[Characteristics of the spontaneously transformed human endothelial cell line ECV304. II. Functional responses of the ECV304 cells].

Functional responses of the spontaneously transformed human endothelial cell line ECV304 were studied in order to asses its applicability as an endothelial cell model for studying angiogenesis and signal transduction. The dependence of proliferation activity of this line on the presence of growth factor was shown. The absent serum in culture medium resulted in blocking of cells in G1-phase of a cell cycle which is not typical for tumor cell lines. Low doses of beta particles emitted during [3H]thymidine decay resulted in blocking the proliferation of these cells in G2M-phase in a dose-dependent manner. Incubation of the cells with another source of beta particles, 3H2O, under condition of equal specific activities of tritium resulted in preferable accumulation of the cells in S-phase. The different efficiency of beta particles of tritium as a part of 3H2O molecule or thymidine demonstrates that various mechanisms are responsible for various check points. The check point of G1/S is absent and that complies with the presence of deletion of chromosome 9 in locus p21. The level of NO produced by constitutive form of NO-synthase in ECV304 cells was relatively low and not modified by inducible NO-synthase inhibitors. The data obtained suggest that ECV304 line cells retained the properties of the initial spontaneously transformed cell line obtained from human umbilical vein (HUVEC) as well as they can be used as a model system for further studies of the properties of vascular endothelial.

[The blocking of the proliferation of the human endothelial cells in culture by beta-irradiation from internal and external sources of the radiation. S-block is induced by the 3H2O beta-particles].

Recently we shown that low doses (0.12-0.46 Gy) of (methyl-3H)-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. The temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the induction of the G2-block estimated by flow cytometry analysis of DNA content and in the induction of the chromosome aberrations (bridges and fragments in anaphase). The aim of this study was the comparative investigation of efficiency of beta-rays emitted 3H from 3H-thymidine and 3H2O by several of the cellular parameters. Here we shown that at the equal conditions of the incubation of the cells in medium with 3H2O induced the accumulation cells in S-phase without decreasing of the mitotic activity and without increasing of the chromosome aberrations level. Unlike from 3H2O the incubation of the cells with 3H-thymidine induced the accumulation cells in G2-phase with decrease of the mitotic activity and with increase of the chromosome aberrations level. Concurrent treatment cells with 3H-thymidine and thymidine abrogate these cellular effects of the 3H-thymidine. Inhibitor ATM-kinase caffeine abrogate as G2-block as S-phase block. These results suggest that the low-dose beta-radiation activates S-phase and G2-phase checkpoints requiring ATM-mediated signal transduction pathway. The factors, which impact on the efficiency of the internal and of the external sources of the irradiation, depend on theirs disposition in relation to radiosensitive target--DNA was discussed.

[Internal and external sources of the radiation induce the blocking of the proliferation of the human endothelial cells in culture. G2-block is induced by beta-particle 3H-thymidine and gamma-rays 137Cs].

We found that low doses (0.12-0.46Gy) of (methyl-) 3H-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. Temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the G2-block estimated by flow cytometry analysis of DNA content. Furthermore, the induced the high level of the chromosome aberrations (bridges and fragments in anaphases). 1Gy of gamma-ray 137Cs and 0.005 Gy of beta-rays induced the same per cent of the aberrant anaphases. Apparently, that the damages of the cellular hereditary structures are responsible for the blocking of the cellular proliferation in G2-phase. We suggest, that the disposition 3H-thymidine into radiosensitive target (DNA) defines the high cytotoxic of the beta-rays.

[Modification of cellular radiosensitivity by NO-synthase inhibitors].

We recently reported that the treatment of V-79 and HeLa cells with nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly reduced the level of the radiation-induced unstable chromosome aberrations. The stereoisomer D-NAME had no effect. We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the NOS-activity. We tested this suggestion on the NO-resistant (ECV-304) and NO-sensitive (HeLa) cells, which were treated with L-NAME or aminoguanidine or D-NAME or cysteamine before gamma-irradiation. There are no significantly differences in radiosensitivity between these cells estimated after exposure by gamma-rays with different doses. However, the radioprotective effect of the NOS-inhibitors manifested only for HeLa. D-NAME had no radioprotective effect neither HeLa nor ECV-304. In contrast NOS-inhibitors, cysteamine treatment EVC-304 reduced the radiation-induced level chromosome aberrations almost twofold. The different mechanisms of the modification of cellular radiosensitivity are discussed.

