Cytoplasmic Beta and Gamma Actin Isoforms Reorganization and Regulation in Tumor Cells in Culture and Tissue.

The cytoplasmic actin isoforms (ß- and γ-actins) contribute greatly to cellular processes such as cel-cell and cell-matrix interactions, as well as cell polarization, motility and division. Distinct isoforms modulations are linked to serious pathologies, so investigations of underlying mechanisms would be of major relevance not only for fundamental research but also for clinical applications. Therefore, the study of the relevant mechanisms of change in the isoform's balance is important for basic research and for clinical studies. The disruption of actin cytoskeleton and intercellular adhesions contribute to the neoplastic transformation, as it is important for the tumor growth, invasiveness and metastasis. Cytoplasmic actins display the functional diversity: ß-actin is responsible for contractility, whereas γ-actin participates in the submembrane flexible cortex organization and direction cell motility. The involvement of ß- and γ-actin in cell architecture, motility, division, and adhesion junctions in normal cells is not equivalent, and the major question was following: whether isoform ratio and the distribution in the cell corresponds to pathological function. Significant data were obtained in the study of tumor and normal cells in culture, as well as on clinical material of human tissues, and via selective regulation of ß- and γ-actin's expression. Investigation of the actins' diversity and function in cancers may help to choose the benefit treatment strategies, and to design new therapies.

Divergent Impact of Actin Isoforms on Division of Epithelial Cells.

We investigated distribution and functions of beta- and gamma-cytoplasmic actins (CYAs) at different stages of non-neoplastic epithelial cell division using laser scanning microscopy (LSM). Here, we demonstrated that beta- and gamma-CYAs are spatially segregated in the early prophase, anaphase, telophase, and cytokinesis. Small interfering RNA (siRNA) experiments revealed that in both beta-CYA- and gamma-CYA-depleted cells, the number of cells was significantly reduced compared with the siRNA controls. Beta-CYA depletion resulted in an enlargement of the cell area in metaphase and high percentage of polynuclear cells compared with the siRNA control, indicating a potential failure of cytokinesis. Gamma-CYA depletion resulted in a reduced percentage of mitotic cells. We also observed the interdependence between the actin isoforms and the microtubule system in mitosis: (i) a decrease in the gamma-CYA led to impaired mitotic spindle organization; (ii) suppression of tubulin polymerization caused impaired beta-CYA reorganization, as incubation with colcemid blocked the transfer of short beta-actin polymers from the basal to the cortical compartment. We conclude that both actin isoforms are essential for proper cell division, but each isoform has its own specific functional role in this process.

Biological Role of Actin Isoforms in Mammalian Cells.

Actin plays an important role in cellular adhesion, muscle and non-muscle contractility, migration, polarization, mitosis, and meiosis. Investigation of specific mechanisms underlying these processes is essential not only for fundamental research but also for clinical applications, since modulations of actin isoforms are directly or indirectly correlate with severe pathologies. In this review we summarize the isoform-specific functions of actin associated with adhesion structures, motility and division of normal and tumor cells; alterations of the expression and structural organization of actin isoforms in normal and tumor cells. Selective regulation of cytoplasmic ß- or γ-actin expression determines functional diversity between isoforms: ß-actin plays the predominant role in contraction and intercellular adhesion, and γ-actin is responsible for the cellular plasticity and motility. Similar data were obtained in different epithelial and mesenchymal neoplastic cell cultures, as well as in immunomorphological comparison of normal human tissues with tumor analogues. Reorganization of the actin cytoskeleton and cell-cell contacts is essential for proliferation control and acquisition of invasiveness in epithelial tumors.

Structural Features of Actin Cytoskeleton Required for Endotheliocyte Barrier Function.

Cytoplasmic actin structures are essential components of the eukaryotic cytoskeleton. According to the classic concepts, actin structures perform contractile and motor functions, ensuring the possibility of cell shape changes during cell spreading, polarization, and movement both in vitro and in vivo, from the early embryogenesis stages and throughout the life of a multicellular organism. Intracellular organization of actin structures, their biochemical composition, and dynamic properties play a key role in the realization of specific cellular and tissue functions and vary in different cell types. This paper is a review of recent studies on the organization and properties of actin structures in endotheliocytes, interaction of these structures with other cytoskeletal components and elements involved in cell adhesion, as well as their role in the functional activity of endothelial cells.

