High doses of synthetic antioxidants induce premature senescence in cultivated mesenchymal stem cells.

Stress-induced premature senescence program is known to be activated in cells by various genotoxic stressors, and oxidative stress is considered to be the main of those. To this end, many studies discover antioxidants as protective anti-aging agents. In the current study, we examined the effects of different antioxidants (Tempol, resveratrol, NAC, DPI) on the mesenchymal stem cells maintained in normal physiological conditions. We used high, but non-cytotoxic antioxidant doses which are widely used in laboratory practice to protect cells from oxidative damage. We show that these substances induce reversible block of cell proliferation and do not cause any genotoxic effects when applied to the quiescent cells. However, the same doses of the same substances, when applied to the proliferating cells, can induce irreversible cell cycle arrest, DNA strand breaks accumulation and DNA damage response activation. As a consequence, antioxidant-induced DNA damage results in the stress-induced premature senescence program activation. We conclude that high doses of antioxidants, when applied to the proliferating cells that maintain physiological levels of reactive oxygen species, can cause DNA damage and induce premature senescence which suggests to re-estimate believed unconditional anti-aging antioxidant properties.

Flow cytometric HyPer-based assay for hydrogen peroxide.

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H2O2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H2O2, by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H2O2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H2O2-reactive dye H2DCFDA and, contrary to the H2DCFDA-based assay, can be employed for the kinetic studies of H2O2 utilization by cells, including measurements of the rate constants of H2O2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H2O2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H2O2.

Redox environment in stem and differentiated cells: A quantitative approach.

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

Reactive Oxygen Species Are Required for Human Mesenchymal Stem Cells to Initiate Proliferation after the Quiescence Exit.

The present study focuses on the involvement of reactive oxygen species (ROS) in the process of mesenchymal stem cells "waking up" and entering the cell cycle after the quiescence. Using human endometrial mesenchymal stem cells (eMSCs), we showed that intracellular basal ROS level is positively correlated with the proliferative status of the cell cultures. Our experiments with the eMSCs synchronized in the G0 phase of the cell cycle revealed a transient increase in the ROS level upon the quiescence exit after stimulation of the cell proliferation. This increase was registered before the eMSC entry to the S-phase of the cell cycle, and elimination of this increase by antioxidants (N-acetyl-L-cysteine, Tempol, and Resveratrol) blocked G1-S-phase transition. Similarly, a cell cycle arrest which resulted from the antioxidant treatment was observed in the experiments with synchronized human mesenchymal stem cells derived from the adipose tissue. Thus, we showed that physiologically relevant level of ROS is required for the initiation of human mesenchymal stem cell proliferation and that low levels of ROS due to the antioxidant treatment can block the stem cell self-renewal.

[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.

Intracellular oxidation of hydroethidine: compartmentalization and cytotoxicity of oxidation products.

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.

[The STAT5 signaling in the expression of alpha-subunit of interleukin-2 receptor in human blood lymphocytes].

The comparative study of the STAT3 and STAT5 activity (as assessed by tyrosine phosphorylation level) and the expression of a α-subunit of interleukin-2 receptor (as examined by cytophotometric evaluation of the number of CD25+ cells) during the phytohemagglutinin (PHA)-induced proliferation of human blood lymphocytes (HBL) have been made. It has been revealed that the level of STAT3 phosphorylation is high in both res ting and competent HBL and remains unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen in both resting and competent HBL. We observed phosphorylation of STAT5 no earlier than 5 h after PHA stimulation and the maximum phosphorylation was detected following 24 h. Exogenous IL-2 induced high level of STAT5 phosphorylation in the competent HBL as early as at 30 min and this level of STAT5 phosphorylation kept in the next 24-48 h. The correlation between alterations in tyrosine phosphorylation level of STAT5 and the expression of CD25 has been established. WHI-P131, an inhibitor of JAK3 kinase, prevents STAT5 activation, cell surface expression of CD25 and lymphocyte proliferation. It has been concluded that JAK3/STAT5 signaling via IL-2 receptor is necessary to maintain the long-term expression of the high-affinity αßγ(c)-receptor of IL-2 and optimal proliferation of HBL.