[Human fetal ventricular cardiomyocytes in vitro: proliferation and differentiation].

In this study, in the primary cell culture of human fetal cardiomyocytes proliferation of myocytes combines with their differentiation. The cells were isolated enzymatically from 19-22 week-old human fetuses and cultured for 14 days. DNA synthesis, ultrastructure and presence of atrial natriuretic peptide (ANP) were examined. In 7 day-old culture, the myocytes make about 60%, in 14 day-old culture--about 50%. Myocytes synthesize DNA and divide mitotically. After a 24 h incubation with 3H-thymidine in 7 day-old culture 1.8 +/- 0.5% of muscle and 25.2 +/- 11.7% of non-muscle cells are labeled, in 14 day-old culture--2.5 +/- 0.5 and 8.1 +/- 1.7% of cells are labeled, respectively. In 7 and 14 day-old cultures the degree of redifferentiation of contractile apparatus in myocytes varies from scattered actin and myosin filaments surrounded by ribosomes to differentiating myofibrils with distinct sarcomeres and Z-discs. Single electron-dense granules, morphologically similar to secretory atrial granules, display ANP-immunoreactivity. Thus, human fetal ventricular cardiomyocytes in cell culture proliferate, differentiate and synthesize ANP for 14 days; this is indicative of vitality of these cells.

[Endothelial vascular grafts (Experimental research)]. / Endotelizirovannye sosudistye protezy. (Eksperimental'noe issledovanie).

The authors present herein their findings obtained in bench-test and experimental studies, which made it possible to work out an original technology of creating an endothelial covering of the inner surface of vascular grafts made of polytetrafluorethylene. The new technology includes the definite sequential processes which are as follows: 1) creation of vascular endotheliocytes; 2) stimulation of growth and reproduction of endotheliocytes; 3) preparation of the graft, including creation of stable positive potential on its inner surface in order to create optimal conditions for endothelization; 4) graft endothelization itself. In order to assess efficacy of endothelial vascular grafts, we carried out a total of 105 experiments on dogs. The experimental conditions made it possible to comparatively study the standard and endothelialized grafts using them in the position of the aortic abdominal portion, carotid and femoral arteries. The new grafts turned out to possess satisfactory performance properties, which precluded formation of thromboses and hyperplasia of the noeintima, simultaneously providing good implantability.

[Heart ventricular cells of neonatal rats in a culture: DNA synthesis, ultrastructure and localization of atrial natriuretic peptide]. / Kletki zheludochkov serdtsa neonatal'nykh krys v kul'ture: sintez DNK, ul'trastruktura i lokalizatsiia predserdnogo natriiureticheskogo peptida.

We determined the optimal conditions suitable for expanding cardiac cells in vitro for their future use in experimental transplantation into injured myocardium of adult animals. Ventricular cardiac cells were isolated enzymatically from 2-3 day-old rats and cultured at different cell densities within 5-7 days to 4 weeks. Mixed cultures of muscle and non-muscle cells were examined by light autoradiography, electron microscopy, and immunogold method. The best results were obtained at a density of 3 x 10(5) cells/ml in the medium, consisting of 90% DMEM and 10% fetal calf serum, during 5-7 days of cultivation. In such cultures myocytes made 62.5 +/- 7.9%. After a 24 h incubation with 3H-thymidine, 22.0 +/- 2.2% of myocytes were labeled. Muscle cells contact with each other and with non-muscle cells, contain myofibrils, contract and display atrial natriuretic peptide (ANP)-like immunoreactivity.

[Modelling of myocardial hypertrophy in vitro for solving problems of medicinal correction]. / Modelirovanie gipertrofii miokarda in vitro dlia resheniia voprosov medikamentoznoi korrektsii.

