[Dependence of the process leading to phosphorylation of retinoblastoma protein (pRb) on the status of the actin cytoskeleton]. / Zavisimost' protsessa, vedushchego k fosforilirovaniiu belka retinoblastomy (pRb), ot sostaiianiia aktinovogo tsitoskeleta.

It has been confirmed that the main actin-dependent period of G1 phase of the cell cycle is the middle of G1. As the critical points in G1 phase are associated with the synthesis of cycling D and E and with the formation of their active complexes with cyclin-dependent kinases (Cdk's), a study of a possible influence of actin filaments on these processes was performed. The activity of G1 kinases was estimated by the degree of phosphorylation of their specific substrate-retinoblastoma protein (pRb). Immunoblot analysis with specific antibodies to pRb revealed hypophosphorylated from of Rb in lysates of resting Swiss 3T3 cells and the appearance of hyperphosphorylated form after 12 h of EGF and serum stimulation. In was shown that actin filaments distruction by H2CB led to a decrease in hyper phosphorylated form appearance, depending on the depth of resting state of the cells and efficiency of their stimulation by growth factors. Thus, these data may suggest the involvement of actin cytoskeleton in functioning of the transcription chain cyclin/Cdk-R1-E2F.

[The role of the actin cytoskeleton in cell transition through the middle of the G1 phase of the mitotic cycle]. / Rol' aktinovogo tsitoskeleta v prokhozhdenii kletkami serediny fazy G1 mitoticheskogo tsikla.

The influence of dihydrocylochalasin B (H2CB), which selectively disrupts the actin cytoskeleton structure, on G1 phase progression after stimulation of quiescent Swiss 3T3 cells by epidermal growth factor (EGF) was investigated. H2CB was added to the culture medium (10 mg/ml) for different periods of time after cell cycle induction by EGF (10 ng/ml). Efficiency of mitogenic stimulation was estimated by 14C-thymidine uptake 21-23 h after EGF addition. It is shown that the main actin-mediated step is in the middle of G1, from 8 to 12 h after stimulation. Disorganization of actin cytoskeleton structure only during this time led to complete and irreversible block of cell entry into the S phase. On the contrary, the same effect of H2CB during the early period of G1 (first 6 h) led to the increase in 14C-thymidine incorporation in DNA. The specificity of actin-dependent period was proven in experiments with another inhibitor of cell proliferation--dancylcadaverine, whose effect was revealed at the earlier time--4-6 h after cell stimulation. Inhibition of protein synthesis during actin-dependent period (8-12 h) led to the same block of cell progression through G1 as it was seen after actin structure disruption. These data suggest that actin-dependent block is associated with the appearance and functioning of such specific regulators of G1 as cyclins (D, E) and their complexes with Cdk's (G1 kinases) which phosphorylate Rb protein (p1 10) associated with transcription factors E2F.

[The intracellular degradation of 125I-EGF and the release of low-molecular degradation products under conditions of actin cytoskeleton disorganization]. / Vnutrikletochnaia degradatsiia 125I-EFR i vyvedenie nizkomolekuliarnykh produktov degradatsii v usloviiakh dezorganizatsii aktinovogo tsitoskeleta.

The influence of cytochalasin B (CB) on the epidermal growth factor (EGF) intracellular degradation and release of low-molecular products of degradation into the culture medium were investigated using two cell lines, A431 and 3T3. Investigated parameters were registered 3.0-4.5 h after the beginning of 125I-EGF endocytosis at 37 degrees C. With A431 cells it was shown that actin cytoskeleton disorganization significantly reduced the rate of 125I-EGF final degradation: a high amount of 125I-EGF, still retained cell-associated, and a reduced amount of low-molecular degradation products were released into the medium in experiments with CB addition. That is in agreement with our previous results (Barkan et al., 1988), revealing a long-term accumulation of EGF-rhodamine fluorescence in a juxtanuclear area of A431 cells after CB treatment. Nevertheless when similar experiments were performed on Swiss 3T3 cells, previously used as a model for demonstrating the inhibitory effect of CB on DNA synthesis stimulation by EGF (Barkan, Nikol'skii, 1986), we could not find such a distinct influence of CB on the process of degradation of internalised 125I-EGF.

[The mitogenic effect of insulin isolated from the mammalian brain on Swiss 3T3 cells]. / Mitogennoe vliianie insulina, vydelennogo iz mozga mlekopitaiushchikh, na kletki Swiss 3T3.

