Investigation of the surface heteroorganic antigens of rat hepatoma cells involved in process of cell proliferation

The investigation of antigenic diversion of hepatoma cells resulting from the expression of heteroorganic kidney antigens has been continued. Tumor-associated heteroorganic antigens 110-115 and 125-130 kDa were detected by immunoserum of narrow specificity in fractions of plasmatic membranes of cells of rat ascitic hepatoma Zajdela and cultured hepatoma HTC; the antigen 75-80 kDa was revealed only for hepatoma Zajdela cells. It has been shown by methods of radioisotope analysis and flow DNA-cytometry that heteroorganic antigens 110-130 kDa can be involved in process of cell proliferation.     

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.     

Comparative investigation of antigens associated with plasmatic membranes of rat hepatoma and myogenic cells using anti-kidney serum

The investigation of antigenic diversion of tumor cells resulting from the expression of heteroorganic antigens has been continued. Tumor-associated heteroorganic antigens with mol. weight 200-210 kDa (identified before as laminin), 105-130, 75-80 and 43 kDa were detected by anti-kidney serum in fractions of plasmatic membranes of cells of rat ascitic Zajdela hepatoma and cultured HTC hepatoma; the antigen 43 kDa was isolated on immunosorbent and identified by mass spectrometry as beta-actin. Anti-kidney serum revealed laminin in fractions of plasmatic membranes of cultured L8 and L6J1 myoblasts, and L6J1 myotubes; apparently, synthesis of laminin by hepatoma and myogenic cells is not connected with their proliferative activity. Besides, anti-kidney serum detected components 38, 42, 44, 48, 62, 78 and 120 kDa, expression of which on myogenic cells surface might be consequence of active cell proliferation and (or) differentiation.     

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.     

Study of surface heteroorganic antigens of rat hepatoma cells participating in the cell proliferation process

The study of the antigen diversion of cells of rat hepatocellular tumors, which is caused by the expression of normal antigens peculiar to renal definitive tissues (so-called “heteroorganic renal antigens”) is continued. Using an immune serum of narrow specificity, in the plasma membrane fractions of Zajdela ascites hepatoma cells and of cultivated HTC hepatoma cells, heteroorganic antigens 110–115 and 125–130 kDa are revealed; the heteroorganic antigen 75–80 kDa is only detected for the Zajdela hepatoma cells. The participation of these heteroorganic antigens in the cell proliferation process is shown by the methods of radioisotope analysis and DNA flow cytometry.     

Detection and identification of the tumor-associated antigens in the fraction enriched with plasma membranes of the Zajdela hepatoma cells

Tumor-associated antigens 45, 57, 80 and 130 kDa were detected using tumor-specific rabbit immune serum in the fraction enriched with plasma membrane of the rat Zajdela hepatoma cells and isolated on the immunosorbent. Revealed proteins were identified as integrin beta-1, ecto-nucleotide pyrophosphatase/phosphodiesterase-3 (E-NPP3), basigin, epithelial cell adhesion molecule (EpCAM), alpha-fetoprotein (AFP) and protein chaperones--glucose-regulated protein 78 (GRP78) and protein disulfide-isomerase (PDI) A1 by mass spectrometry technique. Functions and characteristics of these proteins in tumor cells and some aspects of the Zajdela hepatoma cells origin are discussed.     

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.     

Contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor to the interaction of epidermoid carcinoma A-431 cells with laminin-2/4

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.     

A DNA polymerase activity associated with the skeletal framework of the plasma membranes of a rat hepatoma

A DNA polymerase activity has been identified in the plasma membranes isolated from a rat hepatoma (the Zajdela Ascitic Hepatoma). The enzyme activity was found specifically associated with the detergent insoluble skeleton of the plasma membranes from which it cannot be dissociated by conventional methods. The properties of the enzyme are indistinguishable from those of DNA polymerase-α     

Detection and identification of a tumor-associated heteroorganic antigen of Zajdela hepatoma in non-histone proteins of chromatin

By polyacrylamide gel electrophoresis, a phosphoprotein with mol. weight of 42 kDa was detected in non-histone proteins (NHP) of chromatin of Zajdela ascitic hepatoma cells eluted from phosphocellulose with 0.4-0.5 M NaCl. A protein of the same mol. weight is present in narrow fractions of rat kidney chromatin, but is absent in rat liver. It is suggested that the revealed protein corresponds to the tumor-associated heteroorganic NHP antigen detected earlier in NHP chromatin of rat tumor cells. By MALDI mass spectrometry, this phosphoprotein was identified as ERK2/mitogen-activated protein kinase.     

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.     

Comparative study of chromosomal nonhistone proteins pp23 in cells from different types of tissue]

A specific immunoserum against nonhistone chromosomal protein pp23 from rat kidney was produced. A similar protein was obtained in cells of different tissue specificity (in the liver cells of rats submitted to a single effect of hepatocarcinogens, in rat hepatomas G-27, HTC, Zajdela, in cultivated non-tumor myogenic L6 cells and in kidney cells of newborn rats) by means of immunofluorescent method on using this immunoserum. In all cases a specific fluorescence of the nucleus and nuclear membrane was observed.     

The nuclear matrix of slowly and rapidly proliferating liver cells.

