E. B. Burova

Sublethal Oxidative Stress Induces the Premature Senescence of Human Mesenchymal Stem Cells Derived from Endometrium

The specific responses of mesenchymal stem cells to oxidative stress may play a crucial role in regulation of tissue homeostasis as well as regeneration of organs after oxidative injury. The responses of human endometrium-derived mesenchymal stem cells (hMESCs) to oxidative stress remain still unknown. Herein, we examined the impact of H2O2 on cell viability, induction of premature senescence, and apoptosis. hMESCs were highly resistant to H2O2 compared with human diploid fibroblasts. To test a hypothesis whether hMESCs may undergo oxidative stress-induced premature senescence, cells were briefly exposed to the sublethal H2O2 doses. H2O2-treated cells were permanently arrested, lost Ki67 proliferation marker, and exhibited a senescent phenotype including cell hypertrophy and increased SA-β-Gal activity. Additionally, in stressed cells the expression levels of p21Cip1, SOD1, SOD2, and GPX1 were elevated. hMESCs survived under stress were not able to resume proliferation, indicating the irreversible loss of proliferative potential. While the low H2O2 doses promoted senescence in hMESCs, the higher H2O2 doses induced also apoptosis in a part of the cell population. Of note, senescent hMESCs exhibited high resistance to apoptosis. Thus, we have demonstrated for the first time that hMESCs may enter a state of premature senescence in response to sublethal oxidative stress.

Interferon gamma-dependent transactivation of epidermal growth factor receptor

The present report provides evidence that, in A431 cells, interferon γ (IFNγ) induces the rapid (within 5 min), and reversible, tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). IFNγ‐induced EGFR transactivation requires EGFR kinase activity, as well as activity of the Src‐family tyrosine kinases and JAK2. Here, we show that IFNγ‐induced STAT1 activation in A431 and HeLa cells partially depends on the kinase activity of both EGFR and Src. Furthermore, in these cells, EGFR kinase activity is essential for IFNγ‐induced ERK1,2 activation. This study is the first to demonstrate that EGFR is implicated in IFNγ‐dependent signaling pathways.

Attachment of A-431 cells on immobilized antibodies to the EGF receptor promotes cell spreading and reorganization of the microfilament system

EGF-like sequences, inherent in a number of extracellular matrix proteins, participate in cell adhesion. It is possible that interactions of these sequences with EGF receptors (EGFR) affect actin filament organization. It was shown previously [Khrebtukova et al., 1991: Exp. Cell Res. 194:48-55] that antibodies specific to EGFR induce capping of these receptors and redistribution of cytoskeletal proteins in A-431 cells. Here we report that A-431 cells attach and spread on solid substrata coated with antibodies to EGFR, even in the absence of serum. Thus, EGFR can act as an adhesion protein and promote microfilament reorganization. Binding of the cells to the EGFR-antibody resulted in the formation of a unique cell shape characterized by numerous, actin-based filopodia radiating from the cell body, but without membrane ruffles. There was also a conspicuous circular belt of actin-containing fibers inside the cell margin, and many irregular actin aggregates in the perinuclear area. The morphologies and actin distributions in A-431 cells spread on fibronectin or laminin 2/4 were very different. On fibronectin, cells had polygonal shapes with numerous stress-fibers and thick actin-containing fibers along the cell edges. On laminin-covered substrata, the cells became fusiform and acquired broad leading lamellae with ruffles. In these cells, there were also a few bundles of filaments running the whole length of the cell body, and shorter bundles extending through the leading lamellae towards the membrane ruffles in the cell edge. These effects and those seen with immobilized EGF suggest that different ligand/receptor complexes induce specific reorganizations of the microfilament system.

Calcium alterations signal either to senescence or to autophagy induction in stem cells upon oxidative stress

Intracellular calcium ([Ca2+]i) has been reported to play an important role in autophagy, apoptosis and necrosis, however, a little is known about its impact in senescence. Here we investigated [Ca2+]i contribution to oxidative stress-induced senescence of human endometrium-derived stem cells (hMESCs). In hMESCs sublethal H2O2-treatment resulted in a rapid calcium release from intracellular stores mediated by the activation of PLC/IP3/IP3R pathway. Notably, further senescence development was accompanied by persistently elevated [Ca2+]i levels. In H2O2-treated hMESCs, [Ca2+]i chelation by BAPTA-AM (BAPTA) was sufficient to prevent the expansion of the senescence phenotype, to decrease endogenous reactive oxygen species levels, to avoid G0/G1 cell cycle arrest, and finally to retain proliferation. Particularly, loading with BAPTA attenuated phosphorylation of the main DNA damage response members, including ATM, 53BP1 and H2A.X and reduced activation of the p53/p21/Rb pathway in H2O2-stimulated cells. Next, we revealed that BAPTA induced an early onset of AMPK-dependent autophagy in H2O2-treated cells as confirmed by both the phosphorylation status of AMPK/mTORC1 pathway and the dynamics of the LC3 lipidization. Summarizing the obtained data we can assume that calcium chelation is able to trigger short-term autophagy and to prevent the premature senescence of hMESCs under oxidative stress.

