PubMed Health⌕ Search

Biomedical subjects

N N Nikol'skiĭ

Publications and source records attributed to N N Nikol'skiĭ.

At least 19 recordsLinked to original sources
PubMed2006

[Oxidized glutathione induces activation of the epidermal growth factor receptor and MAP kinases ERK 1,2].

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.

Animals↗
PubMed2005

[Continuous human embryonic stem cell lines].

A new continuous human embryonic stem cell line (HESC-5) derived from a blastocyst is described. The cultured cell passed over 200 population doublings, which exceeds the Hayflick's limit sufficiently. The cells maintained a stable proliferative activity, high activity of alkaline phosphatase, and expression of transcription factor Oct-4 and of surface antigens SSEA-3, SSEA-4 and TRA-1-60 known to be characteristic of embryonic stem cells of the human origin. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm in the new cell line HESC-5, and in the previously described other four stem cell lines confirms the ability of these cells to retain their pluripotency under in vitro condition. In addition, in all the cell lines, a high telomerase activity was revealed, which controls a stable telomere length and, hence, an unlimited ESC proliferation. Unlike other cell lines, HESC-5 was found, under specific conditions, to spontaneously differentiate into hematopoietic cells. A morphological similarity was shown between ESC colonies cultivated both on a feeder layer and in the non-feeder system.

Blastocyst↗
PubMed2005

[Peculiarities of metaphase chromosome methylation pattern in preimplantation human embryos].

Methylation pattern peculiarities revealed by immunocytochemical analysis of metaphase chromosomes from preimplanted human embryos with monoclonal antibodies against 5-methylcytosine are described. Chromosomes of 2-8-cell triploid human embryos are undermethylated, if compared to those from PHA-stimulated fetal cord blood lymphocytes. Hemimethylation (asymmetric labeling of sister chromatids) is typical for the most of embryonic chromosomes at 2-cell--blastocyst stages due most probably to a passive loss of methylation during initial cleavages. Diffuse labeling and sister chromatid exchanges are two other cytogenetic peculiarities revealed by immunofluorescent staining of early human embryos. Hypomethylation of pericentromeric heterochromatin of chromosomes 1, 9, 16 and different methylation status of some homologous chromosomes may distinguish them from metaphase chromosomes of lymphocytes. M-banding pattern typical for chromosomes from adult and cord blood lymphocytes initially appears in embryonic metaphase chromosomes as early as at a 8-cell stage to be established for most part of chromosomes of the karyotype at the morula-blastocyst stage only.

5-Methylcytosine↗
PubMed2004

[Effect of synthetic proteasomal inhibitor MG132 on dynamics of EGF-receptor complexes endocytosis in A431 cells].

The effect of proteasomal activity suppression induced by MG132, a synthetic proteasomal inhibitor of EGF-receptor complexes endocytosis in human epidermoid carcinoma A431 cell line, was studied. Using subcellular fractionation in 17% Percoll gradient, it was demonstrated that the addition of MG132 to the cells 15 min following stimulation of EGF endocytosis resulted in a slight accumulation of 125I-EGF in early endosomes, and in much more significant accumulation of the labeled growth factor in late endosomes/lysosomes, as compared to untreated cells. The release of 125I-EGF degradation products into the incubation medium was significantly (3-12-fold) inhibited in the presence of MG132. At the same time biochemical analysis has demonstrated that the EGF receptor itself is not a direct target of proteasomes, since it is revealed as a full-length protein with native mol. mass (170 kDa) in fractions of early and late endosomes and lysosomes. Possible mechanisms of the MG132 effect on intracellular processing of EGF-receptor complexes are discussed.

Cell Fractionation↗
PubMed2004

[Effect of nocodazole on the activation of transcription factors STAT1 and STAT3 in A431 cells].