The work has been done on primary heart culture from neonatal rat ventricle. Cardiomyocyte hypertrophy was modelled using noradrenaline (NA), angiotensin II (AII) and fetal serum, respectively. Cell hypertrophy of primary heart cultures was assessed by measuring the surface area, the scope of protein synthesis estimated by 3H-leucine autoradiography and the contents of nucleic acids in gallocyanin-chromalum stained cardiomyocytes. The structure of myofibrillar apparatus was studied by rhodamine-conjugated phalloidin and indirect immunofluorescence of muscle alpha-actinin. Treatment with 10(-6) M NA increased 3H-leucine incorporation in 9-day old heart culture by 42% without changing cell size. AII in a dose 1 microM stimulated protein synthesis activity by 1.3 fold and the surface area by 1.7 fold, both in 2- and 9-day old primary heart cultures. The maximum stimulation of cell hypertrophy was provided by the medium supplemented with fetal serum. RNA contents in the cytoplasm of cardiomyocytes increased by 7.8 fold and the myocardial cell size by 2.9 fold in serum-supplemented culture by 9 days of cultivation. In the medium with fetal serum, amounts of cardiomyocytes with tetraploid nuclei reached 33%, against 14% in control. Coculturing of myocardiocytes and fibroblasts rendered effects of fetal serum on the growth of myocardiocytes. Cultivation in the presence of 1 microM enalapril, an ACE inhibitor, suppressed the development of cardiac muscle cells hypertrophy. The effect of enalapril depended on the degree of cellular hypertrophy. Addition of 10 microM amiloride to the medium lowered the protein synthesis by 29% independently on the initial cellular hypertrophy.

[The levels of DNA repair in the cells of diploid and tetraploid cell cultures]. / Issledovanie urovnei reparatsii DNK v kletkakh diploidnykh i tetraploidnykh kletochnykh kul'tur.

Levels of unscheduled DNA synthesis after UV-irradiation were investigated in addition to the activities of DNA-repair enzyme uracil-DNA glycosilase in cells of both diploid (XC-D) and tetraploid (XC-T) cell cultures. It was shown that repair levels were increasing directly and proportionally to the cell ploidy.

[Effect of sanguiritrine on the functional activity of fibroblast lysosomes]. / Deistvie sangviritrina na funktsional'nogo aktivnost' lizosomnogo apparata fibroblastov.

Kinetics of sanguiritrine consumption by L cells of LSM substrain was studied in cell culture. About half of the drug used was absorbed by cells within 20 min. Sanguiritrine inhibited the lysosomal hydrolases (cathepsin D, beta-D-galactosidase and N-acetyl-beta-D-glucosaminidase) activity by 50% at concentration 2.10(-4) M. The drug a concentration 4.10(-4) M inhibited acid lipase by 55% and acid phosphatase by 58%.

[The effect of cryopreservation on the cytogenetic characteristics of a cell subline of skin fibroblasts from the Indian muntjac]. / Vliianie kriokonservatsii na tsitogeneticheskie kharakteristiki kletochnoi sublinii fibroblastov kozhi indiiskogo muntzhaka.

After thawing cells, previously cryopreserved in the presence of dimethyl sulfoxide (DMSO), a decrease in their viability and increase in unscheduled DNA synthesis was observed. In 7 days, these parameters restored to the control level. Cryopreservation without DMSO resulted in the decrease in both cell viability and replicative and unscheduled DNA synthesis. In 14 days, these characteristics were seen to return to the normal level. Cryopreservation of cells without DMSO and their preservation in liquid nitrogen induced the frequency of chromosomal aberrations, mostly chromosomal breaks. The frequency of chromosomal aberrations increased with the duration of cell preservation in liquid nitrogen. The normal level was achieved following 7 days after cell thawing. Cells treated with DMSO only (without cryopreservation) display an increased number of chromosomal and chromatid breaks and translocations. Nonrandom distribution of chromosomal aberrations was observed, with particular chromosomes being involved in the appearance of dicentrics and translocations. The data obtained indicate that cryoprotective activity of DMSO is probably associated with the cell repair systems. The detected antimutagenic and mutagenic activity of DMSO may presumably reflect various conditions for its interaction with cells (with or without cryopreservation), as well as it may be specific for the muntjac cell line used in the present work.

