[Mitochondria-targeted antioxidant SkQR1 selectively protects MDR-negative cells against ionizing radiation].

Radioprotection appeared to be an important problem of today due to atom energetic development and utilization of radiation material in the industry, science and medicine. It has been shown that mitochondrial targeted antioxidant SkQR1 could attenuate radiation injury of human erythroleukemia K562 cells. Pretreatment with SkQR1 before irradiation decreased DNA double strand breaks formation, diminished the number of chromosomal aberrations and suppressed delayed ROS production. Prevention of oxidative stress and normalization of mitochondrial function by mitochondria-targeted antioxidants may be a potential therapeutic strategy not only against immediate consequences of radiation, but, either against its late consequences such as genomic instability. SkQR1 did not protect against radiation-induced damage the K562 subline with high level of multidrug resistance (MDR) due to SkQR1 extrusion with Pgp 170 MDR pump. We suggest that mitochondria-targeted antioxidants might be used for selective protection of normal cells against radiation-induced damage without interference with radiotherapy of MDR-positive tumors.

[Multidrug resistance P-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1].

Mitochondria-targeted antioxidants of the SkQR1 family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQRI is ejected from chemotherapy-resistant cells by P-glycoprotein - one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQRI from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.

[Multidrug resistance p-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1].

Mitochondria-targeted antioxidants of the SkQRI family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQR1 is ejected from chemotherapy-resistant cells by P-glycoprotein--one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQR1 from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells.

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53(-/-) mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53(-/-) xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53(-/-) and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53(-/-) tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal.

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6 -ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis.

Marginal blebbing during the early stages of TNF-induced apoptosis indicates alteration in actomyosin contractility.

Dynamics of alterations of cell surface topography during TNF-induced apoptosis of HeLa cells was examined by phase-contrast videomicroscopy and immunomorphological analysis. The final stage of apoptosis accompanied by cell rounding and general blebbing of the cell surface became after 4-6 h of incubation but much earlier, after 1.5-3 h, essentially flattened lamellipodia at the active edges transformed into the small blebs that were continuously extended and retracted during the next 1-2 h. This phenomenon was called "marginal blebbing". It took place before the cytochrome c release from mitochondria to cytosol. Marginal blebbing was inhibited by drugs that depolymerized actin microfilaments (cytochalasin, latrunculin) or decreased Rho-kinase-dependent contractility of actin-myosin cortex (H7, HA-1077, Y27632). A pancaspase inhibitor, zVAD-fmk, completely prevented marginal and general blebbing, and TNF-induced apoptosis. DEVD-fmk, a specific inhibitor of caspase-3, inhibited both marginal and general blebbing but not cell rounding and death. Thus, marginal blebbing is an early microfilament-dependent apoptotic event. It is suggested that it is initiated by minimal activation of caspase-3 and the following local Rho-kinase-dependent stimulation of actin-myosin cortex contractility. Localization of marginal blebs at the active edge may be associated with special organization of cortex in that zone.

[Cytoskeletal mechanisms of heterocellular contact formation]. / Tsitoskeletnye mekhanizmy formirovaniia geterokletochnykh kontaktov.

We studied the interaction of two main cell types, epitheliocytes and fibroblasts, in a mixed culture. Heterotypic cells had a different cytoskeleton organization and expressed different cell adhesion molecules, cadherins. In spite of this, when the cells contacted in the mixed cultures, a heterophilic contact was formed and the actin cytoskeleton of an epitheliocyte at the site of contact was reorganized: the marginal actin bundle was decomposed and actin structures were formed in its place, that were typical for the fibroblast lamella. No changes were observed in the actin organization of the fibroblast. In architecture, the heterophilic adhesion contacts resembled the contacts between fibroblasts. Both heterophilic and homophilic contacts were transient, rather than constant structures. The formation of heterophilic contacts in mixed cultures can serve as a model of formation of a tissue system consisting of epithelium and mesenchyme.

[Mode of epithelium-fibroblast interaction in mixed heterotypic cell cultures]. / Osobennosti vzaimodeistviia épiteliia i fibroblastov v smeshannykh kul'turakh.

The interaction between epithelium (dog kidney epithelium MDCJ/clone 20) and fibroblasts (diploid human fibroblasts M19 and AG-1523) was studied in mixed heterotypic cell cultures. The mode of cell interaction depends on the manner of their collision. At collision of the epithelium lamella and the lateral side of fibroblast, the lamella was seen to creep under the lateral side to force back the fibroblast. At the frontal collision of epithelium and fibroblast lamellae, the mode of interaction depends on the local situation. With the presence of a free substratum around, the fibroblast formed a new lamella and moved aside from the place of collision. In the case, when the neighboring cells prevented fibroblast from moving, it migrated under the epithelium. In this work, we have first demonstrated the formation of specialized intercellular adhesions between epithelium and fibroblasts. The cultures were studied by phase contrast, interference reflection or video tape recording, using an image processing system (Hamamatsu). For studying adhesion, immuno-fluorescent methods were performed.

