ROLE OF P38 MAP-KINASE IN THE STRESS-INDUCED SENESCENCE PROGRESSION OF HUMAN ENDOMETRIUM-DERIVED MESENCHYMAL STEM CELLS.

Our recent findings clearly demonstrate that human endometrium-derived mesenchymal stem cells (hMESCs) respond to the sublethal oxidative stress by the premature senescence induction via ÀÒÌ/Chk2/p53/ p21/Rb pathway. Furthermore, based on the application of the SB203580 (SB) we suggested p38 MAP-kinase involvement in senescence progression. However, there are several disadvantages concerning this inhibitor: 1) using SB would not be suitable for in vivo experiments due to toxicity issue; 2) the poor kinase selectivity profile of SB complicates interpretation of the obtained data. Here, in order to confirm the implication of p38 in the H2O2-induced senescence of hMESCs, we applied another highly specific inhibitor of p38 ­ BIRB796 (BIRB). In presence of BIRB we revealed cell size decrease, reduction in the levels of reactive oxygen species, partial restoration of proliferation and increase in Rb phosphorylation levels in comparison to H2O2-treated hMESCs. Summarizing the obtained results we can postulate p38 implication in H2O2-induced senescence of hMESCs, and suggest p38 inhibition as a promising approach in prevention of premature senescence.

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.

[OXIDATIVE STRESS-PROMOTED RESPONSES IN HUMAN ENDOMETRIAL STEM CELLS AND LUNG EMBRYONIC FIBROBLASTS].

Human mesenchymal stem cells are an attractive cell source for tissue engineering. During transplantation they may be subjected to oxidative stress due to unfavorable cellular microenvironment, which is characterized by increased levels of reactive oxygen species. Recently, we have demonstrated that oxidative stress responses of human mesenchymal stem cells derived from endometrium (hMESCs) depend upon the oxidizer concentration. Besides, the duration of the H2O2-treatment duration. The effects of the high H2O2 doses on hMESCs and human lung embryonic fibroblasts were compared. In both cell types, H2O2-treatment for 60 min was shown to promote the multiphase cell cycle arrest, as well as to the dose-dependent cell death that occurred equally from all phases of cell cycle. However, the cell death dynamics in hMESCs and fibroblasts were different. Interestingly, in both cell types, shortening of H2O2-treatment duration from 60 to 10 min induced growth retardation, G1-phase accumulation and the cell size increase. Together, these findings allow us to suggest an induction of the premature senescence as a result of the short cell exposure to the high H2O2 doses. Thus, regarding both human endometrial stem cells and human embryonic fibroblasts, shortening of oxidative stress duration induced by high H2O2 doses enables to avoid the cell death and to produce the features of the premature senescence.

[RELATIONSHIP BETWEEN p53/p21/Rb AND MAPK SIGNALING PATHWAYS IN HUMAN ENDOMETRIUM-DERIVED STEM CELLS UNDER OXIDATIVE STRESS].

Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H2O2 activate both p53/p21/Rb and p38MAPK/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However the mutual relations between p53/p21/Rb and MAPK signaling pathways, including ERK, p38 and JNK remain unexplored as yet. Here, we used the specific inhibitors--pifithrin-α (PFT), U0126, SB203580 and SP600125 to "switch off" one of the proteins in these cascades and to evaluate the functional status alterations of the rest proteins. Suppression each of the MAPK significantly increased the p53 phosphorylation levels, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation compared with p38 and JNK. These results suppose the existence of the reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. Analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as the ERK antagonists: JNK is capable to activate ERK, while p38 may block the ERK activation. Together, these results demonstrate complex links between different signaling cascades in stressed hMESC, implicating ERK, p38 and JNK in regulation of the premature senescence via p53/p21/Rb pathway.

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.

[Differences in defense mechanisms against oxidative stress in both human embryonic and endometrium-derived mesenchymal stem cells].

Oxidative stress has been shown to induce either apoptosis or stress-induced premature senescence (SIPS) in different cell types. At present, it is generally accepted that stem cells have high resistance to oxidative stress; however data reported by various authors are controversial. In this study, we investigated stress responses of human embryonic stem cells (hESC) and human mesenchymal stem cells (hMESC) derived from desquamated endometrium to hydrogen peroxide (H2O2). Cell viability was evaluated by MTT assay. LD50 were determined as 300-350, 350-400 and 600-700 µM for hESC, human embryonic fibroblasts and hMESC, respectively. Thus, among the cell lines studied, hMESC demonstrated the most resistance to increased H2O2 concentration. We have found for the first time that sub-lethal doses of H2O2 induce premature senescence phenotype in hMESC, like in HEF, which is characterized by increased expression of cyclin-dependent kinase inhibitor p21(Waf1/Cip1), an irreversible cell cycle arrest, the permanent loss of proliferative potential, cell hypertrophy and SA-ß-Gal staining. While a sub-lethal H2O2 dose (200 µM) promoted in hMESC only SIPS, the higher H2O2 doses induced also apoptosis in the part of the cell population. On the contrary, in hESC, H2O2 regardless of the doses tested (from 50 to 500 µM) triggered apoptosis, that was the only pronounced response of these cells to oxidative damage. The data obtained demonstrate that stem cells of various origins under oxidative stress utilize the different defense mechanisms: hESC rapidly eliminate damaged cells through apoptosis, whereas hMESC may enter SIPS.