[A change in the expression of membrane-associated proteins and cytoplasmic actin isoforms in the progression of human colon tumors]. / Izmenenie ékspressii membranoassotsiirovannykh belkov i tsitoplazmaticheskikh izoform aktina pri progressirovanii opukholei tolstoi kishki cheloveka.

Tumor progression is a complex process that also involves the restructuring of the actin cytoskeleton and the weakening of intercellular adhesive contacts due to the tumor cells that pass through the epithelial-mesenchymal transition (EMT). AIM: Тo identify correlations between clinical features, risk of progression and/or recurrence of human colon adenocarcinomas (CAC), and EMT-related tumor markers. MATERIAL AND METHODS: Descending colon and sigmoid colon adenocarcinoma samples were examined immunohistochemically. Formalin-fixed paraffin-embedded tissue sections were incubated with antigen-specific antibodies, then secondary antibodies labeled with fluorochromes, and the fluorescence intensity of microscopy images was analyzed. RESULTS: The cells of a tumor compared to those of intact colon tissue showed a weak staining of E-cadherin in the cell-cell contact areas. The reduced membrane staining and nuclear localization of ß-catenin were detected in moderately (G2) and poorly (G3) differentiated tumors. There were substantially decreased ß-actin levels in almost all tumor samples and increased γ-actin ones, mainly in the samples belonging to stage IV disease. CONCLUSION: A correlation was found between stage, tumor differentiation grade, risk for relapse or progression of disease, and the impaired expression of different EMT markers: total or partial loss of E-cadherin expression, ß-catenin reorganization in cell-cell contacts, and a change in the ratio of cytoplasmic actin isoforms in the late stages of CAC development. We believe that these molecular markers may have a prognostic potential.

[The reorganization of actin cytoskeleton and microtubule system of human endothelial vein in the intercellular contacts formation].

Endothelial cells are tightly fitted to each other and lining the interior surface of all vessels of living organism to provide vascular permeability regulation and interchange between the blood circulating in vessels and tissue fluids of those organs in which these vessels are located. In vitro endothelial monolayer conserve it's basic barrier function which is native for vessels endothelium. Based on this fact we used endothelial cells growing in vitro as a model system in experimental studies of cytoskeletal and adhesion cell components interaction. In current paper, cultured human vein endothelial cells monolayer was used to quantify cytoskeleton alterations in the of endothelial cells from spreading and formation of the first cell-cell contacts to confluent monolayer formation. The system of actin filaments formed two different cytoskeletal structures in the cells of venous endothelium: 1) cortical actin network; 2) actin stress fibers (bundles) arranged parallel to the substrate. Two actin isoforms, ß- and γ-cytoplasmic (non-muscle) actins, are expressed in endothelial cells. The bundles of actin stress fibers were detected by immunofluorescent staining with antibody against ß-actin, whereas antibodies against γ-actin identified cortical and lamellar networks. For assessment of the actin cytoskeleton organization it's fluorescence intensity on the area of 10 µM2 located (1) near the free edge, and (2) in the zone of cell-cell contacts were analyzed. Fluorescence intensity of ß-actin structures was higher in the areas of cell-cell contact. The fluorescence of γ-actin structures was more intensive at the leading edges of the lamellae, and was the lowest on the stable edges of the cells with formed cell-cell contacts. The endothelial monolayer formation was accompanied by microtubule system alteration: the number of microtubules increased at the cell edge, and besides the microtubules quantity in the area of already formed cell-cell contact was always higher than in free lamella region.

Actin isoforms and reorganization of adhesion junctions in epithelial-to-mesenchymal transition of cervical carcinoma cells.

Malignant cell transformation requires changes in the ability of cells to migrate. The disruption of actin cytoskeleton and intercellular adhesions is an important component of the acquisition of invasive properties in epithelial malignancies. The invasive ability of carcinoma cells is associated with reduced expression of adhesion junction molecules and increased expression of mesenchymal markers, frequently referred to as epithelial-to-mesenchymal transition (EMT). Standard features of the EMT program in cancer cells include fibroblastic phenotype, downregulation of the epithelial marker E-cadherin, induction of Snail-family transcription factors, as well as expression of mesenchymal proteins. We compared the epithelial and mesenchymal marker profiles of nonmalignant HaCaT keratinocytes to the corresponding profiles of cervical carcinoma cell lines C-33A, SiHa, and CaSki. The characteristics of the EMT appeared to be more developed in SiHa and CaSki cervical cancer cells. Further activation of the EMT program in cancer cells was induced by epidermal growth factor. Decreased epithelial marker E-cadherin in CaSki cells was accompanied by increased mesenchymal markers N-cadherin and vimentin. Downregulated expression of E-cadherin in SiHa and CaSki cells was associated with increased expression of Snail transcription factor. Our goal was to study actin reorganization in the EMT process in cell cultures and in tissue. We found that ß-cytoplasmic actin structures are disorganized in the cervical cancer cells. The expression of ß-cytoplasmic actin was downregulated.

