Shatrova An

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.

Evidence for the existence of satellite DNA‐containing connection between metaphase chromosomes

Physical connections between mitotic chromosomes have been reported previously. It was assumed that the interchromosome connection was based on the DNA‐protein thread. However, the data about DNA sequences and protein component in the thread is fragmentary. We demonstrated on the mouse cultured cell line and prematurely condensed chromosomes that: (a) all four mouse satellite DNA fragments (major and minor satellite, mouse satellite 3 (MS3) and mouse satellite 4 (MS4)) were involved in the thread formation; (b) MS4 was involved in the thread to the least extent among all the other fragments; (c) telomere was never a member of the thread; (d) the thread was synthesized at a late G2 phase; (e) RNA helicase p68 and CENP‐B were among the protein components of the interchromosome connection. It was shown by FACS analysis that in mouse and human cell lines: (1) the flow karyotype spectrums were never free from chromosome aggregates; (2) chromosome association did not depend on the chromosome length and each chromosome was free to associate with the other. J. Cell. Biochem. 101: 1046–1061, 2007.

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.

Expression of transient receptor potential vanilloid channels TRPV5 and TRPV6 in human blood lymphocytes and Jurkat leukemia T cells.

Regulation of Ca2+ entry is a key process for lymphocyte activation, cytokine synthesis and proliferation. Several members of the transient receptor potential (TRP) channel family can contribute to changes in [Ca2+]in; however, the properties and expression levels of these channels in human lymphocytes continue to be elusive. Here, we established and compared the expression of the most Ca2+-selective members of the TRPs, Ca2+ channels transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6), in human blood lymphocytes (HBLs) and leukemia Jurkat T cells. We found that TRPV6 and TRPV5 mRNAs are expressed in both Jurkat cells and quiescent HBLs; however, the levels of mRNAs were significantly higher in malignant cells than in quiescent lymphocytes. Western blot analysis showed TRPV5/V6 proteins in Jurkat T cells and TRPV5 protein in quiescent HBLs. However, the expression of TRPV6 protein was switched off in quiescent HBLs and turned on after mitogen stimulation of the cells with phytohemagglutinin. Inwardly directed monovalent currents that displayed characteristics of TRPV5/V6 currents were recorded in both Jurkat cells and normal HBLs. In outside–out patch-clamp studies, currents were reduced by ruthenium red, a nonspecific inhibitor of TRPV5/V6 channels. In addition, ruthenium red downregulated cell-cycle progression in both activated HBLs and Jurkat cells. Thus, we identified TRPV5 and TRPV6 calcium channels, which can be considered new candidates for Ca2+ entry into human lymphocytes. The correlation between expression of TRPV6 channels and the proliferative status of lymphocytes suggests that TRPV6 may be involved in the physiological and/or pathological proliferation of lymphocytes.

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.

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.

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.

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.

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