[Comparison of human endometrial stem cells and fibroblasts resistance to oxidative stress].

The response of human endometrial stem cells (hESCs) to oxidative stress has been investigated by flow cytometry. Two terminally differentiated cell lines were used for the comparison: human embryonic lung fibroblasts and human dermal fibroblasts. The oxidative stress was designed by hydrogen peroxide (H2O2) action in the wide range of concentrations (50-1500 microM) during 24 h. It has been shown that the H2O2 amount per one cell (pM/cell), but not H2O2 concentration in the growth medium, should be taken into account for the accurate evaluation of H2O2 effect on different cell lines. Therefore, in our experiments LD50 reflects the amount of H2O2 per cell, at which 50% cells survived after 24 h. We have demonstrated that hESCs are more resistant to H2O2 than embryonic lung fibroblasts, but less resistant than dermal fibroblasts.

[Effect of formaldehyde in low concentrations on the proliferation and organization of the cytoskeleton of cultured cells].

3-4% (1.07-1.42 M) formaldehyde is one of the most popular and well-known organs, tissues and cells fixer. In this manuscript we have shown that formaldehyde in concentrations of up to 60 microM (0.0002%) does not have any negative effect on the viability of cell lines A431, HEK293 and primary rat fibroblasts, but it is also increases the proliferative activity of A431. The influence on A431 cells might be explained by the activation of epidermal growth factor receptors as a result of their interaction with formaldehyde.

[Metalloprotease-mediated HB-EGF release regulates EGF receptor transactivation in A431 cells under oxidative stress].

We have shown earlier that H2O2 induces EGF receptor transactivation in different cells overexpressing EGF receptor. Mechanism of H2O2-induced EGF receptor transactivation in A431 human epidermoid carcinoma cells was examined in this work. We have demonstrated autophosphorylation of Tyr1045, 1068, 1148, 1173 as well as phosphorylation of Tyr845 of EGF receptor in response to H2O2, as assessed by autophosphorylation specific antibody. Tyrosine phosphorylation of EGF receptor by H2O2 did not involve receptor autophosphorylation at Tyr992. Blocking functions of metalloproteases by broad-spectrum inhibitor GM6001 suppressed H2O2-induced phosphorylation of EGF receptor, suggesting dependence of the transactivation on metalloproteases activity. To elucidate the possible role of EGF receptor agonists in its activation we used HB-EGF and TGF-alpha neutralizing antibody. H2O2-induced EGF receptor phosphorylation was inhibited by HB-EGF, but not TGF-alpha, neutralizing antibody. Taken together, our data suggest that, in human epidermoid carcinoma A431 cells, H2O2 stimulates EGF receptor transactivation via metalloprotease-dependent HB-EGF release and autophosphorylation.

[Interferon gamma-mediated growth regulation of epithelial cells].

Interferon gamma (IFNgamma) is known to inhibit proliferation of certain transformed cell lines. Recently, we have demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to IFNgamma (Burova et al., 2007) and provided direct evidence for the dependence of IFNgamma-induced EGFR transactivation upon EGFR expression level in epithelial cells (Gonchar et al., 2008). This study examines an antiproliferative effect of IFNgamma on human epithelial cells lines A431 and HeLa which express high levels of EGFR, as well as HEK293, which expresses low levels of EGFR. We characterized the IFNgamma-induced changes in these cells by studying cell growth, the cell cycle and induction of apoptosis. The response to IFNgamma differed in the tested cell lines: cell growth was inhibited in both A431 and HeLa cells, but not in HEK293 cells, as shown by cell counts and MTT. The cell cycle phases analyzed by flow cytometry were disturbed in A431 and HeLa cells in response to IFNgamma. In contrast, IFNgamma treatment did not alter distribution by cell cycle phases in HEK293. Our results indicate that IFNgamma exhibit an antiproliferative effect depending on the increased expression of EGFR in A431 and HeLa cells. Further, it was demonstrated that IFNgamma induced the caspase 3 activation in A431 cells, suggesting an involvement of active caspase 3 in IFNgamma-induced apoptosis.