Novel mitochondria-targeted compounds composed of natural constituents: conjugates of plant alkaloids berberine and palmatine with plastoquinone.

Novel mitochondria-targeted compounds composed entirely of natural constituents have been synthesized and tested in model lipid membranes, in isolated mitochondria, and in living human cells in culture. Berberine and palmatine, penetrating cations of plant origin, were conjugated by nonyloxycarbonylmethyl residue with the plant electron carrier and antioxidant plastoquinone. These conjugates (SkQBerb, SkQPalm) and their analogs lacking the plastoquinol moiety (C10Berb and C10Palm) penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in isolated mitochondria or in mitochondria in living human cells in culture. Reduced forms of SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations. In isolated mitochondria and in living cells, the berberine and palmatine moieties were not reduced, so antioxidant activity belonged exclusively to the plastoquinol moiety. In human fibroblasts, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide. At higher concentrations, conjugates of berberine and palmatine induced proton transport mediated by free fatty acids both in model and in mitochondrial membrane. In mitochondria this process was facilitated by the adenine nucleotide carrier. As an example of application of the novel mitochondria-targeted antioxidants SkQBerb and SkQPalm to studies of signal transduction, we discuss induction of cell cycle arrest, differentiation, and morphological normalization of some tumor cells. We suggest that production of oxygen radicals in mitochondria is necessary for growth factors-MAP-kinase signaling, which supports proliferation and transformed phenotype.

[Actins and keratins in the diagnosis of human basal-like breast cancer].

The most common forms of luminal breast cancer (BC) were compared with basal-like and Her2/neu3+ BC. Their primary classification was based on morphological diagnosis and the expression of estrogen receptor (ER), progesterone receptor (PR), and Her2/neu receptors. Monoclonal antibodies to actins and keratins were used for the study. Basal-like BC cells (ER/PR/Her2/ neu-) were regularly stained with antibodies to basal keratins 5/6 and 17, smooth muscle alpha-actin, and p63. Luminal keratin 8 staining was reduced. The solid regions had beta-actin staining with disappearance in the scirrhous component. beta-actin and basal keratins were also observed in metaplastic BC with ER/PR/Her2/neu3+. Immunomorphology using cytoskeletal markers along with the expression of steroid hormone and Her2/neu receptors may be used in the diagnosis of basal-like forms of BC.

Novel mitochondria-targeted antioxidants, "Skulachev-ion" derivatives, accelerate dermal wound healing in animals.

It is shown that the novel mitochondria-targeted antioxidant SkQ1, (10-(6'-plastoquinonyl) decyltriphenylphosphonium) stimulates healing of full-thickness dermal wounds in mice and rats. Treatment with nanomolar doses of SkQ1 in various formulations accelerated wound cleaning and suppressed neutrophil infiltration at the early (7 h) steps of inflammatory phase. SkQ1 stimulated formation of granulation tissue and increased the content of myofibroblasts in the beginning of regenerative phase of wound healing. Later this effect caused accumulation of collagen fibers. Local treatment with SkQ1 stimulated re-epithelization of the wound. Lifelong treatment of mice with SkQ1 supplemented with drinking water strongly stimulated skin wounds healing in old (28 months) animals. In an in vitro model of wound in human cell cultures, SkQ1 stimulated movement of epitheliocytes and fibroblasts into the "wound". Myofibroblast differentiation of subcutaneous fibroblasts was stimulated by SkQ1. It is suggested that SkQ1 stimulates wound healing by suppression of the negative effects of oxidative stress in the wound and also by induction of differentiation. Restoration of regenerative processes in old animals is consistent with the "rejuvenation" effects of SkQ1, which prevents some gerontological diseases.

[Distribution of actin isoforms in normal, dysplastic, and tumorous human breast cells].

