones—nonadhesive sphere colonies and two types of monolayer clones differing in proliferative potential, shape of colonies, and cell composition. Upon reaching a critical size, the spheres disintegrated, with separation of single cells and islands of different sizes, some of them being attached with monolayer formation. Three clonal cell lines were obtained: 1C as a result of expansion of a spherical clone and 4G and 10E from monolayer clones. We established that there is tumorigenicity of the 1C cell line, which, at a dose of 107 cells, led to the development of ascites and to the death of 50% of animals. The presented results indicate the existence in the ZH-fl cell population of tumor-initiating cells generating spherical clones—floating multicellular islets that, in culturing in the complete growth medium, are partly differentiated and are attached with monolayer formation.     

Uptake in vitro of nucleic acid precursors and nucleic acids by Zajdela ascitic hepatoma cells.

Zajdela ascitic hepatoma cells are shown to take up pyrimidine bases at much lower rates than obtained in slices from normal rat liver. The rates of uptake of adenine and uridine by the Zajdela cells are, however, as high as in the slices. Like the slices, again, the Zajdela cells take up E. coli RNA and DNA at very low rates but, unlike the slices, thses cells degrade rapidly the RNA taken up. The Zajdela cells resemble parenchymal cell suspensions derived from normal rat liver in regard to the uptake of pyrimidine bases and the ability to degrade heterologous RNA.