CHLOROQUINE DOES NOT CANCEL E1A+cHa-Ras TRANSFORMANTS' DEATH INDUCED BY mTOR KINASE INHIBITOR pp242.

We have studied the dependence of cell viability on cell autophagy in control and senescent E1A+cHa-Ras transformed rat embryo fibroblasts. pp242, a TORC1/C2 kinase inhibitor, was used as a trigger of cell autophagy. Cell senescence was induced in the cells by sodium butyrate. pp242 inhibitor occurred to dramatically reduce the functional activity of mitochondria in intact cells causing their death by mitophagy and apoptosis. The presence of chloroquine that blocks lysosome and autophagosome fusion does not cancel pp242 effects. Senescent cells were more resistant to pp242 than control ones. However, their viability was significantly reduced in the presence of chloroquine and pp242. Thus, our results allow us to consider that the usage of chloroquine and pp242 combination is an effective way of cell death induction in intact and senescent Ras-transformants.

CHLOROQUINE DOES NOT CANCEL E1A+cHa-Ras TRANSFORMANTS' DEATH INDUCED BY mTOR KINASE INHIBITOR pp242. .

We have studied the dependence of cell viability on cell autophagy in control and senescent E1A+cHa-Ras transformed rat embryo fibroblasts. pp242, a TORC1/C2 kinase inhibitor, was used as a trigger of cell autophagy. Cell senescence was induced in the cells by sodium butyrate. pp242 inhibitor occurred to dramatically reduce the functional activity of mitochondria in intact cells causing their death by mitophagy and apoptosis. The presence of chloroquine that blocks lysosome and autophagosome fusion does not cancel pp242 effects. Senescent cells were more resistant to pp242 than control ones. However, their viability was significantly reduced in the presence of chloroquine and pp242. Thus, our results allow us to consider that the usage of chloroquine and pp242 combination is an effective way of cell death induction in intact and senescent Ras-transformants.

MEK/ERK-PATHWAY IS REQUIRED TO MAINTAIN CYTOPROTECTIVE AUTOPHAGY PROCESS IN IRRADIATED E1A+cHa-Ras TRANSFORMANTS.

Autophagy is a conservative process of misfolded protein and damaged organelle degradation that serves to support cellular viability. Autophagy is often induced in response to stress, DNA damage, retinoids, starvation and growth factor withdrawal. The aim of the present work was to study autophagic response of E1A+cHa-Ras-transformed cells to irradiation and to analyze the role of MEK/ERK pathway in regulation of autophagy induced by irradiation. MEK/ERK suppression has been found to decrease the viability of irradiated cells. Inhibition of MEK/ERK pathway leads to the changes in the autophagy induced by irradiation connected with disturbances of final stages followed by accumulation of adaptor protein p62/SQSTM1 in autophagic cavities within cytoplasm. Thus, the data obtained allow to suggest that active MEK/ERK pathway is required to support, the cytoprotective autophagy which is induced in response to irradiation of transformed E1A+cHa-ras cells.

[Antiapoptotic gene bcl-2 prevents cellular senescence program reactivation induced by histone deacetylase inhibitor sodium butyrate in E1A and cHa-ras transformed rat fibroblasts].

We have investigated the role of apoptosis resistance gene bcl-2 in the activation of cellular senescence program induced by histone deacetylase inhibitor (HDACi) sodium butyrate (NaBut) in transformed rat fibroblasts. This study was conducted in a resistant to apoptosis induction cell line of rat embryo fibroblasts transfor- med by oncogenes E1A, cHa-ras and bcl-2 (ERasBcl). The parent cell line transformed with only EJA and cHa-ras (ERas) was used as a control. It has been found that NaBut reduces proliferation rate of ERasBcl cells significantly weaker than of ERas transformed cells, despite the fact that the G1 cell cycle arrest was observed in both cell lines. After NaBut treatment, hypertrophy of the apoptosis resistant transformants ERasBcl also was reduced compared to parent cell line ERas, due to less activation of mTORC1, which is known to control the synthesis of protein and ribosome biogenesis. The degree of mTORC1 activation was as.sessed by its target proteins phosphorylation: the ribosomal S6 protein and 4E-BP1--inhibitor of translation initiation factor eIF4E. Since cell senescence process may be associated with changes in autophagy regulation, we analyzed the dynamics of one of the main autophagosome formation markers--protein LC3. The accumulation of lipid-bound form LC3-II changes significantly in ERasBcl cells after NaBut treatment and has transient nature. The set of analyzed cellular senescence markers suggests that a high level of apoptosis resistance gene bcl-2 expression prevents the realization of tumor-suppressor senescence program induced by HDACi sodium butyrate treatment.