Mitogenic properties of the insulin derived from pig brain were compared with the action of pancreatic (standard) pig insulin and epidermal growth factor (EGF) using the culture of Swiss 3T3 cells. The brain insulin, likely as the pancreatic insulin, induced uptake of 14C-thymidine by resting cells in a dose-dependent manner at concentration of 0.5-2.0 micrograms/ml in culture medium. However, at equal concentrations of these hormones the proliferating effect of the brain insulin appeared to be 2-fold higher than the effect of the pancreatic hormone. At the same time the mitogenic action of both hormones was lower than that of EGF (10 ng/ml). The additive effect of the brain insulin and EGF, administered in combination, was more pronounced than the effect of the pancreatic insulin combined with EGF. The data obtained suggest a possible participation of brain insulin in the process of nerve cell proliferation.

[Certain biochemical characteristics of an insulin-like substance from the bivalve mollusk Anodonta cygnea]. / Nekotorye biokhimicheskie kharakteristiki insulinopodobnogo veshchestvadvustvorchatogo molliuska Anodonta cygnea.

An insulin-like substance (ILS) was isolated from the visceral organs of the bivalve mollusc Anodonta cygnea by chromatography on a sulfocationite CU-23 and purified by reverse phase liquid chromatography. ILS was shown to be made up to several fractions with Mr ranging from 9 to 20 kDa which have identical amino acid composition but different hydrophobicity and N-terminal amino acids. It was supposed that the heterogeneity of ILS fractions is due to its genetical or posttranslational polymorphism. ILS has a low (0.02%) affinity for the mammalian insulin receptor and a low immune affinity for mammalian insulin and possesses a mitogenic activity which is commensurate with that of the epidermal growth factor. The data obtained suggest that Anodonta cygnea ILS represents a separate branch of a relatively ancient family of insulin-like hormones and growth factors responsible for metabolism and proliferation of invertebrate tissues.

[The stimulation of DNA synthesis in resting Swiss 3T3 cells by non-histone chromosomal proteins]. / Stimuliatsiia sinteza DNK v pokoiashchikhsia kletkakh Swiss 3T3 negistonovymi khromosomal'nymi belkami.

Nonhistone chromosomal proteins (NHC), isolated from the kidney and liver of intact rats, the liver of rats treated with hepatocarcinogen DEN and the rat hepatoma, stimulate DNA synthesis in Swiss 3T3 cells in resting culture. The maximum stimulating effect was obtained in the presence of narrow NHC fractions eluted with 0.4-0.5 M NaCl from the phosphocellulose column and identified as hetero-organic NHC protein antigens of the kidney origin associated with hepatocellular tumors.

[Endocytosis and intracellular transport of epidermal growth factor after destruction of the actin cytoskeleton by cytochalasin B]. / Endotsitoz i vnutrikletochnyi transport épidermal'nogo faktora rosta pri destruktsii aktinovogo tsitoskeleta tsitokhalazinom B.

Endocytosis of the epidermal growth factor (EGF) was investigated in three cell lines--A431, 3T6 and Swiss 3T3--after their incubation with cytochalasin B (CB). CB was introduced into culture medium (10 mkg/ml) 1.5-2 hours before addition of 125I-EGF (20-40 ng/ml). The label uptake rate was measured after a 35-40 minutes incubation of cells with 125I-EGF. It appeared that disorganization of microfilamentous network caused by CB exerted no influence on the binding of EGF to the surface membrane receptors and its internalization. Nevertheless, the experiments performed on A431 cells using a fluorescent label--rhodamine--bound to EGF (EGF-R) indicate that CB, though not influencing the initial steps of endocytosis, inhibits the next step--the intracellular transport of EGF-receptor complexes from the trans-Golgi region to lysosomes. As was shown elsewhere (Barkan, Nikol'sky, 1986), CB inhibits the mitogenic effect of EGF on resting Swiss 3T3 cells. So, the process of EGF-receptor uptake and delivery to the trans-Golgi region is evidently not enough to stimulate the cell proliferation; next steps of transport and degradation of ligand-receptor complexes are presumably needed.

[Why does a cell monolayer replicate the metal tray pattern of the thermostat?]. / Pochemu kletochnyi monosloi vosproizvodit risunok metallicheskoi polki termostata?