Tetraploidization or autophagy: The ultimate fate of senescent human endometrial stem cells under ATM or p53 inhibition

ABSTRACT Previously we demonstrated that endometrium-derived human mesenchymal stem cells (hMESCs) via activation of the ATM/p53/p21/Rb pathway enter the premature senescence in response to oxidative stress. Down regulation effects of the key components of this signaling pathway, particularly ATM and p53, on a fate of stressed hMESCs have not yet been investigated. In the present study by using the specific inhibitors Ku55933 and Pifithrin-α, we confirmed implication of both ATM and p53 in H2O2-induced senescence of hMESCs. ATM or p53 down regulation was shown to modulate differently the cellular fate of H2O2-treated hMESCs. ATM inhibition allowed H2O2-stimulated hMESCs to escape the permanent cell cycle arrest due to loss of the functional ATM/p53/p21/Rb pathway, and induced bypass of mitosis and re-entry into S phase, resulting in tetraploid cells. On the contrary, suppression of the p53 transcriptional activity caused a pronounced cell death of H2O2-treated hMESCs via autophagy induction. The obtained data clearly demonstrate that down regulation of ATM or p53 shifts senescence of human endometrial stem cells toward tetraploidization or autophagy.

Inhibition of the EGF receptor and ERK1/2 signaling pathways rescues the human epidermoid carcinoma A431 cells from IFNγ-induced apoptosis.

Interferon gamma (IFNγ) has been demonstrated to inhibit tumor growth in vivo as well as proliferation of multiple types of cultured transformed cells. In this study, we showed that IFNγ promoted progressive death in A431 cells, overexpressing EGF receptor (EGFR). Based on the data provided by evaluating cell morphology, MTT assay, FACS analysis, and cleaved caspase-3 staining we concluded that the major cause of IFNγ-induced A431 cell growth inhibition was not cell cycle arrest, but apoptosis. We investigated a role for the EGFR and ERK1/2 MAPK signaling pathways in IFNγ-induced apoptosis of A431 cells. IFNγ-induced cell death was accompanied by both an increase of the ERK1/2 MAPK activation and a simultaneous reduction of the EGFR activation. Activation of ERK1/2 was crucial for IFNγ-induced cell death because MEK1/2 inhibitors, PD0325901 and U0126 efficiently protected cells from apoptosis by suppressing caspase-3 activation. Even though EGFR tyrosine kinase inhibitor AG1478 also rescued A431 cells from IFNγ-induced apoptosis, unlike MEK1/2 inhibitors, it initiated G1 arrest. Together, these results suggest that sustained inhibition of both EGFR and ERK1/2 leads to significant protection of the cells from IFNγ-induced apoptosis, indicating important roles for the EGFR tyrosine kinase and ERK1/2 MAP-kinases in regulating A431 cell death.

Antioxidant-Dependent Prevention of H2O2-Induced Premature Senescence inHuman Endometrial Stem Cells

Human endometrium-derived mesenchymal stem cells (hMESC) undergo the premature senescence in response to sublethal H2O2 doses what may complicate their successful transplantation into recipients with age-related disorders accompanied by increased levels of oxidative stress. Therefore, basic research is needed to improve and develop new therapeutic strategies based on the stem cell prevention from premature senescence in oxidative stress conditions. The effects of antioxidants on hMESC remain as yet unexplored. The present study aimed to evaluate an ability of antioxidants, N-acetyl-Lcysteine (NAC) and diphenylene iodonium (DPI) to prevent H2O2-induced premature senescence of hMESC

Different protective mechanisms of human embryonic and endometrium-derived mesenchymal stem cells under oxidative stress

Oxidative stress has been shown to cause 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 disputed. 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, 370–400, and 600–700 μM for hESC, human embryonic fibroblasts, and hMESC, respectively. Thus, of the studied cell lines, hMESC exhibited the greatest resistance to increased H2O2 concentration. We found for the first time that a sublethal concentration of H2O2 induced premature senescence phenotype in hMESC, like in HEF, that was characterized by increased expression of cyclin-dependent kinase inhibitor p21Waf1/Cip1, an irreversible cell cycle arrest, the permanent loss of proliferative potential, cell hypertrophy, and the SA-β-Gal staining. Whereas the sublethal H2O2 concentration (200 μM) promoted in hMESC only SIPS, higher H2O2 concentrations also induced apoptosis in a small part of the cell population. On the contrary, in hESC, H2O2, regardless of the tested concentrations (from 50 to 500 μM), triggered apoptosis, which was the only pronounced response of these cells to oxidative damage. The obtained data demonstrate that stem cells of different origins under conditions of oxidative stress use different protective mechanisms: hESC rapidly eliminate damaged cells through apoptosis, whereas hMESC are subjected to 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 the p53/p21/Rb and p38/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However, the interrelations between the p53/p21/Rb and MAPK signaling pathways, including ERK1/2, p38, and JNK, remain yet unexplored. 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 of the proteins. Each MAPK suppression significantly increased the p53 phosphorylation level, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation rather than p38 and JNK. These results suggest the existence of a reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. By analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as ERK antagonists: JNK is able to activate ERK, while p38 may block ERK activation. Together, these results demonstrate the existence of complex links between different signaling cascades in stressed hMESC, implicating ERK, p38, and JNK in regulation of premature senescence via the p53/p21/Rb pathway.

Effect of Formaldehyde at a Low Concentration on Proliferation and Organization of Cytoskeleton of Cultured Cells

Formaldehyde at a concentration of up to 3–4% (1.07–1.42 M) is one of the most widespread and well-known fixatives of organs, tissues, and cells. In the present work, it was shown that formaldehyde at a concentration of up to 60 μM (0.0002%) did not produce negative effect on the viability of cells of lines of A431, HEK293, and primary fibroblasts, but increased the proliferative activity of the A431 cells. This action on the A431 cells can be explained by the activation of a receptor of the epidermal growth factor as a result of its interaction with formaldehyde.