[P66shc action on resistance of colon carcinoma RKO cells to oxidative stress].

P66shc protein is an alternative transcript product of SHC1 gene. While two other isoforms (p52shc and p46shc) have adaptor function in RAS signaling pathway, p66shc regulates reactive oxygen species (ROS) level. P66shc genome knockout significantly extends lifespan in mice. Though p66shc was determined to translocate into mitochondria and led to increase in intracellular ROS, the mechanism by which the protein take part in signaling pathways that regulates resistance to cellular stresses remains poorly studied. P66shc has an important role in carcinogenesis and its increased expression correlates with poor prognosis in colon cancer. In this work we have applied RNA interference using lentiviral constructions that express short hairpin RNA (shRNA) against N-terminal CH2 domain of p66shc isoform. Using this approach p66 but not p52 and p46 SHC1 isoform expression was selectively suppressed in colon carcinoma RKO cells. RKO cells with p66shc knockdown have shown to be more resistant to oxidative stress induced by hydrogen peroxide or serum starvation. Fragmentation of mitochondria that depends on mitochondrial ROS accumulation during oxidative stress was significantly decreased in this cells. The data obtained are in agreement with hypothesis that p66shc participates in ROS accumulation in mitochondria and by this means promotes induction of apoptosis.

[Preparation and characterization of mouse embryonic fibroblasts with K72W mutation in somatic cytochrome C gene].

Mouse embryonic fibroblasts (MEF) with point mutation in somatic cytochrome C gene were generated and characterized. It was shown that substitution of lysine for tryptophan in position 72 (K72W) decreased the proapoptotic functions of cytochrome C in response to staurosporin treatment without disrupting its respiratory functions. The presence of this mutation did not affect the pattern of cytochrome C gene expression or its localization inside the cell. These cell cultures therefore represent an interesting model for study apoptotic signaling and physiological functions of cytochrome C.

[The role of intermediate filaments in forming cellular processes induced in fibroblasts by the tumor promoter TPA]. / Rol' promezhutochnykh filamentov v formirovanii kletochnykh otrostkov, indutsirovannykh u fibroblastov opukholevym promotorom TFA.

A tumor promoter phorbol 12-myristate 13-acetate (PMA) induces characteristic reversible changes in the cell shape in certain fibroblastic lines. This reaction to PMA may be regarded as a prototype of reorganizations involving formation of stable cytoplasmic processes. Two specific drugs, Taxol and Colcemid, were used to study the role of microtubules and vimentin-containing intermediate filaments (IF) in the development of PMA-induced reorganizations. A short (I h) exposure to PMA induced formation of processes in the control cells rather than in the Colcemid treated cells having depolymerized microtubules and the IF that collapsed around the nucleus. A longer (3-4 h) exposure to PMA of the colcemid-treated cells induced a partial reversal of the IF collapse; those parts of peripheral lamellae that contained IF were transformed into narrow noncontractile processes. It is suggested that the local interaction of the IF with the actin system is an essential step in the formation of processes from lamellae.

[Preparation and use of monoclonal antibodies against seal alkaline phosphatase]. / Poluchenie i primenenie monoklonal'nykh antitel proteiv shchelochnoi fosfatazy tiulenia.

Monoclonal antibodies (termed as APP.1 and related to subclass IgG1) against seal alkaline phosphatase, have been obtained. APP.1 did not influence the enzymatic activity of alkaline phosphatase. The dissociation constant for the APP.1 interaction with Greenland seal alkaline phosphatase was equal to 8.5 x 10(-10) M. It was found that APP.1 interact with intestinal isoenzymes of common and fur seal, calf and deer alkaline phosphatases. An APP.1 complex with seal alkaline phosphatase was obtained and successfully applied in immunoenzymatic analysis. The use of this complex made it possible to diminish the limit of detectability of antibodies against peptide fragments of HIV-1 and HIV-2 proteins. Moreover, this complex allowed the identification of cytokeratin-8 and vimentin in human kidney slices and embryonic fibroblast-like cells, respectively.

[Formation of an actin cytoskeleton and adhesive structures during the spreading of cultured cells]. / Formirovanie aktinovogo tsitoskeleta i adgezionnykh struktur v khode rasplastyvaniia kul'tiviruemykh kletok.

Fibroblast spreading was studied using immunofluorescent method that provided visualization of actin structures and adhesion contacts in the same cell. Four stages of actin system formation were observed. 1. Actin concentration in ruffles at the cell periphery. Formation of numerous dot-like contacts along the whole perimeter of the cell. 2. Formation of a circumferential actin bundle. Focal contacts are located at the outer edge of the bundle. 3. Gradual transformation of the circumferential bundle into actin network with triangular meshes. Peripheral (rather than internal) filaments of the network are associated with the focal contacts. 4. Appearance of the system of long straight actin bundles (stress fibers) associated with dash-like focal contacts. The stress fibers are supposed to arise from the triangular actin network which in its turn arises from the circumferential bundle. It is suggested that the formation of actin cytoskeleton is a process driven by the development of tensions in actin structures attached to the focal contacts at the cell periphery.

[Formation of processes during fibroblast spreading in an in vitro medium with cytochalasin B]. / Formirovanie otrostkov pri rasplastyvanii fibroblastov v srede s tsitokhalazinom B in vitro.