[Actin interaction with the plasma membrane fraction of cells of murine fibroblast L sublines differing by growth type in culture]. / Vzaimodeistvie aktina s fraktsiei plazmaticheskikh membran kletok sublinii myshinykh fibroblastov L, razlichaiushchikhsia po tipu rosta v kul'ture.

The association between murine fibroblast L plasma membranes and actin was studied by means of low-shear viscometry of membrane-actin mixtures. Membrane fractions of 3 genetically related sublines of L cells were used differing in cytoskeleton structural organization. The suspension cell subline LS membranes showed actin gelatin activity. On the contrary, the membranes of monolayer cell sublines L-929 and LSM were seen to bind actin and to cause decrease in viscosity. Possible mechanisms of such interactions are discussed.

[Effect of the segregation and accumulation of chloroquine and daunorubicin by L cells (LSM subline) on the activity of lysosomal hydrolases]. / Vliianie segregatsii i akkumuliatsii khlorokhina i daunorubitsina kletkami L (sublinii LSM) na aktivnost' lizosomnykh gidrolaz.

Kinetics of chloroquine and daunorubicin (DNR) uptake by cultured L cells (subline LSM) has been studied. With their constant concentrations in the medium the uptake of both chloroquine and DNR was characterized as a two phase process. Within 1.5-2 hours, these cells accumulated as much as 90 per cent of the total chloroquine and DNR amounts taken up during the whole incubation period. The segregation and accumulation of these substances took place in lysosomes. Chloroquine and DNR concentrations within lysosomes exceed those in the medium by 1100 and 5000 times, respectively. The chloroquine and DNR accumulation in lysosomes inhibited activities of some lysosomal hydrolases tested: cathepsins B and D, N-acetyl-beta, D-glucosaminidase and acid phosphatase. Unlike, the activity of acid lipase was not affected by chloroquine, and was sufficiently stimulated (by 55%) by DNR. The mechanism of inhibition of lysosomal enzymes by chloroquine and DNR is not yet known, although some suggestions are made. Possible consequences of lysosomal activity inhibition for cell metabolism are discussed in addition to a possible role of lysosomotropic agents as regulators of lysosomal functional activity.

[Effect of hyperthermia on protein synthesis in monolayer and suspension cultures of mouse L cells]. / Effekt gipertermii na sintez belkov v monosloinoi i suspenzionnoi kul'turakh kletok myshi L.

Changes in protein synthesis that occurred under the influence of heat shock (HS) in monolayer (L929) and suspension (LS) mouse cell cultures were studied. The rates of protein synthesis determined as 35S-methionine incorporations were seen reduced from the initial level up to 40-60 and 6-13% after HS at 42 and 44 degrees C, respectively. Simultaneously the rate of actin and tubulin syntheses decreased, the decrease being more pronounced in LS cells. According to electrophoresis and autoradiography data, after hyperthermia both the cell cultures were able to synthesize heat shock proteins (HSP), primarily HSP70. After a 40 min HS towards L929 and LS cells at 43 degrees C, the shares of their HSP70 bands in the total label loaded on the gel constituted, resp., 8.8 and 5.4%. The data suggest that L929 cells, with their synthetic activity lower than in LS cells, appear more resistant to HS and are able eventually to synthesize larger amounts of HSP70, compared to the latter.

[Lysosomotropic agents as regulators of the activity of lysosomal hydrolases]. / Lizosomotropnye agenty kak reguliatory aktivnosti lizosomnykh gidrolaz.

Activities of lysosomal enzymes acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-galactosidase, acid lipase and cathepsins B and D were studied after accumulation of neutral red, acridine orange chloroquine and daunorubicin in lysosomes of fibroblasts of the LSM substrain. All the drugs studied proved to be inhibitors of these enzymes except of daunorubicin, which stimulated acid lipase activity. Lysosomotropic drugs are considered as possible regulators of the activity of lysosomes.