[The sorting of heterotypic cells in mixed cultures]. / Sortirovka geterotipnykh kletok v smeshannykh kul'turakh.

Bovine tracheal epithelium, FBT cells, transformed mouse kidney epithelium, MPTR cells, and normal mouse embryo fibroblasts were used to demonstrate cell sorting in mixed monolayer cultures grown on solid substrates. Sorting was caused by contact cell competition for the substrate territory. Central position after sorting was occupied by most adhesive cells capable of displacing other groups of cells from the substrate.

Movement of cell-associated matrix during contact competition of cells in mixed cultures.

Co-cultured contacting heterotypic cell groups compete with one another for the attachment to the substratum. In the course of this competition one cell group can progressively push another from the substratum. Alterations to the distribution of cell-associated matrix structures in the course of contact competition were examined by immunomorphological methods in the paired cultures of two epithelial lines and those of fibroblasts and epithelia. It was found that matrix structures formed by the retreating cells withdrew from the substratum simultaneously with the cells. These results show that two forms of matrix structures should be distinguished: the usually immovable matrix upon which the cells crawl and the movable matrix that the cells carry with them when they move.

[The reorganization of the extracellular matrix in mixed monolayer cultures]. / Reorganizatsiia vnekletochnogo matriksa v smeshannykh monosloinykh kul'turakh.

Heterotypic cells in combined monolayer sheets can push each other from the substratum due to a competition in attachment of pseudopodia of contacting heterotypic cells: different types of epithelium can push each other and fibroblasts from the substratum in mixed cultures. The formation of extracellular matrix in mixed cultures was studied with antisera to fibronectin and laminin by immunofluorescent microscopy. It was shown that one group of cells in the sheet pushed another group together with their extracellular matrices. It is supposed that the interaction of contacting heterotypic cells and associated extracellular matrices may play an important role in distribution of different cell types in embryogenesis and carcinogenesis.

[The competition of 2 types of epithelial layers in mixed cultures]. / Konkurentsiia dvukh tipov épitelial'nykh plastov v smeshannykh kul'turakh.

The interaction of epithelial cells was studied in mixed cultures by phase contrast, time-lapse microcinematography, transmission and scanning electron microscopy. Epithelial cells, united into monolayer sheets with smooth apical surfaces, can be competing for substrate lamellae at the lower surfaces and force out each other from the substrate up to full elimination. The competition of cells for territory may be an important factor in morphogenesis in vivo.

Competition of contacting heterotypic epithelial sheets for the territory in culture.

Co-cultured epithelial cells of various lines formed mixed cohesive sheets. Contact interactions of different cells in the sheets were studied by phase contrast and interference reflection microscopy, time-lapse microcinematography, transmission and scanning electron microscopy. It was found that heterotypic cells in the combined sheets were locked together by the complexes of specialized contacts and were metabolically coupled, as shown by the dye transfer. At the same time heterotypic cells continued to extend pseudopods at the contacting lateral surfaces and to attach these pseudopodies to substratum. Due to competition of pseudopod attachments one group of cells in the sheet often progressively pushed another group from the substratum. This competition of cells for substratum may provide an important mechanism for morphogenetic reorganisation of epithelial sheets and of other groups of interacting cells in vivo.

[Interaction of different types of epithelium in a mixed culture]. / Vzaimodeistvie raznotipnogo épiteliia v smeshannoi kul'ture.

Interaction of several lines of epithelial cells was studied in a mixed culture: FBT (bovine fetal trachea), MDSK (dog kidney), IAR-2 (rat liver), MPTR (SV-40 transformed mouse kidney). During mixed cultivation epithelia cells of different types were capable to force out each other from the substrate to full elimination. This capacity correlated with the pattern of cell contacts with the substrate. It is supposed that epithelial cells can form lamellae at the lower surfaces competing for substrate. Those cells which have lamellae with continuous marginal focal contacts and numerous focal contacts eliminate the cells having lamellae with few focal contacts.

[Contact interactions of epithelial layers]. / Kontaktnye vzaimodeistviia épitelial'nykh plastov.

Cultured together epithelial cells of mouse kidney (line MPTR) were seen to form a mixed cohesive sheet. The mutual interaction of different cells in the sheet was studied by phase contrast microscopy, time-lapse microcinematography, scanning electron microscopy and by some other morphological tests. The upper surface of the mixed sheet was continuous on the border of the two cell types. Despite this border between different cell types was continuously moving from FBT towards MPTR, as if MPTR was "pushed aside" by FBT. In some cases such a moving caused a total exclusion of MPTR from the sheet. Excluding cells differed from the excluded ones in their better spreading and better adhesion to the underlying substrate. Possible mechanisms of such an exclusion are discussed.

[Cultivation of embryonic mouse liver epithelium]. / Kul'tivirovanie épiteliia émbrional'noi pecheni myshi

Mouse embryo liver epithelial cells maintained in vitro were connected with each other by highly permeable cell-to-cell contacts, synthesized alpha-fetoprotein and possessed the property of contact inhibition of phagocytosis. Methods of cultivation of these cells are presented in this paper.

[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.