RESUMEN

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.

Asunto(s)

Antioxidantes/farmacología , Senescencia Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Antioxidantes/administración & dosificación , Antioxidantes/síntesis química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Femenino , Humanos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

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.

Asunto(s)

Técnicas Biosensibles/métodos , Citometría de Flujo/métodos , Fluoresceínas/química , Colorantes Fluorescentes/química , Peróxido de Hidrógeno/análisis , Células Madre Mesenquimatosas/metabolismo , Apoptosis , Ciclo Celular , Células Cultivadas , Humanos , Células K562 , Cinética , Células Madre Mesenquimatosas/citología

RESUMEN

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.

Asunto(s)

Células Madre Adultas/citología , Células Madre Embrionarias/citología , Especies Reactivas de Oxígeno/metabolismo , Células Madre Adultas/metabolismo , Antioxidantes/metabolismo , Diferenciación Celular , Células Cultivadas , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Células HeLa , Humanos , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción

RESUMEN

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.

Asunto(s)

Senescencia Celular , Endometrio/metabolismo , Células Madre Mesenquimatosas/metabolismo , Estrés Oxidativo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Endometrio/citología , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Imidazoles/farmacología , Células Madre Mesenquimatosas/citología , Naftalenos/farmacología , Fosforilación/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Proteínas de Unión a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores

RESUMEN

The expression of an α-subunit of interleukin-2 receptor (IL-2Rα) was assessed by quantifying activation-induced upregulation of CD25 in IL-2-independent Jurkat leukemic cell line. It has been found that in growing Jurkat culture within 24 h, phytohemagglutinin (PHA, 5 µg/ml) or PHA in combination with 12,13-phorbol dibutirate (PDBu, 10(-8)M) increase the number of CD25+ cells to 32.3 ± 3.4% (n = 11) and 44.8 ± 8.6% (n = 6) respectively. Interleukin-2 (IL-2, 200 U/ml) alone or in combination with PDBu did not induce CD25 expression in Jurkat cells. All the tested stimulatory agencies affected neither the proliferation status no the growth of Jurkat cell cultures. In contrast to human blood T cells, WHI-P131, a selective pharmacological inhibitor of JAK/STAT signaling and CD25 expression, did not decrease the number of induced CD25+ cells in Jurkat culture. Flow cytometry analysis revealed a dose-dependent decrease in the proportion of cells in G1 phase and an increase in the proportion of cells in G2/M phase in WHI-P131-treated Jurkat cultures. It has been also found that WHI-P131 induces G2/M arrest in the absence of PHA or PDBu. We have concluded that (1) the IL-2-independent T cells of Jurkat line had not loss the mechanism for IL-2Rα expression in response to T cell receptor activation, (2) in the transformed T cells, WHI-P131 can arrest cell cycle at G2/M phase and has effects on targets other than IL-2 receptor-associated tyrosine kinase JAK3.

Asunto(s)

Subunidad alfa del Receptor de Interleucina-2/agonistas , Forbol 12,13-Dibutirato/farmacología , Fitohemaglutininas/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Interleucina-2/farmacología , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/genética , Quinasas Janus/metabolismo , Células Jurkat , Recuento de Linfocitos , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Factores de Transcripción STAT/antagonistas & inhibidores , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Transducción de Señal

RESUMEN

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.

Asunto(s)

Células Madre Mesenquimatosas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Humanos , Células Madre Mesenquimatosas/citología

RESUMEN

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.

Asunto(s)

Endometrio/citología , Fibroblastos/citología , Pulmón/citología , Células Madre Mesenquimatosas/citología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Endometrio/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Pulmón/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

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.

Asunto(s)

Colorantes Fluorescentes/farmacología , Mitocondrias/metabolismo , Membranas Mitocondriales/fisiología , Fenantridinas/farmacología , Superóxidos/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Etidio/análogos & derivados , Etidio/química , Citometría de Flujo , Colorantes Fluorescentes/química , Humanos , Microscopía Fluorescente , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Fenantridinas/química , Superóxidos/química

RESUMEN

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.