[The surface expression of CD25 at different stages of proliferative response in human lymphocytes. I. The role of JAK and Src tyrosine kinases as revealed by inhibitory analysis].

The long-lasting expression of an alpha-subunit of interleukin-3 receptor (IL-2Ralpha) was found to accompany the PHA-induced proliferation of human blood lymphocytes (HBL), so that to the end of the second day of mitogenic stimulation only, the large blasts may express the high affinity alphabetagamma(c) receptor for IL-2. With the selective pharmacological drugs to JAK (WHI-P131) and Src (PP2) it is shown that the non-receptor tyrosine kinases are involved in the surface CD25 expression. It is revealed that the PP-2-inhibitable expression of CD25 is timely associated with the initial stage of T cell activation, whereas WHI-P131-inhibitable expression was present during the whole G0/G1/S transition. These data indicate that at the early, antigen-dependent stage the expression of IL-2Ralpha is induced via Src-dependent signaling pathway, and prolonged increase in IL-2Ralpha expression is regulated by IL-2/IL-2 receptor interaction via JAK-dependent signaling pathway.

[The surface expression of CD25 at different stages of proliferative response in human lymphocytes. II. The role of interleukin-2].

The expression of alpha-subunit of interleukin-2 receptor (IL-2Ralpha) was assessed by quantifying activation-induced upregulation of CD25 in human blood lymphocytes (HBL) stimulated by interleukin-2 (IL-2). It was established that exogenous IL-2 induced no surface expression of CD25 neither proliferation at 48 h of IL-2 action. In component HBL, pretreated by sub-mitogenic doses of phytohemagglutinin (PHA), 5-15 % of cell population was revealed to represent the CD2t+ cells, and in the competent cells only, exogenous IL-2 induced the surface expression of CD25 as well as the growth and the proliferative response, which was comparable with those to mitogenic doses of PHA. The JAK3 inhibitor WHI-P131 eliminated IL-2-dependent CD25 expression without influencing the CD25 expression in competent cells. Unlike, PP2 was found to inhibit the IL-2-dependent CD25 expression in a lesser extent than WHI-P131, however this drug was effectively inhibited CD25 expression in PHA-pretreated, competent HBL. These data suggest that Src-dependent signaling participate in the early IL-2Ralpha expression that precedes the IL-2-dependent cell cycle progression of activated HBL. It is concluded that in normal T cells, the IL-2Ralpha expression in firstly induced by antigen (mitogen) and thereafter it is held IL-2 through JAK-dependent signaling pathway.

[Decrease in tumorigenic activity of murine hepatoma cells after treatment with antioxidants and melatonin].

We studied the effect of antioxidants such as N-acetylcysteine (NAC, 10 mM) and alpha-lipoic acid (ALA, 1.25 mM) and of the hormone melatonin (1 microM) on the ability of murine hepatoma cells MH22a to develop tumors in syngenic mice (C3HA) after subsutaneous injection. Tumor formation and development slowed down and mouse mortality decreased when the injected cells were pretreated by NAC, ALA or melatonin during 20 h. Melatonin had the most marked effect. Tumors appeared in 100 % cases after 10 days in control mice when untreated cells had been injected; injection of cells pretreated by NAC or ALA resulted in tumor formation only in 40 and 53 % of mice, respectively. When cells were pretreated with melatonin the tumors appeared only in 18-20 days after injection. Until the end of the observation (36 days) 67 % of control mice died, but when the cells were pretreated by NAC or ALA mouse death-rate was 20 and 53 %, respectively. In the case of melatonin we did not observed any dead mice at all. We showed that treatment by antioxidants delayed (NAC) or completely inhibited (ALA) cell cycle of hepatoma cells. Cell cycle was restored after removal of the antioxidants. Melatonin did not change cell cycle phase distribution. We conclude that there is no direct correlation between loss of tumorigenic properties and changing of proliferative activity of hepatoma cells. Different mechanisms of antioxidants and melatonin action resulting in transient tumor phenotype normalization are discussed.

