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.

[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.

[MAP-kinase cascade analysis in transformed cell with different abilities to make G1/S block under serum starvation]. / Analiz MAP-kinaznykh kaskadov u transformantov, razlichaiushchikhsia po sposobnosti osushchestvliat' blok kletochnogo tsikla na granitse faz G1/S posle syvorotochnogo golodaniia.

We have studied the ability of ERK1,2, JNK1,2 and p38 kinases to be regulated after serum deprivation in E1A + E1B-19 kDa- and E1A + E1A + c-Ha-ras-transformed rat embryo fibroblasts. It was demonstrated that oncogene transformation resulted in an increase of total kinase content independently of the type of complementing oncogene. However, for ERK1,2 kinases phosphorylation was found to depend on the type of complementing oncogene. Besides, unusual biphasic character for ERK1,2 kinases phosphorylation was checked in control fibroblasts REF52 and in transformed E1A + E1B-19 kDa cells, which undergo G1/S arrest after a 24 h serum starvation. According to the immunoblotting data, phosphorylated forms of ERK1,2 kinases are not detected after 15-30 min of serum deprivation, but their content is restored up to the control level within several hours. At the same time, the level of ERK1,2 phosphorylation in E1A + c-Ha-ras cells did not change after serum withdrawal. Besides, serum deprivation did not lead to significant changes in the level of phosphorylation of both type stress kinases--JNK2 and p38 in all types of studied cells. We discuss possible mechanisms of biphasic alteration in ERK1,2 phosphorylation level under condition of serum deprivation of REF52 cells and E1A + E1B-19 kDa-transformed fibroblasts, able to be arrested in G1 phase.

[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.

[The clinical significance of MDR-1 gene expression in the hemopoietic cells of patients with acute leukemias in different phases of the disease]. / Klinicheskoe znachenie ékspressii gena MDR-1 v gemopoéticheskikh kletkakh bol'nykh ostrymy leikozami v raznykh fazakh zabolevaniia.

AIM: Analysis of cytostatic therapy effects on expression of gene MDR-1 in hemopoietic cells of patients with acute leukemia (AL) in complete clinicohematological remission (CCHR). MATERIALS AND METHODS: The study included 48 AL patients. 27 of them were untreated, 25 were resistant to or had recurrent AL. 4 patients were followed up. Bone marrow mononuclear fraction was investigated. Expression of MDR-1 gene in the cells was evaluated at hybridization. RESULTS: High expression of MDR-1 gene occurred in leukemic blast cells either upon achievement of CCHR or at least 6 months after its onset. When using schemes of chemotherapy containing two potential inductors of gene MDR-1, expression of this gene was registered significantly more frequently than in using schemes based on one inducing drug (p < 0.05). Frequency of occurrence of enhanced expression of gene MDR-1 in leukemic blasts significantly correlated with frequency of CCHR (p < 0.05). Correlation between occurrence of the gene's expression in normal hemopoietic cells in CCHR and occurrence of early AL recurrences was not found. CONCLUSION: The findings may facilitate design of new AL treatment programs.

[Hyperexpression of the multiple drug resistance gene (MDR-1) in chronic myeloleukemia patients]. / Giperékspressiia gena mnozhestvennoi lekarstvennoi ustoichivosti (MDR-1) u bol'nykh khronicheskim mieloleikozom.

AIM: To elucidate prognostic value of MDR-1 gene expression in patients with chronic myeloid leukemia (CML). MATERIALS AND METHODS: The MDR-1 gene expression was studied by in situ hybridization in hemopoietic cells of 63 Ph-positive CML patients in different phases of the disease. The survival of the patients and duration of the chronic phase (CP) were evaluated using the Caplan-Meyer method. RESULTS: MDR-1-positive patients had a shorter survival (p < 0.01) and CP (p < 0.05) than negative ones. MDR-1 gene overexpression has no impact either on the survival or duration of AP and BP (p < 0.05). Moreover, the MDR-1 gene overexpression is not dependent either on the previous treatment or other prognostic markers. CONCLUSION: Overexpression of MDR-1 gene is an independent prognostic factor and an additional parameter to Sokal's scores.

[Immunomodulating therapy in perforated peritonitis]. / Immunokorrigiruiushchaia terapiia pri razlitom peritonire.

The immunological responsiveness was studied in 160 patients with generalized peritonitis. In the postoperative period, 51 patients underwent immunomodulating therapy. The highest effect was produced by transfusion of 300-400 ml leukoconcentrate.

[Use of leukoconcentrate in the treatment of postoperative suppurative complications in cardiosurgical patients with immunosuppression]. / Ispol'zovanie leikokontsentrata pri lechenii posleoperatsionnykh gnoinykh oslozhnenii u kardiokhirurgicheskikh bol'nykh s immunodepressiei.

Skin tests with a complex of antigens have revealed anergy in the postoperative period in 17 out of 116 patients with acquired heart diseases. Postoperative purulent complications were observed in 13 out of them. They were effectively treated by repeated transfusions of concentrations of the donor compatible leukocytes.

[Prediction of infectious complications in surgical patients]. / Prognozirovanie infektsionnykh oslozhnenii u khirurgicheskikh bol'nykh.

An analysis of results of intracutaneous tests with a set of microbe allergens in 121 patients has shown that absence of a skin reaction to all the allergens under test demonstrates a disturbed immune response (incidence of infectious complications was 77%, both lethal outcomes included). The selective hypersensitivity to the injection of certain allergens was due to the carrier state. The normal reaction of slow type hypersensitivity was found in 101 patients (86,4%) and showed no disorders in immune reactivity which was confirmed by the postoperative course without complications. The test of the slow type hypersensitivity is considered to be highly informative and to allow prognosis of postoperative complications.