[REGULATION OF THE mTOR SIGNALING PATHWAY IN MACROPHAGES IN VARIOUS PATHOLOGIES].

Macrophage is a key cell of immune system, it participates in antiviral, antimicrobial and antitumor defense of the organism, also in regeneration and reparation of tissues. Macrophage coordinates functioning of immune system, participates in tumor growth progression. The process of inflammation consists of two stages. Cytotoxical potential of immunocompetent cells will be realized in the first stage, to avoid a bacterial infection. The second stage of inflammatory process is associated with reparation and regeneration. During inflammation, according it stages, macrophages change functional state, switching from cytotoxical M1 to M2, that associated with reparation. We suppose, that rapamysin, a suppressor of mTOR, causes completely different effects on tumor associated macrophages and cells of microglia. Rapamycin transforms tissue macrophages into M1 phenotype, promoting the tumor regression. While in microglial cells of the central nervous system it induces transformation into M2 phenotype, facilitating the course of the neurodegenerative disease and slowing down the aging.

[Role of MEK/ERK pathway in the regulation of HDACI-induced senescence of transformed rat embryo fibroblasts].

A key regulator of cellular senescence, mTORC1 complex, is the target of many signaling cascades including Ras/Raf/MEK/ERK-signaling cascade. In this paper we investigated the role of MEK/ERK-branch of this cascade in the process of cellular senescence induced by histone deacetylase inhibitor (HDACI) sodium butyrate (NaBut), in transformed rat embryo fibroblasts. Suppression of MEK/ERK activity by inhibitor PD0325901 does not prevent activation of mTORC1 complex induced by NaBut treatment. After the suppression of MEK/ERK, activity of mTORC1 increased as well as complex mTORC2. Activation of mTOR-containing complexes accompanied by the reorganization of the actin cytoskeleton with the formation of actin stress fibers and the appearance of some markers of cellular senescence. In contrast to NaBut-induced senescence accumulation of proteins was not observed, which may be due to increased activity of the degradation processes. Furthermore, the induction of senescence in conditions suppressed MEK/ERK leads to a drastic decrease in cell viability. Thus, NaBut-induced senescence upon suppressed activity of MEK/ERK-branch of MAP kinase cascade has a more pronounced tumor-suppressor effect associated with stronger activation of both mTOR-complexes, reorganization of the actin cytoskeleton and protein degradation.

[TOR-centric concept of regulation mitogenic, metabolic and energetic signal processing in cell].

Kinase TOR (target of rapamycin), discovered as a target of antibiotic rapamycin, the evolutionarily conservative serine/threonine kinase that integrates numerous extra-cellular and intracellular signals, regulating cell growth, protein synthesis and metabolism. Mammalian kinase (mTOR) exists in two complexes: the rapamycin-sensitive TORC1 and rapamycin resistant mTORC2, controlling in the cell different programs. Identification of mTOR as integral component PI3/Akt way that deregulated during carcinogenesis, as well as the existence of a cross-talk between the tumor-suppressor p53 cascade and mTOR demonstrates its unique role in the process of neoplastic growth. This review discusses the various aspects of the regulation of the kinase mTOR, the relationship with the general cell-signaling pathways and its use as a target for the cancer, diabetes, obesity, neurodegenerative changes and hereditary syndromes of aging.

[Involvement of MAP-kinase cascades into the regulation of sodium butyrate induced premature cell senescence].

We studied the role of p38 kinase and JNK1,2 in the activation of the complex mTORC1 and the program of senescence induced by histone deacetylase inhibitor, sodium butyrate (NaBut), in mouse embryonic fibroblasts transformed by E1A+cHa-Ras oncogenes. It was found that transformants from knockouts for the genes p38, were able to implement the program of NaBut-induced senescence, according to the data of the cell cycle arrest, inhibition of proliferation, hypertrophic changes associated with the activation of mTORC1 and SA-beta-galactosidase activity. According to the behavior of these markers, cell knockouts for the genes jnk1,2 were unable to implement NaBut-induced senescence. Induction of senescence closely correlates with the activation of the complex mTORC1, as it was shown by inhibiting mTORC1 with rapamycin. We believe that JNK 1,2 kinases are required for mTORC1 activation and acquiring the markers of premature senescence, induced by NaBut in the E1A+cHa-Ras transformants.

p21(Waf1) is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate.

