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.

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.

[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.

[Delayed biological consequences induced by gamma-radiation in human tumorigenic diploid line SK-UT-1B]. / Otdalennye biologicheskie posledstviia gamma-radiatsii v chelovecheskoi opukholevoi diploidnoi linii SK-UT-1B.

The progeny of SK-UT-1B cells that survived gamma-irradiation with 4 Gy up to the 80th passage was examined. Descendants of irradiated cells lost p53 transactivation properties. Simultaneously, in the presence of nocodazole coordination between M and S phases was disrupted. Meanwhile, descendants of irradiated cells maintained the accurate spindle assembly checkpoint. These data suggest that p53 transactivation function may be required for coordination of M and S phases, rather than for spindle assembly checkpoint. Since it is known that p53 regulates both these processes on the basis of data obtained, we suggest that functions of p53 required for coordination of M and S-phases and for spindle assembly checkpoint are separated. Besides, the data obtained indicate that radiation-induced chromosomal rearrangements are associated with activation of DNA recombination process.

Analysis of p53-dependent mechanisms in the maintenance of genetic stability in diploid tumourigenic line SK-UT-1B of human uterine leiomyosarcoma.

The cells of tumourigenic line SK-UT-1B combine features characteristic both of normal (diploid karyotype, a low level of polyploid cells, absence of chromosomal marker) and tumour cells (high level of chromosomal instability, high malignancy). We suggest that maintenance of diploid karyotype in this line is controlled via the p53/p21 pathway. We demonstrate that the amount of p53 increases following gamma-irradiation and accumulated p53 protein seems to be functional as p53-luc and p21/Waf-luc reporter plasmids were found to be activated. However, gamma-irradiation-induced increase of p53 was not accompanied by increase of p21/Waf on the protein level. Apparently this is one of the reasons for G1/S and G2/M checkpoint control disruption. The absence of these checkpoints could not prevent the proliferation of cells with intrachromosomal rearrangements. The only effective checkpoint in SK-UT-1B is the p53-dependent M checkpoint, which directed the cells with changed chromosome numbers to apoptosis and therefore strictly guarded the diploidy of the cell population. This indicates that p53 can control the preservation of genetic stability at different levels via different pathways.

[Repaired and unrepaired DNA damage induced by x-irradiation of nonproliferating rat hepatocytes]. / Repariruemye i nerepariruemye povrezhdeniia DNK, voznikaiushchie pri rentgenovskom obluchenii neproliferiruiushchikh gepatotsitov krys.

An effective system of repair of double-strand DNA breaks was found in the stock culture of rat hepatocytes by the method of elastoviscosimetry. The inhibitors of DNA resynthesis, hydroxyurea and arabinosyl cytosine, which transformed single-strand breaks into double-strand ones were used to estimate the level of spontaneous affection of the hepatocyte genome. With a reference to this level the authors have demonstrated that after a dose of 2 Gy and 6-24-hour repair, the additional number of lesions was registered. These lesions were shown to be incised by nucleases similar to those involved into the conventional repair process.

[Modification of DNA damage in rat hepatocytes following whole-body roentgen radiation using cysteamine and 8-bromocaffeine]. / Modifitsiruemost' povrezhdenii v DNK gepatotsitov krys posle obshchego rentgenovskogo oblucheniia s pomoshch'iu tsisteamina i 8-bromkofeina.

Using the elastoviscosimetric method it was shown that the level of residual DNA double-strand breaks in rat hepatocytes depended on conditions of total-body X-irradiation: it increased in the presence of a sensitizer, 8-bromocaffeine and decreased in the presence of a protector, cysteamine. This correlation confirms a major role of fixed DNA double-strand breaks in the formation of a lethal effect in a cell.