The distribution of beta- and gamma-cytoplasmic actins was compared in the normal cells and dysplastic malignant breast epithelial cells. In the normal luminal epithelium, beta- and gamma-cytoplasmic actins were located in different cell compartments: gamma-actin was more expressed in the apical parts of epithelial cells while beta-actin was in their basolateral domain. Polarized distribution of actinic isoforms was partially preserved in the papillomas and fibroadenomas; a more pronounced coexpression of isoforms was detected in the dysplastic proliferates. In ductal and lobular in situ carcinoma cells, gamma-actin filamentous structures were absent while the gamma-cytoplasmic actin network throughout the cytoplasm was increased. It is generally accepted that the enhanced motility of cancer cells as to the nonmalignant situation is crucial in the process of cancer invasion. The authors' findings suggest that specific monoclonal antibodies to beta- and gamma-cytoplasmic actins may be used as supplementary markers that can differentiate benign and malignant breast neoplasms.

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.

[Effect of actomyosin contractility on focal contacts of myofibroblasts and structure of stress fibers]. / Vliianie aktomiozinovoi sokratimosti na fokal'nye kontakty miofibroblastov i strukturu stress-fibrill.

Myofibroblasts from rat lung were cultivated. These cells in addition to beta- and gamma-cytoplasmic actins, expressed alpha-smooth muscle actin (alpha-SMA) and formed a system of "supermature" focal contacts, which were connected with thick stress-fibers expressing alpha-SMA and myosin II. Reduction of actin-myison contractility by inhibitors BDM and ML-7 lead to stress fiber reorganization, e.g., decrease in their thickness, a selective disappearance of alpha-SMA expression and myosin translocation from bundles to the cytoplasm. Using immunofluorescence, interference-reflection microscopy and morphometry, we have demonstrated that an inhibition of actin-myosin contractility also leads to dispersion of myofibroblastic focal contacts. Phase-contrast and DIC video-enhanced microscopy of live cells showed morphological reorganization at the leading edge after inhibitory treatment. Thus, actin-myosin contractility controls the structure of "supermature" focal contacts of myofibroblasts and alpha-SMA expression in stress fibers.

Scatter factor induces segregation of multinuclear cells into several discrete motile domains.

The effects of scatter factor, HGF/SF, on multinuclear MDCK epitheliocytes were examined. Multinuclear cells were obtained by blocking cytokinesis by low concentration of cytochalasin D; these large cells had discoid shape and did not move much on the substrate. Incubation of these cells with HGF/SF induced their profound reorganization: their cytoplasm was reversibly segregated into several individually moving motile flattened domains, termed lamelloplasts and connected with one another by cylindrical domains termed cables. One or several nuclei were present in many lamelloplasts, but some lamelloplasts were anuclear. Nuclei were absent from the cables. Lamelloplasts continuously formed actin-rich ruffles at their edges; their cytoplasm contained small actin bundles and numerous focal adhesions. In contrast, cable, had no ruffles or focal adhesions. Dense networks of vimentin and keratin intermediate filaments were present in lamelloplasts; bundles of filaments of both types were seen in the cables. Segregation was accompanied by redistribution of centrosomes from perinuclear zone into lamelloplasts. As a result each lamelloplast in segregated cell acquired individual complex of centrosome and radiating microtubules. The cables contained numerous parallel microtubules but never had centrosomes. This reorganization of microtubular system was essential for segregation as alterations of shape and actin cytoskeleton were prevented by microtubule specific drugs: colcemid and Taxol (paclitaxel). It is suggested that mechanism of segregation is based on activation of two types of opposite actin reorganization: formation of actin networks in lamelloplasts and their dismantlement in the cables. Spatial distribution of the domains in which these opposite types of reorganizations occur may be regulated by microtubular system. It is also suggested that mechanisms of HGF/SF-induced segregation may be closely related to the mechanisms of important physiological reorganizations of cells, such as polarization of pseudopodial activities in motile cells and cytokinesis.

Brefeldin A distorts discoid and polarized morphological organizations of epithelial cells in culture.