Asunto(s)

Apoptosis/genética , Senescencia Celular/genética , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Endometrio/citología , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Especificidad de Órganos , Estrés Oxidativo

RESUMEN

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.

Asunto(s)

Regulación de la Expresión Génica , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-2/metabolismo , Linfocitos/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/metabolismo , Janus Quinasa 3 , Linfocitos/citología , Linfocitos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fitohemaglutininas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/genética

RESUMEN

Tumorigenicity of murine hepatoma cells (MH22a) and their sensitivity to lysis by natural killers (NKs) have been studied after exposure to polychromatic visible and infrared light (VIS-IR, 480-3400 nm, 40 mW/cm2), similar to the terrestrial solar spectrum without its minor UV component, in order to elucidate the involvement of this important environmental and physiotherapeutic factor in regulation of the anti-tumor defense system. The MH22 cells were in vitro exposed to VIS-IR light and their sensitivity to lytic activity of NKs was evaluated. We found that sensitivity of MH22a cells to lysis by NKs after exposure to VIS-IR light at a dose of 4.8 J/cm2 increased 1.5-2 times, while it did not change after exposure to a dose of 9.6 J/cm2 at all ratios (1 : 5-1 : 50) of the number of NKs (effectors) to that of hepatoma cells (targets). The increase in the sensitivity of hepatoma cells to NKs was accompanied by structural changes of cell surface: the capability of supramembraneous glycoproteins (glycocalix) to sorb the vital dye alcian blue (AB) was significantly lower as compared with the unexposed cells of control group. However, no changes in AB sorption was revealed in hepatoma cells exposed to the light at a dose 9.6 J/cm2. Tumorigenicity of photo-irradiated MH22a cells has been studied in the in vivo experiments. Light-exposed (4.8 and 9.6 J/cm2) and intact hepatoma cells were transplanted into syngenic mice C3HA. Tumor volumes 25 days after transplantation proved to be smaller after exposure to the light at both doses than in the control group (4-4.5 times and 2.5-4 times, respectively), which correlated with the increase in the sensitivity to lisys by NKs and decrease in the AB sorption only after light exposure at dose 4.8 J/cm2. Using the flow cytometry method we could show that VIS-IR light at the applied doses did not interfere with the distribution of hepatoma cells over the cycle phases and thus deceleration of the tumor growth was not associated with cytostatic effect of VIS-IR light. To evaluate effect of polychromatic light on the growth of the preformed tumors, the 5-day course of daily light exposures of tumor bearing mice C3HA was carried out in 10 days after subcutaneous transplantation of 2 x 10(5) cells of syngene hepatoma when the tumors had developed in 100% animals. Like in the case of transplantation of the light-exposed cells, irradiation of the tumor bearing mice at doses 4.8-9.6 J/cm2 resulted in deceleration of tumor growth (2.1-2.9 and 2.2 times respectively) for 4 weeks as compared with non-irradiated mice.

Asunto(s)

Carcinoma Hepatocelular/radioterapia , Células Asesinas Naturales/inmunología , Neoplasias Hepáticas/radioterapia , Células Tumorales Cultivadas/efectos de la radiación , Azul Alcián/metabolismo , Animales , Pruebas de Carcinogenicidad , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Citotoxicidad Inmunológica/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Glicocálix/química , Glicocálix/efectos de la radiación , Rayos Infrarrojos , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de la radiación , Luz , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Carga Tumoral , Células Tumorales Cultivadas/trasplante

RESUMEN

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.

Asunto(s)

Endometrio/metabolismo , Fibroblastos/metabolismo , Estrés Oxidativo , Células Madre/metabolismo , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Endometrio/citología , Endometrio/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Humanos , Peróxido de Hidrógeno/farmacología , Pulmón/citología , Pulmón/embriología , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Piel/citología , Piel/metabolismo , Células Madre/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Proliferación Celular , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Quinasas Janus/fisiología , Linfocitos T/inmunología , Familia-src Quinasas/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Humanos , Interleucina-2/inmunología , Quinasas Janus/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Fitohemaglutininas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Familia-src Quinasas/antagonistas & inhibidores

RESUMEN

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.