Cells freshly seeded into the closed culture flasks or dishes and placed on the metal tray with holes of the thermostat or incubator are seen to form the layer with uneven density: with high density corresponding to the flask bottom regions above the metal and low density corresponding to the flask bottom region above the holes in the tray. The effect was shown using several cell lines with different degrees of transformation and saturation density, including Swiss 3T3. The main cause of this effect is the difference between the temperature inside the thermostat and the lower temperature of the flasks with culture medium (rather than between the metal framework and the air), together with a high heat conduction of the metal. The reverse difference in the temperatures (higher temperature of the culture flasks) leads to the formation of the reverse pattern of the cell layer, with higher density corresponding to the holes. The temperature differences exert their influence presumably during the first 10-15 minutes after the cells seeding, when the process of cell sedimentation is involved possibly by creating the microcurrents in the medium.

[Effect of dihydrocytochalasin B on the induction of DNA synthesis by epidermal growth factor, insulin and serum in Swiss 3T3 cells]. / Vliianie digidrotsitokhalazina B na induktsiiu sinteza DNK épidermal'nym faktorom rosta, insulinom i syvorotkoi v kletkakh Swiss 3T3.

The addition of a microfilament-disorganizing agent--dihydrocytochalasin B B (5-10 micrograms/ml)--to to quiescent confluent or sparse (in 0.5% serum) Swiss 3T3 cells, 1-2 hours prior to stimulation, inhibited the initiation of DNA synthesis induced by an epidermal growth factor (7.5-10 ng/ml) and insulin (0.5-1.0 micrograms/ml), but exerted a low effect on serum stimulation. DNA synthesis was measured 21-23 hours after the growth factor administration by 14C-thymidine incorporation in acid-insoluble material and the ratio of this fraction to exogenous thymidine uptake. Moreover, the polar solvent dimethylsulfoxide, present in culture medium at low concentration (0.1-0.5%), also caused a decrease in the basal level of 14C-thymidine incorporation in resting cells, and a less decrease in the induced incorporation.

[Amine inhibition of serum-induced DNA synthesis in 3T3 and 3T6 cell cultures]. / Podavlenie aminami sinteza DNK, indutsirovannogo syvorotkoi v kul'ture kletok 3T3 i 3T6.

Several amines were shown to inhibit growth stimulation in quiescent confluent and sparse cultures of Swiss 3T3 and 3T6 cells by changing for a fresh medium containing 10-20% serum. Proliferation was inhibited by dansylcadaverine (0.1 mM), chloroquine (0.05 mM), 5-methoxytryptamine (0.1 mM), cystamine (0.1 mM), dimethylurea (100 mM), methylurea (100 mM), and in some experiments--by urea (100 mM). The inhibitory activity was not associated with a direct influence of amines on DNA synthesis or thymidine phosphorylation. The findings suggest that amines may influence the process of clustering of growth factor-receptor complexes, or the fusion of plasma membrane and intracellular vesicles containing some components required for cell proliferation.

[Minimally transformed 3T3 cell lines as an object for research on the mechanisms of proliferation]. / Minimal'no transformirovannye kletochnye linii 3T3 kak ob"ekt issledovaniia mekhanizmov proliferatsii.

The present review gives a detailed description of mouse cell lines 3T3 (Swiss 3T3, Balb/c 3T3, and NIH 3T3) including their establishment, evolution, growth properties and formation of the specific monolayer with low saturation density. Questionable views of the 3T3 cell origin are discussed. The main features of these continuous cell lines are shown, such as a high requirement for serum growth factors, substrate dependence, inhibition of cell division in dense monolayer and absence of oncogenic potentions, which are similar to those of normal diploid cells, thus making these pseudonormal lines useful in studies of cell proliferation and oncogenesis. Different methods for obtaining quiescent 3T3 cells are observed, and, more in detail, the achievement of the resting state in confluent monolayer and at low serum concentration in sparce culture. The data reporting the efficiency of growth stimulation using serum, purified growth factors and hormones are analysed. Special attention is paid to the susceptibility of 3T3 lines to culture conditions, and to dependence of experimental data obtained upon keeping stability of these conditions.

[Effect of cultivation conditions on the growth properties of Swiss 3T3 cells]. / Vliianie uslovii kul'tivirovaniia na rostovye svoistva kletok Swiss 3T3.

