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.

Transcriptome of Lung Cancer Cells Resistant to the Cytotoxic Activity of Macrophages.

The role of the immune system in tumor progression has been the subject of research for more than 100 years since Paul Ehrlich hypothesized that the presence of the immune system limits the occurrence of cancer. One of the mechanisms hindering the initiation and progression of the tumor is the cytotoxic activity of macrophages; however, in some cases, it is not sufficient to control tumorigenesis. This may be due to both the development of resistance of tumor cells to the antitumor activity of macrophages and the development of a tolerant phenotype of macrophages that do not have sufficient antitumor activity. In this work, the lung cancer cells resistant to the cytotoxic action of macrophages were obtained and characterized for the first time, and the genes associated with the observed changes were identified. Understanding the mechanisms of resistance of tumor cells to the cytotoxic activity of macrophages and the peculiarities of its manifestation in a tumor environment is critically important for improving the effectiveness of the existing methods of cancer treatment and developing novel methods for tumor immunotherapy.

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.

Components of the Hepatocellular Carcinoma Microenvironment and Their Role in Tumor Progression.

This review summarizes recently published data on the mechanisms of tumor cell interaction with the tumor microenvironment. Tumor stroma influences the processes of hepatocarcinogenesis, epithelial-to-mesenchymal transition, invasion, and metastasis. The tumor microenvironment includes both cellular and noncellular components. Main cellular components of hepatocellular carcinoma (HCC) stroma are tumor-associated fibroblasts, hepatic stellate cells, immune cells, and endothelial cells that produce extracellular components of tumor microenvironment such as extracellular matrix, various proteins, proteolytic enzymes, growth factors, and cytokines. The noncellular components of the stroma modulate signaling pathways in tumor cells and stimulate invasion and metastasis. The tumor microenvironment composition and organization can serve as prognostic factors in HCC pathogenesis. Current approaches in HCC targeted therapy are aimed at creating efficient strategies for interrupting tumor interactions with the stroma. Recent data on the composition and role of the microenvironment in HCC pathogenesis, as well as new developments in antitumor drug design are discussed.

[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.

E-Cadherin repression increases amount of cancer stem cells in human A549 lung adenocarcinoma and stimulates tumor growth.

Here we show that cancer stem cells amount in human lung adenocarcinoma cell line A549 depends on E-cadherin expression. In fact, downregulation of E-cadherin expression enhanced expression of pluripotent genes (c-MYC, NESTIN, OCT3/4 and SOX2) and enriched cell population with the cells possessing the properties of so-called 'cancer stem cells' via activation of Wnt/ß-catenin signaling. Repression of E-cadherin also stimulated cell proliferation and migration in vitro, decreased cell amount essential for xenografts formation in nude mice, increased tumors vascularization and growth. On the other hand, E-cadherin upregulation caused opposite effects i.e. diminished the number of cancer stem cells, decreased xenograft vascularization and decelerated tumor growth. Therefore, agents restoring E-cadherin expression may be useful in anticancer therapy.

[Prospects for the use of cells possessing myogenic potential in the treatment of skeletal muscle diseases: a review of research. Part 1 - satellite cells].

Musculoskeletal functions disorders may develop as a consequence of injuries and various types of congenital / acquired diseases, among which a special place belongs to muscular dystrophy. The technology with use of cells possessing myogenic potential is considered as one of the most promising approaches to solve the problem of effective restoration of skeletal muscles structure and function. In part I of the article the characteristic features, functions and phenotypic characteristics of satellite cells (SC) are reviewed as key factors of skeletal muscle tissue regeneration. Presented analysis of research results (preclinical and clinical) concerning therapeutic possibilities of technology using SC. In the second part of review will be presented data of the therapeutic use of stem cells of muscle and non-muscle origin for the treatment of skeletal muscles diseases.

[Prospects for the Use of Cells Possessing Myogenic Potential in the Treatment of Skeletal Muscle Diseases: a Review of Research. Part 2--Populations of stem cells of muscle and non-muscle origin].

