[Energy corrective and antioxidative actions of cytoflavin during postischemic period of human dermal fibroblasts in vitro].

The influence of metabolic drug Cytoflavin (CF) with antihypoxic and antioxidative properties on human dermal fibroblasts in a model of ischemia-reoxygenation in vitro was studied. It was revealed that the restoration of ATP synthesis in fibroblasts in the postischemic period was considerably accelerated (in 2.1 times) by the addition of CF to the culture medium. The drug had a cell protective effect of reducing cell mortality during the reoxygenation after ischemia by 2-2.7 times. CF effectively reduced the level of reactive oxygen species (ROS) in fibroblasts after H2O2 treatment which allowed maintaining their survival at the level of control cells. Pretreatment of the cells with CF for one day ensured the maintenance of normal levels of ROS during the investigated time period in the fibroblasts subjected to H2O2 treatment, and reduced H2O2-induced cell death by almost a third compared to control cells. The introduction of CF in culture medium after ischemia showed no influence on Hsp70 synthesis, but led to decrease in GRP78 synthesis, raised after ischemia, to the control level, indicating a resolve of the endoplasmic reticulum (ER) stress and functional normalization of ER.

[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.

[Tumor antigens].

Antigenic distinctions between malignant and normal cells are the key problem of cancer immunology. The researches in this direction allow clarifying not only the mechanism of cell malignancy, tumor progression and the way it escapes from immuno-surveillance of an organism, but also introduce a substantial contribution in clinical tumor immunology and immunotherapy. To date, a lot of the facts about antigens of the tumor cells are accumulated. In the introduced review are given classification and description of tumor antigens. We propose to unite tumor-associated antigens which are characteristic of normal nonhomologous to tumor tissues in a group of heteroorganic antigens. According to definition, such well-known antigens as cancer/testis antigens and onconeural antigens are included in this group.

[Recognition and lysis by natural killers of tumor cells with participation of laminin].

According to the data obtained in the present work, the receptor complex of mouse natural killers (NK) includes laminin, antibody to which blocks EK-activity (NKA regardless of the presence of complement. Preincubation of mouse splenocytes with anti-laminin serum led to a decrease in their NKA towards tumor cells-targets (CT), the NKA activity decreasing 2 times with respect to cultivated cells of rat hepatoma HTC, while 10 times - to cultivated cells of human erythroblastosis K562. Pretreatment of aplenocytes with noraml nonimmune serum did not lead to a change of NKA. Quite different was the pattern after the tumor cell preincubation with anti-laminin serum: pretreatment of CT K562 led to a twofold decrease in sensitivity of these cells to NK-lysis, whereas the pretreatment of CT K562, on the contrary, made them twice sensitive to NK-lysis. Electrophoretic separation of protein of CT plasma membranes with subsequent immunoblotting with anti-laminin immune serum revealed the presence oflaminin on HTC cell plasma membrane, which was identified as laminin 8/9 by the mass-spectrometry method, while no laminin was detected on K562 cells. Preincubation of splenocytes with laminin did nor affect NKA with respect to CT K562 and HTC. Pretreatment of CT K562 and HTC with laminin decreased the NKA to zero. The obtained data allow suggesting a doubtless participation of laminin and its receptors in CT cytolysis by NK.

[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.

[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.

[Tumor-associated hetero-organic antigens as markers of deranged cell differentiation and malignancy]. / Opukholeassotsiirovannye geteroorgannye antigeny kak markery disdifferentsirovki i malignizatsii kletok.

We used rat liver tissue slices and isolated hepatocytes to demonstrate that single exposures to a "weak" carcinogen 4-dimethylaminoazobenzol (DAB) or to a "strong" one, such as N-diethylnitrosamine (DENA), cause identical antigenic restructuring to occur. It presented as formation of membranous heteroorganic antigens of renal origin on hepatocyte surfaces. Antigens were associated with Zajdela's hepatoma; their synthesis lasted longer after exposure to DENA. They, however, were not identified in intact rat liver. Their synthesis, following single exposure, was assayed immunocytofluorimetrically. It is suggested that the antigens be used as markers of cell dysdifferentiation and malignancy and testing substances for carcinogenicity.

[The detection of membrane tumor-associated antigens of Zajdela's hepatoma on the surface of cultured rat cells]. / Vyiavlenie membrannykh opukholeassotsiirovannykh antigenov gepatomy Zaidela na poverkhnosti kul'tiviruemykh kletok krys.

Membrane tumor-associated antigens of the rat hepatoma Zajdela were revealed using immune serum against cell membranes of this hepatoma. By means of SDS-polyacrylamide gel electrophoresis and the following immunoblotting, it was shown that the membrane tumor-associated antigens of hepatoma Zajdela consisted, at least, of 10 components with molecular weights near 22, 25, 31, 33, 42, 46, 56, 65, 96 and 180 kDa. Specific binding of the immune serum with antigens of cultured hepatomas 27 and HTC cell membranes, of myogenic cells and rat kidney fibroblasts may be suggestive of the expression of some tumor-associated hepatoma Zajdela antigens on the surface of these cultured cells.

[The membrane hetero-organic antigens of tumors of hepatocellular and myogenic origins]. / Membrannye geteroorgannye antigeny opukholei gepatotselliuliarnogo i miogennogo proiskhozhdeniia.

Cell-surface antigens of some tumors and homologous definitive tissues were investigated with organospecific immunoserum against rat kidney membranes using methods of immunofluorescence and enzyme immunoassay. Myogenic cells were also tested to resolve the role of the membrane hetero-organic antigens in the process of proliferation. Membrane hetero-organic antigens, commonly attributed to definitive kidney tissue, were found on the cell surface of rat rhabdomyosarcoma RA-2 and cultured cells of rat hepatomas HTC and 27. However, hetero-organic antigens on the surface of active proliferating myogenic cells were not found. It is concluded that membrane hetero-organic antigens, usually attributed to definitive kidney tissue, represent one of characteristic manifestations of neoplastic cell transformation rather than results of intensive proliferation of the cells.