A possibility of using Swiss 3T3 cells, adapted to the growth in the Eagle basal medium and bovine serum, in studies of cell proliferation and quiescent state was shown on the basis of their growth characteristics. Proliferative activity of cultures was estimated by measuring the intensity of DNA synthesis (incorporation of labeled thymidine and flow cytofluorometric analysis), mitotic index and cell number counts. Growth rate and saturation density of the culture were analyzed depending on serum concentration, substrate quality and medium changes both in growth and quiescent states. In spite of repeated medium changes such adapted cells had saturation density within 4.10(4)--7.10(4) cells/cm2, standard for this line. Besides, a distinct inhibition of cell proliferation at confluence or after incubation with low serum (0.5%) and a possibility of the following stimulation of cell divisions by adding a fresh medium containing different concentrations of serum were demonstrated. The increased rate of adipose conversion was detected in resting confluent 3T3 cells cultivated in closed vessels, as compared to cells growing in tissue culture dishes in the CO2 incubator.

[Rapid method for the intravital assessment of proliferative activity in monolayer cultures]. / Bystryi metod prizhiznennoi otsenki proliferativnoi aktivnosti v monosloinykh kul'turakh.

A simple and quick method is proposed for measuring the mitotic activity in monolayer cell cultures. The method is based on counts of mitotic figures in several microscopic fields without fixing and staining the cells. The counting procedure takes little time, and culture dishes (or flasks) may be then used for other experiments. The proliferation activity of Swiss 3T3 cells was estimated by this technique and compared with the results provided by the flow cytofluorimetric analysis.

[Factors responsible for increasing the cytogenetic effectiveness of cyclophosphamide administered to irradiated animals. Cytogenetic effect of mutagens not requiring metabolic activation on bone marrow cells of irradiated rats]. / Izuchenie prichin, obuslovlivaiushchikh usilenie tsitogeneticheskoi éffektivnosti tsiklofosfana pri vvedenii ego obluchennym zhivotnym. Issledovanie tsitogeneticheskogo deistviia mutagenov, ne trebuiushchikh metabolicheskoi aktivatsii, na kletki kostnogo mozga obluchennykh krys.

The cytogenetic action of mutagens, that do not require metabolic activation (embichin and sarcolysine), and X-radiation on rat bone marrow was studied 1 month after preliminary irradiation with the dose of 4.25 Gy. The frequency of chromosome aberrations induced by the above-mentioned effects was the same as that in nonirradiated rats.

[Factors responsible for increasing the cytogenetic effectiveness of cyclophosphamide administered to irradiated animals. Mutagenic effect of cyclophosphamide on irradiated and intact cells in culture]. / Izuchenie prichin, obuslovlivaiushchikh usilenie tsitogeneticheskoi éffektivnosti tsiklofosfana pri vvedenii ego obluchennym zhivotnym. Mutagenno deistvie tsiklofosfana na obluchennye i intaktnye kletki v kul'ture.

The frequency of cyclophosphamide-induced chromosome aberrations in preirradiated cultures of L-cells and embryonal rat fibroblasts has been estimated. The mutagen was subjected to nonenzymatic activation in solution at 37 degrees C. In contrast to the results previously obtained on animals we failed to observe any influence of preirradiation on the cytogenetic effect of cyclophosphamide.

[Activation of cyclophosphane when stored as a solution]. / Aktivatisiia tsiklofosfana pri khranenii ego rastvora.

There was found an effect of nonenzymic activation of cyclophasphane when keeping its solution during 24 hours at 37 degrees C. The products, formed as a result of its hydrolysis, induce chromosome rearrangement in the L-fibroblast culture in vitro while cyclophosphane dissolved immediately before the injection into the cell culture does not show a marked cytogenetic effect.

[Ultraviolet fluorescence of irradiated cultured cells]. / Izuchenie ul'trafioletovoi fluorestsentsii obluchennykh kletok v kul'ture.

Using cytofluorometry in the UV region of spectrum, a dose-dependent increase in ultra-violet fluorescence (UVF) intensity of L-fibroblasts was shown after X-irradiation at dose range of 80 to 500 rad, with maximum effect at 400 rad. The time dynamic observation of fluorescence intensity changes after irradiation at does 400 rad has demonstrated the highest values of UVF on the first days (1--3 days) with the following decrease within a month, and returning to the control level by the end of the study.