Cell senescence is characterized by senescent morphology and permanent loss of proliferative potential. HDAC inhibitors (HDACI) induce senescence and/or apoptosis in many types of tumor cells. Here, we studied the role of cyclin-kinase inhibitor p21(waf1) (Cdkn1n gene) in cell cycle arrest, senescence markers (cell hypertrophy, SA-ßGal staining and accumulation of γH2AX foci) in p21(Waf1+/+) versus p21(Waf1-/-) mouse embryonic fibroblast cells transformed with E1A and cHa-Ras oncogenes (mERas). While short treatment with the HDACI sodium butyrate (NaB) induced a reversible G(1) cell cycle arrest in both parental and p21(Waf1-/-) cells, long-term treatment led to dramatic changes in p21(Waf1+/+) cells only: cell cycle arrest became irreversible and cells become hypertrophic, SA-ßGal-positive and accumulated γH2AX foci associated with mTORC1 activation. The p21(Waf1+/+) cells lost their ability to migrate into the wound and through a porous membrane. Suppression of migration was accompanied by accumulation of vinculin-staining focal adhesions and Ser3-phosphorylation of cofilin, incapable for F-actin depolymerization. In contrast, the knockout of the p21(Waf1) abolished most of the features of NaB-induced senescence, including irreversibility of cell cycle arrest, hypertrophy, additional focal adhesions and block of migration, γH2AX foci accumulation and SA-ßGal staining. Rapamycin, a specific inhibitor of mTORC1 kinase, decreased cellular hypertrophy, canceled coffilin phosphorylation and partially restored cell migration in p21(Waf1+/+) cells. Taken together, our data indicate a new role of p21(Waf1) in cell senescence, which may be connected not only with execution of cell cycle arrest, but also with the development of mTOR-dependent markers of cellular senescence.

[Sodium butyrate do not induce the program of premature senescence in transformants with JNK1,2 knockout].

We studied the role of JNK1,2 stress-kinases in the regulation of premature senescence program, stimulated by the inhibitor of histone deacetylase, sodium butyrate (NaB). It was found, that the transformants EIA + cHa-ras selected from embryonic mouse fibroblasts with knockout jnk1,2 stress-kinase genes did not block the cell cycle after sodium butyrate treatment. The data on the cell cycle distribution and cell growth curves showed that even long term (during five days) NaB influence did not suppress proliferation. We did not also reveal any cellular hypertrophy and increase in SA-beta-galactosidase activity after NaB treatment. The data presented suggest that JNK stress-kinases are involved in sodium butyrate-induced senescence in E1A + cHa-Ras mouse transformants, and they are indicative of that JNK1,2 have tumor suppressor properties.

[Role of p38alpha kinase in activation of premature senescence program in transformed mouse fibroblasts].

We investigated the role of p38alpha stress-kinase in regulation of premature senescence program, stimulated by histone deacetylase inhibitor--sodium butyrate (NaB)--after application to rodent transformed cell lines. Investigation was performed on the E1A + cHa-ras transformants selected from mice embryonic fibroblasts null at the p38alpha kinase gene or null fibroblasts at the PPM1D gene, which encoded phosphatase Wip1. Absence of Wip1 led to constitutive activation of p38alpha kinase. It was revealed that after NaB treatment both cell lines completely stopped proliferation due to irreversible cell cycle arrest in G1/S phase. In both cell lines sodium butyrate induced sustained block of prolifaration due to irreversible cell cycle arrest in G1/S phase. Following sodium butyrate treatment cells expressed marker of senescence--beta-galactosidase activity (SA-beta-Gal). Long-term (during several days) NaB treatment of cells led to partial restoration of actin cytoskeleton, focal adhesion contacts and heterochromatin focus formation (SAHF) in the nucleus of senescent cells. Obtained data allow us to suppose that irreversible process of cellular senescence activated by sodium butyrate can occur in the absence of functionally active p38 kinase by means of other ways of cell cycle suppression.

[Induction of premature senescence program by an inhibitor of histone deacetylase sodium butyrate in normal and transformed rat fibroblasts].

We investigated a possibility to induce the premature cell senescence in rat embryo fibroblasts and E1A + cHa-ras transformants. We found that after the treatment with sodium butyrate, an inhibitor of histone deacetylases, both normal and transformed cells completely stopped to proliferate and accumulated at G1/S and G2/M phases of the cell cycle. The cloning efficiency data show that the cell cycle arrest induced by sodium butyrate is irreversible and correlates with the accumulation of active phosphorylated form of stress kinase p38, and with the expression of marker of senescence--beta-galactosidase activity (SA beta-Gal). The program resembling the premature senescence after sodium butyrate treatment is supposed to develop both in normal and transformed cells. The irreversible block of proliferation in E1A + cHa-ras transformants may be regarded as an example of activation of anticancer program like that of premature senescence in the tumor rodent cells.