Brefeldin A (BFA), an agent disorganizing organelle traffic, was used to find out the role of this traffic in the maintenance of shape and cytoskeletal organization of cultured epithelial MDCK cells. Cell shape was assessed using dispersion and elongation indices (Dunn and Brown, 1986, J Cell Sci 83: 313-340). BFA reversibly transformed discoid MDCK cells with circular actin pattern into moderately polarized cells with straight actin bundles. In other experiments the same MDCK cells were first transformed into highly polarized fibroblast-like cells by incubation with 'scatter factor' (HGF/SF) and then treated with BFA. In these conditions BFA significantly decreased the degree of polarization. It is suggested that both highly polarized and discoid morphological organizations are controlled by organelle traffic.

The role of the microtubular system in the cell response to HGF/SF.

The effects of the microtubular drugs colcemid and taxol on the morphological changes induced by hepatocyte growth factor/scatter factor (HGF/SF) in MDCK cells were studied. Dynamic changes in the area and shape of individual cells were assessed by morphometric methods whereas alterations of the cytoskeleton were assessed by immunomorphological methods. The results suggest that there are two components in the response to HGF/SF: (a) activation of the extension of lamellae leading to cell spreading; and (b) reorganization of microtubules leading to polarization of cell shape. The latter response is highly sensitive to microtubular drugs, especially taxol. HGF/SF induced spreading in taxol-treated MDCK cells but these cells retained a non-polarized discoid shape and a pattern of actin microfilament bundles characteristic of the untreated cells. Colcemid and taxol did not prevent HGF/SF-induced migration of cells in Boyden chambers but completely inhibited the outgrowth of multicellular strands and tubules from cell aggregates in collagen gels. These results show that enhanced lamella formation in response to HGF/SF without polarization of cell shape is sufficient to induce cell motility. In contrast, microtubule-dependent polarization is essential for complex morphogenetic responses such as tubulogenesis in collagen gels.

Motility of intracellular particles in rat fibroblasts is greatly enhanced by phorbol ester and by over-expression of normal p21N-ras.

Particle motility in cultured rat fibroblasts was studied using video-enhanced differential interference contrast microscopy. The average velocity of large bright particles (apparent diameter about 0.5-0.7 micron) was measured in control cells and in cells treated with agents which affected targets related to signal transduction pathways. A Rat-2-derived fibroblast line transfected with a construct containing multiple copies of the N-ras proto-oncogene under the control of dexamethasone-sensitive promoter was used as a main experimental model. Dexamethasone treatment was shown to induce high levels of N-ras expression in these cells. This treatment greatly increased the average particle velocity. At the same time dexamethasone did not influence the particle motility in the non-transfected parent cells and in the cells transfected with a construct which did not contain N-ras. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), also induced an approximate eightfold increase in the particle rate after several hours of incubation, while sphingosine, an inhibitor of PKC, prevented this activation. Sphingosine alone reduced the particle motility after a 20 min incubation. The particle movements were inhibited also by colcemid. These data show that the activation of N-ras and PKC produced dramatic activation of microtubule-dependent particle motility. A possible role of this activation in signal-induced alterations of cell morphology is discussed.

[Changes in the morphology of fibroblasts of different degrees of transformation under the action of 12-tetradecanoylphorbol-13-acetate]. / Izmeneniia morfologii fibroblastov razlichnoi stepeni transformatsii pri deistvii 12-tetradekanoilforbol-13-atsetata.

A study was made of morphological changes in mouse sarcoma cells of two clones, as well as in BALB/c 3T3 mouse fibroblasts and in mouse embryonal fibroblasts, upon the action of the promotor 12-O-tetradecanoylphorbol-13-acetate (TPA). In all the cell types, with the exception of mouse embryo fibroblasts, TPA induced specific changes in the shape and cytoskeleton, the most obvious changes being observed in cells with higher malignant characteristics.

[Changes in the form and cytoskeleton of cultured fibroblasts as affected by 12-tetradecanoylphorbol-13-acetate]. / Izmenenie formy i tsitoskeletona kul'tiviruemykh fibroblastov pod vliianiem 12-tetradekanoilforbol-13-atsetata.

A new type of reorganization of cytoskeleton induced by 12-o-tetradecanoyl-phorbol-13-acetate motility: division of the cell into an actin-rich active part and stable processes with numerous microtubules. Such a phenomenon was observed under a short-term influence of TPA on different lines of cultured fibroblasts: NRK, Balb/C 3T3, C-103, C-84, CAK-7. The effect of TPA was reversible and suppressed by cytochalasin B and colcemid. TPA is supposed to induce changes in the interaction between actin cortex and microtubule system.