[Cell senescence induced by histone deacetylase inhibitor sodium butyrate in rodent transformed cells resistant to apoptosis].

The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts transformed by E1A+E1B19 kDa oncogenes has been studied. These transformants are resistant to apoptosis in response to gamma-irradiation and growth factor deprivation. The process of cell senescence was investigated by the analysis of cell growth curves, G1/S and G2/M cell cycle arrest, and senescent associated beta-galactosidase expression. The irreversibility of sodium butyrate antiproliferative activity was analyzed by clonogenic assay. We show that sodium butyrate suppresses proliferation and induces senescence in the E1A+E1B19 kDa transformed cells. Interestingly, NaB induces growth arrest due to accumulation of cells in G2/M phase, these cells are not tetraploid but mainly binuclear. Thus, in case of NaB induced senescence in E1A+E1B19 kDa transformed fibroblasts, the observed suppression of cell proliferation may be the result of cytokinesis failure leading to formation of binuclear and multinuclear cells incapable to proliferate.

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.

[Specific function of STAT5 in regulation of proliferation of chronic leukemia K562 cells: inhibitory effect of WHI-P131].

In this study, we examined the possible role of JAK/STAT signaling pathway in regulation of proliferation of chronic leukemia cells K562. The thyrosine phosphorylation of STAT3 and STAT5 was used as a marker of an activation status of STAT proteins. We demonstrate that in growing culture of K562 both STAT3 and STAT5 are constitutively activated. To determine the significance of STATs activity in maintaining the high level of K562 proliferation we tested two JAK inhibitors: AG-490 (JAK2 and JAK3 inhibitor) and WHI-P131 (a new specific JAK3 inhibitor). We showed that in long-tern cultures (48 h) of K562 cells with AG-490 or WHI-P132 the cells remain viable. It was found that treatment with WHI-P131 (30-100 microM) decreased the thyrosine phosphorylation of STAT5 being without effect on the high level of STAT3 phosphorylation. In proliferating K562 cells, AG-490 (25-50 microM) did not influence STAT3 and STAT5 phosphorylation. The flow cytometry analysis revealed a dose-dependent decrease in G1 and S phases and an increase in G2/M phases in WHI-P131-treated K562 cell cultures and no changes in cell cycle structure in AG-490-treated cells. Thus, our findings indicate a preferential role for STAT5 (not constitutively active STAT3) in proliferation of leukemia to other JAK drugs which stimulate apoptosis and decrease proliferation, WHI-P131 prevents K562 cells growth by arresting in G2/M phases of cell cycle.

[Generation of dopamine neurons from human embryonic stem cells in vitro].

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.

[Effect of histone deacetylases inhibitor sodium butyrate (NaB) on transformants E1A + cHa-Ras expressing wild type p53 with supressed transactivation function].

Induction of cellular senescence by various antitumour agents is a promising strategy of cancer treatment. We assessed the ability of sodium butyrate (NaB), a histone deacetylase inhibitor (HDACi), to reactivate the cellular senescence program in either E1A + cHa-Ras-transformed rat embryo fibroblasts with wild-type p53 (ERas(WT)) and in the isogenic cell line where p53 was inactivated due to expression of the potent genetic suppressor element GSE56 (ERas(GSE56)). NaB treatment increased p53 transcriptional activity and induced an irreversible G1/S cell cycle arrest in ERas(WT), but not in ERas(GSE56) cells. By the transient transfections method using reporter luciferase (p53-LUC) constructions, it was shown that p53-LUC activity as a marker of p53 transactivation function did not increase after X-rays exposure of transformants ERas(GSE56). p53 activity in transformants ERas(WT) increased both after irradiation or upon NaB treatment. Interestingly, the expression of senescence-associated beta-galactosidase (SA-beta-Gal), widely used as a marker of senescence, as well as loss of clonogenic ability, were observed in both cell lines following NaB treatment. Thus, our results suggest that induction of p53 transcription activity could be the key determinant of HDACi-induced cell cycle arrest and senescence in transformed cells and provide an additional evidence of SA-beta-Gal invalidity as a sufficient senescence marker.