The STAT transcription factors (signal transducers and activators of transcription), STAT1 and STAT3, are involved in signal transduction from growth factors and different cytokine receptors. STAT1 and STAT3 activation mechanisms are not sufficiently investigated, but they are known to depend upon both cell type and stimulus for either of them. Recently, we have shown that nocodazole blocked EGF-induced STAT1 transport to the nucleus. Here, we have compared STAT1 and STAT3 activation in response to IFNgamma, IFNalpha and epidermal growth factor (EGF) in A431 cells. We have shown the STAT1 activation by all these agents; unlike, STAT3 was activated by EGF only. STAT1 and STAT3 activation upon EGF is blocked by both nocodazole and Src-kinase family inhibitor. STAT1 activation upon IFNgamma influence is blocked by nocodazole, but does not depend on the activity of Src-family kinases. The increased STAT3 phosphorylation results from a combined action of Src-kinase inhibitor and IFNgamma. IFNalpha-induced activation of STAT1 was not inhibited by either nocodazole or Src-kinase inhibitor. Taken together, the data obtained suggest that the activation of both STAT1 and STAT3 in A431 cells is accomplished by different mechanisms.

Antineoplastic Agents↗
PubMed2003

[EGF-induced signal transduction in clonal cells of epidermoid carcinoma A431].

A431 epidermoid carcinoma cells have an increased expression of EGF receptor. In contrast to many other cell lines and primary cells, these respond to EGF in high (more than 1 ng/ml) concentrations by cell cycle arrest, apoptosis and detachment. Clonal variants of A431 (1a and 8a), able to grow in the presence of EGF in high concentration, were previously developed in our laboratory (Gudkova, Sorokin, 1989). Here we tested upper pathways of signal transduction from EGF receptor in the clonal variants, as compared to A431. We found no reasonable difference in the expression of EGF receptor, as well as in its EGF-induced phosphorylation in A431 and clonal variants. There were also no changes in the amount and activation of ERK MAP kinase in different cell lines. In contrast, the amount of STAT1 transcription factor, known to play pro-apoptotic and antiproliferative roles, was strictly diminished in both the clonal variants tested (1a and 8a), as compared to the parental line A431. However, EGF-induced tyrosine phosphorylation of STAT1 decreased only in 8a. Increased phosphorylation of Akt protein kinase, the key component of PI-3 kinase of the anti-apoptotic and proliferative signaling pathway, was also observed in clonal variants. The data obtained demonstrate that resistance to EGF can be acquired in cells having similar levels of EGF receptor expression and phosphorylation, but different in STAT1 or PI-3 kinase signal transduction pathways. These pathways may presumably represent two antagonistic key elements regulating A431 proliferation and apoptosis.

Apoptosis↗
PubMed2003

[STAT1 and STAT3 activation by oxidative stress in A431 cells involves Src-dependent EGF receptor transactivation].

Different cellular signal transduction cascades are affected by environmental stressors (UV-radiation, gamma-irradiation, hyperosmotic conditions, oxidants). In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of STATs in A431 carcinoma cells. Oxidative stress, initiated by addition of H2O2 (1-2 mM) to A431 cells, activates STAT3 and, to a lesser extent, STAT1 in dose- and time-dependent manner. Maximum phosphorylation levels were observed after a 2 minutes stimulation at 1-2 mM H2O2. Phosphorylation was blocked by AG1478, a pharmacological inhibitor of the epidermal growth factor receptor tyrosine kinase, implicating intrinsic EGF receptor tyrosine kinase in this process. Consistent with this observation, H2O2-stimulated EGFR tyrosine phosphorylation was abolished by specific Src kinase family inhibitor CGP77675, implicating Src in H2O2-induced EGFR activation. An essential role for Src and JAK2 in STATs activation was suggested by three findings. 1. Src kinase family inhibitor CGP77675 blocked STAT3 and STAT1 activation by H2O2 in a concentration-dependent manner. 2. In Src-/-fibroblasts, activation of both STAT3 and STAT1 by H2O2 was significantly attenuated. 3. Inhibiting JAK2 activity with the specific inhibitor AG490 reduced the level of H2O2-induced STAT3 phosphorylation, but not STAT1 in A431 cells. These data show essential roles for Src and JAK2 inactivation of STAT3. In contrast, H2O2-mediated activation of STAT1 requires only Src kinase activity. Herein, we postulate also that H2O2-induced STAT activation in carcinoma cells involves Src-dependent EGFR transactivation.