The paper reviews research results of myogenic potential, specific properties, functions, and phenotypic features of a number of stem cells populations of muscle and non-muscle origin, their ability to participate in the restoration of the physiological homeostasis of skeletal muscle. The results of scientific research allow to predict with high probability the establishment of an effective cell technology to treat degenerative muscle diseases.

[Use of autogenous fibroblasts of human oral mucosa for gum recession treatment].

Minimally invasive soft tissue augmentation is a challenging problem for dentists. Use of autogenous fibroblast of oral mucosa is a promising method. The paper represents the results of preclinical, clinical and laboratory studies on the use of autogenous oral mucosa fibroblasts (AOMF) for gum recession correction. The results confirm statistically significant gum thickness augmentation and gum recession decrease after three weekly AOMF injections. The effect lasted for at least 9 months.

Downregulation of VEGF-C expression in lung and colon cancer cells decelerates tumor growth and inhibits metastasis via multiple mechanisms.

Experimental and clinical studies positively correlate expression of vascular endothelial growth factor (VEGF)-C in cancer cells with accelerated tumor progression and/or unfavorable clinical outcome. However, many aspects of tumor-promoting activity of VEGF-C and consequences of its downregulation for tumor progression remain poorly understood. To clarify these points, we created a set of VEGF receptor 3-positive lung carcinoma A549 and colon carcinoma HCT116 cell sublines with stable repression of VEGF-C synthesis. Analysis of the behavior of these cells revealed multiple effects of VEGF-C downregulation, which, in addition to deceleration of cell proliferation and invasion in vitro and inhibition of lymphangiogenesis in tumor and surrounding tissues observed earlier, included previously undescribed effects, in particular, partial restoration of epithelial phenotype, reduction in the percentage of tumor-initiating cells (cancer stem cells) in the cell population and inhibition of metastasis of orthotopic lung cancer xenografts to other lung lobes. These results are consistent with the idea of high potentiality of VEGF-C as a cancer drug target.

p53 hot-spot mutants increase tumor vascularization via ROS-mediated activation of the HIF1/VEGF-A pathway.

The function of p53 tumor suppressor is often altered in various human tumors predominantly through missense-mutations resulting in accumulation of mutant proteins. We revealed that expression of p53 proteins with amino-acid substitutions at codons 175 (R175H), 248 (R248W), and 273 (R273H), representing the hot-spots of mutations in various human tumors, increased the number of vessels in HCT116 human colon carcinoma xenografts and, as a result, accelerated their growth. Stimulation of tumor angiogenesis was connected with about 2-fold increase in intracellular level of reactive oxygen species (ROS). Antioxidant N-acetyl-l-aspartate (NAC) decreased vessels number in tumors formed by cells with inactivated p53 and inhibited their growth. Effect of ROS on angiogenesis in tumors expressing hot-spot p53 mutants was correlated with their ability to increase a content of HIF1 transcriptional factor responsible for up-regulation of VEGF-A mRNAs.

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.

[The protective role of p53 in Ras-induced transformation of REF52 cells]. / Zashchitnaia funktsiia p53 pri ras-indutsirovannoi transformatsii kletok REF52.

A study was made of the effect of activated oncogene N-RAS on the function of tumor suppressor p53 and the proliferating ability of rat embryo fibroblasts REF52. The proliferation rate and the portion of S-phase cells increased in the first three days of N-RAS expression. After 5-7 days, the p53 function was enhanced, as manifest in increased p53 lifespan and nuclear content and induced transcription of p53-responsive genes. In particular, Cdk2 p21WAF1/CIP1, an inhibitor of cyclin-dependent kinase 2, was produced to a higher level and arrested the cell cycle in G1. Cells with abrogated or dramatically inhibited N-RAS expression were generated at this stage. Having a selective advantage, these cells gradually displaced N-RAS-expressing cells arrested in G1, so that one month after oncogene induction the culture mostly consisted of morphologically normal, actively proliferating Res-negative cells. Neither cell cycle arrest nor reversion to the normal phenotype were observed in N-RAS expressing cells devoid of the p53 function. Thus, p53 prevented stable N-RAS-induced transformation of REF52 cells, arresting the cell cycle and expediting revertant selection.