The nuclear matrix of slowly proliferating rat liver is compared with rapidly proliferating regenerating liver and Zajdela ascites hepatoma cells. While no differences are detected in overall ultrastructure, composition or polypeptide profiles of normal liver versus regenerating liver matrices, significant alterations are observed in the polypeptides of Zajdela hepatoma nuclear matrices.     

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.     

Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells

Full length mRNA to a drug-inhibited cell surface NADH oxidase, tNOX or ENOX2, is present in both non-cancer and cancer cells but is translated only in cancer cells as alternatively spliced variants. ENOX2 is a growth-related protein of the external plasma membrane surface that is shed into the circulation and is inhibited by a series of quinone site inhibitors with anticancer activity. To test the possibility that ENOX2 expression might be important to early stages of non-cancer cell development, the expression of the protein was monitored in chicken embryos during their development. Polyclonal antisera to a 34 kDa human serum form of ENOX2 cross-immunoreactive with the drug-responsive NADH oxidase of chicken hepatoma cells was used. The protein was identified based on drug-responsive enzymatic activities and analyses by western blots. The drug-responsive activity was associated with plasma membranes and sera of early chicken embryos and with chicken hepatoma plasma membranes but was absent from plasma membranes prepared from livers or from sera of normal adult chickens and from late embryo stages. The findings suggest that ENOX2 may fulfill some functions essential to the growth of early embryos which are lost in late embryo stages and absent from normal adult cells but which then reappear in cancer.     

Gangliosides of plasma membranes from normal rat liver and Morris hepatoma.

Plasma membranes were isolated from normal rat liver and Morris hepatoma 5123tc by discontinuous sucrose gradient centrifugation. There was an average two and one-halffold enrichment of gangliosides in plasma membranes from normal liver and hepatoma as compared with their respective whole cells. The amount of total gangliosides in plasma membranes from hepatoma was eight times greater than that found in normal liver. This increase resulted from a fivefold increase in hematosides, an eightfold increase in monosialogangliosides and a twenty-twofold increase in disialogangliosides. Trisialogangliosides were present in normal liver but not in hepatoma.     

Beta-1 and beta-4 integrins, and laminin 67 kDa receptors in interaction between A431 cells and laminin isoforms

Laminins, as basal membrane glycoproteins, are able to stimulate cell adhesion and migration, and to influence gene expression. The laminin molecule has a set of bioactive sites that interact with different integrin and nonintegrin receptors, and, as a result, the reaction of the same cell type to different laminin isoforms may be different. The aim of this study was to determine the contributions of both integrins with beta 1 and beta 4 chains and 67 kDa laminin receptor in the interaction of A431 cells with two laminin isoforms: laminin-1 and laminin-2/4. The obtained data show that integrin alpha 6 beta 4 is more specific for interaction with laminin-2/4 than with laminin-1 and takes part in the stage of attachment of A431 cells to laminin. 67 kDa receptor promotes cell spreading on laminin-2/4 and inhibits cell spreading on laminin-1. An assumption was made about the complex action of receptors for interaction of A431 cells with laminins ("integrin alpha 6 beta 4--67 kDa receptors" complex).     

Laminin alpha5-chain is expressed early and widely during the development of the anterior segment of rat eye

The distribution of a novel laminin alpha5-chain in the basement membranes of the anterior segment of rat eye was studied. Frozen sections of embryonic day (E)16--17, post-natal day (P)2, 5, 10, 15 and 30 and adult rat eyes were immunostained for laminin chains alpha2, alpha5, beta1, beta2 and gamma1 and for laminin-5, as well as for EHS-laminin, to visualize all basement membranes. Laminin alpha5-, beta1- and gamma1-chain immunoreactivities were found in the basement membranes of the inner and outer layers of optic cup, lens epithelium, further corneal epithelium and skin of the eyelids in E16--17 rat eyes. In P2 and older rat eyes, laminin alpha5-, beta1- and gamma1-chains were all seen in the basement membranes of the corneal and conjunctival epithelium, Descemet's membrane, lens epithelium, ciliary processes, blood vessels and skin of the eyelids. There was a change in the expression pattern of laminin alpha5, beta1- and gamma1-chains in Descemet's membrane from the endothelial side of the membrane (P2--P15 eyes) to both sides of the membrane after P30. Immunoreactivity for laminin-5 was weak in the basement membrane of E16--17 epidermis, but strong in the basement membrane of corneal, conjunctival and eyelid epithelium in P2 and older rat eyes. Laminin alpha2- and beta2-chains were seen in conjunctival and uveal blood vessels in P15 and older rat eyes. The laminin beta2-chain emerged into the basement membrane of conjunctival epithelium in P30 and older rat eyes, suggesting a role for the laminin beta2-chain in the maturation of conjunctiva. The results suggest that laminin alpha5-chain, possibly in laminin-10 (alpha5beta1gamma1), is early and widely expressed in the basement membranes of developing and adult rat eye and, further, that laminin alpha5-chain is a major laminin alpha-chain, partly in coexpression with the alpha3-chain of laminin-5 in the basement membranes of the anterior segment of the eye in developing and adult rats. © 1998 Chapman & Hall     

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.