[Antiapoptotic oncogene bcl-2 induces a program of senescence in E1A + c-Ha-ras-transformants treated with adriamycin].

Introduction of bcl-2 gene in EIA + c-Ha-ras-transformed rat embryo fibroblasts, which are unable to be arrested after damaging influences and possess high proapoptotic sensitivity, results not only in suppression of cell death but also in re-establishment of cell cycle block following DNA damage and serum starvation. Flow cytometry showed that E1A + c-Ha-ras + bcl-2-transformants treated with DNA-intercalator adriamycin are capable of being arrested at G1/S boundary for a long time (for less than 5 days). According to the growth curve data, the number of Bcl-2-overexpressing cells remanins constant for a week of cultivation with adriamycin. Clonogenic efficacy of E1A + c-Ha-ras + bcl-2-cells is brought to no already in 16 h after adriamycin addition. Apoptotic death, revealed by oligonucleosomic fragmentation of DNA, as well as cell death, occurring due to mitotic catastrophe, after adriamycin treatment are almost absent in Bcl-2-overexpressing transformants, as compared with parental E1A + c-Ha-ras-transformants. Bcl-2 introduction in E1A + c-Ha-ras-transformants is accompanied by a rise of SA beta-Gal (Senescence Associated beta-Galactosidase) activity, which is commonly considered to be a marker of cell senescence. Adriamycin treatment of E1A + c-Ha-ras + bcl-2-transformants results in a much higher rise in SA beta-Gal activity, as compared with untreated cells. Co-immunoprecipitation experiments demonstrated the introduction of Bcl-2 to result in formation of Bcl-2 complexes with early region E1A oncoproducts, which are thought to be responsible for proapoptotic susceptibility of E1A-expressing transformants. The data obtained lead to suggestion that bcl-2 transfer to E1A + c-Ha-ras-transformants may induce a switch from the cell death program on the program of senescence after DNA damage, due, presumably, to Bcl-2 interaction with the apoptosis activator the viral oncoprotein E1A.

[The MDR gene and cellular sensitivity to various effects]. / Gen mdr i chuvstvitel'nost' kletok k razlichnym vozdeistviiam.

mdr-Transfected K-562 cells revealed a relatively high resistance to cytotoxic monokines and ionizing radiation as compared to parental cells. Taken together with what is known about the resistance of mdr-expressing cells to multiple cytotoxic drugs, our results point to malignant cells having universal mechanisms of chemo-, bio- and radioresistance.

[Effect of ionizing radiation on functional activity of macrophagesi]. / Vliianie ioniziruiushchei radiatsii na funktsional'nuiu aktivnost' makrofagov.

It was shown that in vitro irradiation (8 Gy) of murine peritoneal macrophages suppressed their spontaneous cytotoxity and induced growth-stimulating activity. Exposure to 4 Gy induced mRNA proximal factors--TGF-beta and TNF-alpha and boosted growth-stimulating activity. These effects should be considered when evaluating efficacy of radiotherapy for tumors.

[The effect of activated macrophage products on neurite growth in an organotypic culture of chick embryo sensory ganglia]. / Vliianie produktov aktivirovannykh makrofagov na rost neiritov v organotipicheskoi kul'ture chuvstvitel'nykh gangliev kurinykh émbrionov.

Murine peritoneal macrophages, activated by BCG vaccine, and human peripheral blood monocytes, activated by lipopolysaccharides, exerted neurite stimulating or neurite inhibiting effects in various periods of activation. The supernatants of these preparations were active in organotypic culture of chick embryo dorsal root ganglia. The inhibition of neurite growth on the 1st day of cultivation was followed by the neurite-stimulating effect. The fluctuation of neurite-inhibition and neurite-stimulation effect of macrophage supernatants suggest the availability of certain changes in cytokine composition in different periods of macrophage activation.

[Synthesis and expression of transforming growth factor-beta in activated macrophages]. / Sintez i èkspressiia transformiruiushchego faktora rosta-beta aktivirovannymi makrofagami.

It has been established that once macrophages become activated, they pass through different stages of functional activity. Mouse macrophages activated by BCG "exerted" pronounced cytotoxic effects for 2-5 days to be followed later by growth-stimulating ones. However, in other experiments, the cytotoxic effect was either absent or occurred at later stages which was probably due to a certain functional state of macrophages before activation. The synthesis of TGF-beta increased 1-2 days after activation with BCG vaccine, lipopolysacharide and gamma radiation. An increase in mRNA TGF-beta i expression was observed only 5 days after activation of macrophages.