[Effect of inactivating the p33ING1 tumor suppressor on the function of cell cycle "checkpoints" and genome stability]. / Vliianie inaktivatsii opukholevogo supressora p33ING1 na funktsiiu "sverochnykh tochek" kletochnogo tsikla i stabil'nost' genoma.

Novel candidate tumor suppressor p33ING1 is known to regulate activity of the p53 protein. The effect of p33ING1 inactivation on the functioning of the cell cycle "checkpoints" and the frequency of chromosomal aberrations was examined. Transduction of the p33-GSEas genetic suppressor element, known to reduce the p53 activity, into p53-positive rat and human cells resulted in: (1) partial abolishment of ethylmetansulphonate- or colcemid-induced arrest of the G1-to-S transition in the G0-synchronized cultures; (2) abolishment of the block in the S phase by the DNA synthesis inhibitor, N-phosphonacetil-L-aspartate (PALA); (3) an increase of the number of spontaneous chromosomal breaks and sister-chromatid exchanges; (4) increased frequency of colchicine-induced polyploidy. Similar effects were observed upon transduction of the p53-GSE22 genetic suppressor element, known to reduce p53 transcriptional activity. Presumably, the effect of p33ING1 inactivation on the cell cycle checkpoints and genetic stability is associated with a decrease in p53 activity.

P53-dependent effects of RAS oncogene on chromosome stability and cell cycle checkpoints.

Mutations activating the function of ras proto-oncogenes are often observed in human tumors. Their oncogenic potential is mainly due to permanent stimulation of cellular proliferation and dramatic changes in morphogenic reactions of the cell. To learn more on the role of ras activation in cancerogenesis we studied its effects on chromosome stability and cell cycle checkpoints. Since the ability of ras oncogenes to cause cell transformation may be dependent on activity of the p53 tumor-suppressor the cells with different p53 state were analysed. Ectopic expression of N-ras(asp12) caused in p53-deficient MDAH041 cell line an augmentation in the number of chromosome breaks in mitogenic cells, significant increase in the frequency of metaphases showing chromosome endoreduplication and accumulation of polyploid cells. Similar effects were induced by different exogenous ras genes (N-ras(asp12), H-ras(leu12), N-ras proto-oncogene) in Rat1 and Rat2 cells which have a defect in p53-upstream pathways. In contrast, in REF52 and human LIM1215 cells showing ras-induced p53 up-regulation, ras expression caused only slight increase in the number of chromosome breaks and did not enhance the frequency of endoreduplication and polyploidy. Inactivation in these cells of p53 function by transduction of dominant-negative C-terminal p53 fragment (genetic suppressor element #22, GSE22) or mutant p53s significantly increased the frequency of both spontaneous and ras-induced karyotypic changes. In concordance with these observations we have found that expression of ras oncogene caused in p53-defective cells further mitigation of ethyl-metansulphonate-induced G1 and G2 cell cycle arrest, but did not abrogate G1 and G2 cell cycle checkpoints in cells with normal p53 function. These data indicate that along with stimulation of cell proliferation and morphological transformation ras activation can contribute to cancerogenesis by increasing genetic instability.

Disruption of actin microfilaments by cytochalasin D leads to activation of p53.

Activation of p53 plays a central role in the cell's response to various stress signals. We investigated whether p53 is activated upon disruption of actin microfilaments, caused by cytochalasin D (CD). We show that treatment with CD leads to accumulation of p53 in the cells and activation of p53-dependent transcription. Treatment with CD led to arrest of G1-to-S transition in cells retaining wild-type p53, while cells with inactivated p53 showed partial rescue from it. CD also induces apoptosis in p53+/+, but not in p53-/- cells. The obtained data suggest that disruption of the actin microfilaments activates p53-dependent pathways.