[Actin and tubulin synthesis in monolayer and suspension cultures of murine L cells]. / Sintez aktina i tubulina v monosloinoi i suspenzionnoi kul'turakh kletok myshi L.

The rates of total protein, actin and tubulin synthesis were studied for monolayer (L-929) and suspension (LS) cultures of mouse L cell. Data on pulse 34S-label incorporation into the cellular protein pool show that LS characterized by a short cell cycle have, comparatively to L-929, higher rates of protein synthesis and phosphorylation. According to PAGE data, the level of actin and tubulin synthesis in suspension line exceeds that in monolayer one. The correlation between growth conditions, biosynthetic parameters and dynamics of cytoskeleton is discussed.

[Changes in the karyotype and surface properties of L cells during the adaptation of a suspension culture to monolayer growth]. / Izmenenie kariotipa i poverkhnostnykh svoistv L-kletok pri adaptatsii suspenzionnoi kul'tury k rostu v monosloe.

Distribution of cells (L929, LS, LSM) in the two-phase polymer system was studied in addition to characterization of their karyotypes. In the course of LSM cell passages, the increased ratio of cells with a reduced number of chromosomes was found. The results obtained show that in the process of adaptation of the suspension cell culture to the growth as monolayer (the culture of LSM cells) the changes of the cell surface do not dependent on the chromosome number.

[Dependence of the redistribution of ligand-receptor complexes in cultured cells on the type of growth and on the cell cycle phase]. / Zavisimost' pereraspredeleniia ligand-retseptornykh kompleksov u kul'tiviruemykh kletok ot tipa rosta i fazy kletochnogo tsikla.

The process of active redistribution of ligand-receptor complexes on the surface of both suspension (LS) and adapted for growing in monolayer (LMS) sublines of mouse fibroblasts L was studied. The binding of ligands to its specific receptors on the surface of the LS cells induced an accumulation of ligand-receptor complexes on one pole of a cell with the formation of a typical cap. Under the same conditions in the LSM cells the cleaning of processes and lamello-plasma surfaces from the ligand-receptor complexes was registered. The binding of ligands to the surface of the LSM cells, detached with EDTA, induced the same capping process, as in the case of the LS cells. No differences were found in the redistribution capacity of the ligand-receptor complexes in synchronized cultures of the LS and LSM cells in the G1 and S phase of the cycle. But such a redistribution was not registered on the surface of cells in metaphase and anaphase. The accumulation of ligand-receptor complexes was found in the region of cleavage between daughter cells in telophase. These results are in a good agreement with the well-known data on the changes in the cytoskeleton organization during transition from a monolayer to a suspension state and during mitosis.

[Effect of the segregation of neutral red, acridine orange and ammonium chloride by L cells (subline LSM) on lysosomal hydrolase activity]. / Vliianie segregatsii neitral'nogo krasnogo, akridinovogo oranzhevogo i khloristogo ammoniia kletkami L (sublinii LSM) na aktivnost' lizosomal'nykh gidrolaz.

Kinetics of Neutral red (NR) and Acridine orange (AO) uptake by cultured L cells (subline LSM) has been studied. It was found that the uptake of both NR and AO, with their constant concentrations in the medium was characterized as a two-phase process. During 2 hours, these cells concentrated as much as 90% of the total amount of NR and AO taken up during the whole incubation period. The segregation and accumulation of NR, AO as well as NH4Cl took place in lysosomes. NR and AO concentrations within the cells exceed by 600 and 400 times, respectively, those in the medium. NR, AO and NH4+ accumulation in cells resulted in inhibition of the activity of the following lysosomal hydrolases: cathepsins B and D, acid lipase, N-acetyl-beta,D-glucosaminidase, beta-galactosidase, acid phosphatase and galactosyltransferase, the latter being a marker of Golgi apparatus. The effect of lysosomal enzyme activity inhibition on the cell economy, and a possible role of lysosomotropic agents as regulators of the lysosomal apparatus functional activity are discussed.