The spreading of normal mouse fibroblasts in a solid substrate involves extension and attachment of numerous pseudopodia resulting in the formation of lamellar cytoplasm in the cell periphery which is attached to the substrate. During the spreading of fibroblasts in the presence of cytochalasin B (CB), a system of arbor-like branched processes forms de novo, rather than lamellar cytoplasm. Unlike the normal lamellar cytoplasm, the arbor-like processes are unable to clear their surface from concanavalin A-patched receptors and do not reveal microfilament bundles in their attachment sites. The cells spreading in the presence of both CB and colcemid do not form well-organized branched structures but extend numerous unstable pseudopodia. The formation of lamellar cytoplasm can be regarded as a combination of several functionally different processes: a) rudimentary pseudopodial CB- and colcemid-resistant reactions, b) CB-sensitive lamelliation, and c) colcemid-sensitive stabilization.

[Possible role of centrioles in stabilizing cytoplasmic microtubules]. / Vozmozhnaia rol' tsentriolei v stabilizatsii tsitoplazmaticheskikh mikrotrubochek.

Small fragments of the peripheral cytoplasm were obtained from cytochalasine B-treated mouse embryo fibroblasts and studied for distribution of microtubules by indirect immunofluorescence. Microtubules were demonstrated to progressively depolymerize in these fragments which did not contain any tubules after 6 hours of incubation in the growth medium. This effect was specific for microtubules, since the distribution of intermediate filaments remained unchanged during incubation. The fragments remained viable during incubation, inasmuch no changes were detectable in the membrane potential of the mitochondria stained with rhodamine 123. Progressive destruction of microtubules in the tiny cell fragments is likely to be related to the lack of centrioles in such fragments.

[Active region of the lamellar cytoplasm and focal contacts--the most cytochalasin-sensitive structures of fibroblasts]. / Aktivnaia oblast' lamelliarnoi tsitoplazmy i fokal'nye kontakty--naibolee chuvstvitel'nye k tsitokhalazinam struktury fibroblastov.

Effects of low doses of cytochalasin B (CB, 2 micrograms/ml) and cytochalasin D (CD, 0.2 microgram/ml) on the spreading of normal mouse fibroblasts in culture were investigated. CB desorganized the cortical layer of actin microfilaments to cause partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Low doses of CB did not inhibit the control, on the upper surface of these lamellas distal zones with convex outer edges ruffles were lacking. The disappearance of these ruffling active edges was accompanied with the loss of ability to clear the surface of lamellas from the concanavalin A receptors cross-linked by a corresponding ligand. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of cross-linked surface receptors.

[Distribution and movement of concanavalin A receptors on the surfaces of cells in suspension]. / Raspredelenie i dvizhenie retseptorov k konkanavalinu A na poverkhnosti kletok v suspenzii.

Normal mouse fibroblasts, transformed mouse fibroblasts (L strain), the ascite hepatoma cells (strain 22), the Krebs II ascite cells, and mouse spleen lymphocytes were incubated in suspension in the medium containing concanavalin A which is capable of crosslinking definite groups of surface receptors. Surface-attached ligands were revealed by both direct and indirect immunofluorescent methods. The incubation of all the cell types with that ligand-induced patching rather than capping of corresponding surface receptors. Colcemid had no effect on the ligand-induced redistribution of receptors.

[Effect of cytochalasin B on the distribution of concanavalin A receptors of the surfaces of normal mouse fibroblasts]. / Vliianie tsitokhalazina B na raspredelenie retseptorov konkanavalina A na poverkhnosti normal'nykh fibroblastov myshi.

Normal mouse fibroblasts are spread over the glass in the cultural medium containing cytochalasin B (CB). The cultures were incubated with concanavalin A (Con A), which binds some surface components. Con A-labeled surface receptors were visualized by the indirect immunogluorescent method. When living cultures were incubated with Con A, small patches of Con A-bound receptors were formed over the surface. 5 hours after seeding, the control cells formed plates of the lamelloplasm, whose external edge was active, with pseudopodia being formed along it. The patches of receptors were cleared selectively, from the surface of psedopodia and of the lamelloplasm, to the central parts of the cell surface. In contrast, CB-treated cells, during spreading, formed thin ribbon-like pseudopodia. The properties of these CB-treated pseudopodia differed from those formed by control cells; the former were unable to remove patches from the pseudopodial surface. The effect of CB was reversible: 30 minutes after the removal of CB, small parts of the lamelloplasm appeared, from which patches of receptors were removed. These experimental data suggest that CB-treated cells may form pseudopodia with a deficient structure and modified functional properties.

[Effect of agents disrupting microtubules on the distribution of receptors on the surface of cultured cells]. / Vliianie agentov, razrushaiushchikh mikrotrubochki, na raspredelenie retseptorov poverkhnosti kul'tiviruemykh kletok.

Substrate-attached normal mouse fibroblasts, transformed mouse fibroblasts (L-strain) and epithelial cells (MPTR strain) were incubated with two ligands that are cross-linking different group of the surface receptors: concanavalin A and cationic ferritin. Surface-attached ligands were revealed by the indirect immunofluorescent methods. The incubation of control cells with these ligands induced a patching of corresponding surface receptors, and a clearing of these receptors from the surface zones located on the lamellar cytoplasm near the cell edges actively protruding pseudopodia. Effects of three antitubulins (colcemid, colchicine and vinblastin) on the ligand-induced redistribution of receptors were examined and compared with the previously described effects of these drugs on the distribution of active cell edges.

[Insensitivity of stationary cultures of transformed mouse fibroblasts to agents stimulating DNA synthesis in normal cell cultures]. / Nechuvstvitel'nost' gustykh kul'tur transformirovannykh myshinykh fibroblastov k deistviiu agentov, stimuliruiushchikh sintez DNK v kul'turakh normal'nykh kletok

Stationary cultures of the mouse transformed cells L and S-40 sensitive to topoinhibition were found to be insensitive to the action of hyaluronidase, RNAase, and colcemid in doses known to stimulate multiplication of normal mouse fibroblasts. These cultures were still insensitive to the action of medium change and removal of a part of the monolayer.