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.
Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line.
Earlier, we demonstrated transactivation of the epidermal growth factor receptor (EGFR) in response to interferon gamma (IFNgamma) in epidermal carcinoma A431 cells. It was shown that IFNgamma-induced EGFR transactivation is impossible in some cancer epithelial cells. Here, we hypothesize that IFNgamma-dependent EGFR transactivation in these cells correlates with EGFR quantity on the cell surface. To test this suggestion, a line of stably transfected HEK293 cells (HEK293delta99 cells) expressing high level of mutant EGFR lacking 99 C-terminal residues has been established. HEK293delta99 cells demonstrated EGFR transactivation in response to IFNgamma unlike the parent HEK293 cells, in which transactivation lacked. In HEK293delta99 and A431 cells, the time courses of EGFR activation induced by IFNgamma have the same pattern. In HEK293delta99 cells like A431, IFNgamma-induced EGFR transactivation requires EGFR kinase activity and occurs via autophosphorylation mechanism. Taken together, these data provide direct evidence of the dependence of IFNgamma-induced EGFR transactivation upon EGFR expression level in epithelial cells.
Ligand-independent activation (transactivation) of the epidermal growth factor receptor (EGFR) was demonstrated upon cell stimulation with cytokines, activators of G-protein-coupled receptors and various stressors. Recently, we showed transactivation of EGFR and activation of transcription factor STAT3, rather than STAT1, induced by glutathione disulfide (GSSG) and glutoxim in epidermoid carcinoma A431 cells (Burova et al., Dokl. Akad. Nauk., 2005, 404: 1-3). Glutoxim (PHARMA-VAM, Moscow) is a pharmacological synthetic analogue of GSSG, whose therapeutic use as an immunomodulator has been permitted. In this study, we investigated dynamics of EGFR activation upon A431 cell stimulation with GSSG and glutoxim. The time course of activation has a sinuous pattern. It has been shown that the intrinsic EGFR tyrosine kinase is responsible for the receptor phosphorylation induced by GSSG and glutoxim. Here, we also demonstrated the activation of ERK 1,2 upon treatment of A431 cells and HER14 cells (HIN 3T3 fibroblasts transfected with full-length EGFR) with these drugs. ERK 1,2 activation was abolished by AG1478, a pharmacological inhibitor of EGFR tyrosine kinase, implicating intrinsic EGFR tyrosine kinase in this process.
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.
Dynamics of nuclear translocation of transcription factor Stat1 in human epidermoid carcinoma A431 cells in response to the epidermal growth factor (EGF) was examined by immunofluorescent microscopy and in cytoplasmic and nuclear extracts by Western blot. In has been shown that a prolonged presence of EGF induces a rapid tyrosine phosphorylation and nuclear translocation of Stat1 (within 5 min). The maximum amount of this protein in the nucleus was reached 30 min after the cell treatment to be maintained at the same level for 5 h. To study the dynamics of the export of Stat1 from the nucleus, a gentle treatment of cells with acetate buffer, pH 4.5, was used for extracting the surface-bound EGF. In this case, a complete dephosphorylation of Stat1 in the cytoplasm was observed in 30 min and the export of the Stat1 from the nucleus lasted for 1-6 h. These studies suggest the existence of an EGF-dependent dynamic equilibrium between the import and export of Stat1 in A431 cells.
Here we demonstrate that the epidermal growth factor (EGF) induces association of prosomes (20S-proteasomes) with its receptor in A-431 cells. Additionally, ligand-dependent association of ribonucleoprotein particles (alpha-RNP), containing small ALU-like RNA, with the EGF receptor was demonstrated. A suggestion has been put forward on the involvement of prosomes and alpha-RNP in the EGF signal transmission to different stages of gene expression.
The interaction between epidermal growth factor (EGF) and specific membrane receptors in A-431 cells results in stimulation of its tyrosine kinase (TK) activity. The intrinsic tyrosine kinase phosphorylates both the receptor itself and different intracellular substrates. Upon EGF binding, clustering and internalization of EGF-receptor (EGF-R) complexes are observed. In the present study by incubating A-431 cells with EGF at 4 or at 37 degrees C, eluting surface-bound EGF we were able to compare the TK-activity of internalized EGF receptors to those localized on the plasma membrane. By the method of the phosphorylation of exogenic substrate poly (Glu/Tyr) 4:1 in vitro it was shown that the total TK-activity was equal to 3 microM PO4- incorporated/mg protein/min. Under conditions, when 40% EGF-R complexes were internalized their activity consisted of 46% of the total after inactivation 60% surface-bound receptors. We draw a conclusion that the TK-activity of EGF-receptors is remained during their internalization.
Using cultured A431 cells, a comparative analysis was performed of endocytosis stimulated by the epidermal growth factor (EGF) just following the ligand influence (early endocytosis) and after a 3 hour incubation of A431 cells with EGF (delay endocytosis). It is shown that at the early endocytosis the total amount of 125I-EGF, associated with cells, is decreased less than at the delay endocytosis. The decrease in total amount of cell-associated 125I-EGF was accompanied with the increase in the EGF concentration in the incubation medium. The data obtained suggested a lower rate of internalization for delay endocytosis. The Scatchard analysis of 125I-EGF-binding has shown that both high affinity EGF-receptor and low affinity EGF-receptor undergo the down-regulation. The Percoll gradient fractionation of EGF-loaded cells has shown that at the delay endocytosis 125I-EGF is displaced to the fraction of heavy endosomes and lysosomes slower than at the early endocytosis. It is suggested that a delay of EGF-receptor complexes transition from endosomes to lysosomes may arise during delay endocytosis.
The mechanism by which transcription factor STAT1 is translocated from the cytoplasm to the cell nucleus is not clear. We put forward a hypothesis suggesting an important role of the cytoskeleton in signal transduction. The results of the present work show that the treatment of cells with nocodazole, a microtubule-disrupting drug, inhibits completely STAT1 import to the nucleus. However, the treatment of cells with cytochalasin B, which is known to depolymerize microfilaments, exerted no detectable effect on the transport of STAT1. The sensitivity to nocodazole treatment suggests that STAT1 may utilize a transport pathway that involves the tubulin cytoskeleton. These data throw light on some mechanism of a rapid and effective nonvesicular transport of STAT1.