[The expression of CD25 in phytohemagglutinin- or interleukin-2-stimulated human peripheral blood lymphocytes].

The timely expression of a high affinity receptor for interleukin-2 (IL-2R) in human peripheral blood lymphocytes stimulated by various mitogens was examined by cytophotometric evaluation of the number of CD25 bearing cells (CD25+). In resting lymphocyte culture both phytohemagglutinin (PHA, 10 (microg/ml) and 12,13-phorbol dibutirate (PDBu, 10-8 M) and ionomycin (IM, 5 x 10(-7) M) induce the long-lasting increase (during 48 h) in the number of CD25+ cells. Only in competent (not in resting) lymphocytes, pretreated by submitogenic doses of PHA (1 microg/ml), interleukin-2 (IL-2) is capable to induce the time-dependent CD25 expression. When comparing the time course of CD25+ expression and the blasttransformation it was found that the CD25 markers were revealed on small stimulated lymphocytes and all the large blasts were the CD+ cells with high-affinity alphabetagamma(c) receptor for IL-2. In conclusion, the expression of alpha-subunit of IL-2R takes place during the IL-2-dependent stage of T cell proliferation and may be directly induced by IL-2 via IL-2R.

[Effect of alpha-lipoic acid on fibroblasts 3T3 and 3T3-SV40. Comparison with N-acetylcysteine action].

In this study we investigated the effect of alpha-lipoic acid (ALA) in concentration range 0.7-5.0 mM on the intracellular level of reduced glutathione, the cell cycle phase distribution, the structure of microfilaments and microtubules of normal (3T3) and transformed (3T3-SV40) fibroblasts. We obtained that ALA increased the glutathione content in transformed cells, but did not change its level in normal cells, induced cell cycle arrest of 3T3 cells (but not 3T3-SV40 cells), and disrupted actin microfilaments in cells of both lines. The effect of ALA was compared with N-acetylcysteine (NAC) action. The whole complex of findings allows us to affirm that each of these antioxidants acts on its own target molecules in normal and transformed cells and activates different signal and metabolic pathways in these cells. But at the same time the intermediate steps of ALA and NAC action can be common (alteration of the intracellular level of glutathione, reorganization of actin cytoskeleton, etc.).

[Long-term regulation of Na+, K+-ATPase pump in human lymphocytes: the role of JAK/STAT- and MAPK-signaling pathways].

In interleukin-2 (IL-2)-induced human blood lymphocytes, the Na+/K+ pump function (assessed by ouabain-sensitive Rb+ influx), the abundance of Na+, K+-ATPase alpha1-subunit (determined by Western blotting) and the alpha1- and beta1-subunits mRNA of Na+, K+-ATPase (RT-PCR), as well as the phosphorylation of STAT5 and STAT3 family proteins and ERK1/2 kinase have been examined. A 3.5-4.0-fold increase in the expression of alpha1- and beta1-subunits mRNA of Na+, K+-ATPase was found at 24 h of IL-2 stimulation. The inhibitors of JAK3 kinase (B-42, WHI-P431) was shown to decrease both the phosphorylation of STATs and the rise in the oubain-sensitive rubidium influx as well as the increased abundance of Na+, K+-ATPase alpha1-subunit. The inhibition of the protein kinases ERK1/2 by PD98059 (20 microM) suppressed the alpha1-subunit accumulation. All the kinase inhibitors tested did not alter the intracellular content ofmonovalent cations in resting and IL-2-stimulated lymphocytes. It is concluded that MAPK and JAK/STAT signaling pathways mediate the IL-2-dependent regulation of the Na+, K+-ATPase expression during the lymphocyte transition from resting stage to proliferation.

[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.