Asunto(s)

Proliferación Celular , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Interleucina-2 , Linfocitos T/inmunología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Humanos , Interleucina-2/inmunología , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/fisiología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Fitohemaglutininas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/fisiología

RESUMEN

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.

Asunto(s)

Receptores ErbB/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metaloproteasas/metabolismo , Estrés Oxidativo , Línea Celular Tumoral , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Peróxido de Hidrógeno/farmacología , Transducción de Señal , Activación Transcripcional

RESUMEN

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.

Asunto(s)

Antivirales/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Interferón gamma/farmacología , Antivirales/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Células Epiteliales/citología , Receptores ErbB/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células HEK293 , Células HeLa , Humanos , Interferón gamma/metabolismo

RESUMEN

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.

Asunto(s)

Proteínas Portadoras/farmacología , Diferenciación Celular/efectos de los fármacos , Dopamina , Células Madre Embrionarias/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Neuronas/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Diferenciación Celular/fisiología , Línea Celular , Modelos Animales de Enfermedad , Células Madre Embrionarias/citología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Neuronas/citología , Neuronas/trasplante , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/terapia , Tirosina 3-Monooxigenasa/biosíntesis

RESUMEN

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.

Asunto(s)

Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Interleucina-2/inmunología , Linfocitos T/inmunología , Proliferación Celular , Humanos , Interleucina-2/farmacología , Activación de Linfocitos , Mitógenos/farmacología , Fitohemaglutininas/farmacología , Linfocitos T/efectos de los fármacos , Factores de Tiempo

RESUMEN

The key protein of the global cellular response to DNA damage is proteinkinase ATM. Ataxia-telangiectasia (AT), a genetic disorder due to mutations in both alleles of ATM gene, is characterized by numerous neurological abnormalities, increased frequency of malignant tumors, premature ageing and increased radio sensitivity. AT is the most frequently found disease displaying inherited radio sensitivity. The data accumulated by this time on the role of the protein ATM in regulation of cellular response to DNA damage and detailed description of its proteins-targets allows to analyze repair potential and manifestation of premature ageing markers both in the cells obtained from AT patients and in the cells of their heterozygous parents. Primary skin fibroblasts obtained from AT patients and their heterozygous parents were analyzed by the flow cytometry and comet assay. It has been shown that cells of the patient AT8SP do not initiate cell cycle blockade after ionizing irradiation during all the experiment, unlike the healthy donor cells where cell cycle blockade is observed. Irradiated cells of the heterozygous parents demonstrated less brightly expressed changes in cell cycle parameters than healthy donor's cells did. The ability to repair DNA double-strand breaks (DSBs) after irradiation is reduced in the cells of AT patients and their heterozygous parents in comparison with the healthy donor's cells. Cells of the healthy donor were capable to repair not less than 90 % of DNA damage for 2.5 h. The repair efficiency in the cells of AT patients came only to about 30 % of DNA damage and in the cells of heterozygous carries of the disease was approximately 50 %. The difference in the dynamics of DNA damage repair process in different proband's families is in accordance with the reports about great phenotypic variety of the given disease.

Asunto(s)

Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Adolescente , Ataxia Telangiectasia/patología , Proteínas de la Ataxia Telangiectasia Mutada , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de la radiación , Ensayo Cometa , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Femenino , Fibroblastos/efectos de la radiación , Rayos gamma , Humanos , Masculino , Proteínas Quinasas/genética

RESUMEN

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 G(2) 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.

Asunto(s)

Cromosomas/genética , ADN Satélite/genética , Metafase/genética , Animales , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Proteína B del Centrómero/metabolismo , Cromosomas/metabolismo , ARN Helicasas DEAD-box/metabolismo , ADN Satélite/metabolismo , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Ratones , Mitosis/genética , Telómero/genética , Telómero/metabolismo