Cell Line, Tumor↗
PubMed2003

[Dependence of EGF receptor and STAT factor activation on redox of A431 cells].

Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.

Acetylcysteine↗
PubMed2003

[The role of phosphatidylinositol-3-kinases P85/P110 and hVPS34 in endocytosis of EGF-receptor complexes].

The previous data (Zheleznova et al., 2001) did not enable the authors to conclude which particular wortmannin sensitive PI-3-kinase--p85/p110 (I class PI-3-K) or hVPS34 (III class PI-3-K)--may be involved in the regulation of EGF-receptor endocytosis. In the present work, we have shown that upon stimulation of EGF-receptor endocytosis additional structures stained with antibody against p85 appear in A431 cells, but the p85-positive compartment never co-localized with EGF-receptor-containing compartments either in control or in wortmannin-treated cells. At the same time, wortmannin treatment prevented association of hVPS34 with endosomal membranes. We have also found that early endosomal markers--Rab5 and EEA1 (membrane association of the latter depends on Rab5 and hVPS34)--co-localized with EGF-receptor in the juxtranuclear region during late stages of endocytosis, both in control and upon wortmannin treatment. These observations favor our suggestions that the transition of EGF-receptors from early to late endosomes may occur directly in this juxtranuclear region and be tightly associated with the formation of so called multivesicular bodies (MVB), which are late endosomes per se. We suggest that wortmannin may have no effect on early EEA1-dependent stage of the receptor endocytosis but blocks a transition of EGF-receptor complexes into the late endosomes by inhibiting activity of hVPS34 and removing it from membranes. The hVPS34 product PI-3-K, according to the known data, is involved in the formation of internal vesicles of MVB. Accumulation of EGF-receptors in these vesicles is believed to be necessary for the receptor degradation.

Androstadienes↗
PubMed2003

[Effect of EGF on ubiquitination and proteasome-dependent degradation of phospholipase C gamma1 in A431 cells].

Phospholipase C gamma 1 (PLC gamma 1), an enzyme participating in phosphoinositide turnover, is one of the key elements in cell signaling. Here it is shown that treatment of A431 carcinoma cells with proteasome inhibitors Mg132 and lactacystin results in increasing the PLC gamma 1 intracellular level. Simultaneously, several additional bands with lower electrophoretic mobilities were detected on immunoblots, using anti-PLC gamma 1 antibodies. PLC gamma 1 ubiquitinilation was shown using immunoprecepitation. In control A 431 cells, PLC gamma 1 is ubiquitinilated, but the addition of EGF greatly induces the ubiquitinilation of the protein. Association of PLC gamma 1 with ubiquitin-ligase c-Cb1 was shown. Dynamics of ubiquitinilation under EGF treatment is in a close agreement with that of association of PLC gamma 1 and c-Cb1. It is concluded that PLC gamma 1 is ubiquitinilated and degraded by proteasomes. PLC gamma 1 ubiquitinilation is an EGF-dependent process.

Acetylcysteine↗
PubMed2003

[Isolation and characterisation of continuous human embryonic stem cell lines].

A long-term cultivation (5-8 months) of human blastocyst-derived embryonic cells (hES) was performed. Several properties of hESs were examined to prove the state of continuous cell lines. These cells have passed through 100-175 population doublings with the average population doubling time equal to 37.0 +/- 1.5 h. Isolated hESs, referred to as HESC-1, HESC-2, HESC-3, HESC-4, cultivated on mitotically inactivated mouse embryonic fibroblasts (STO continuous cell line), formed multilayer colonies of various shape. The cells maintained stable proliferative activity, high activity of alkaline phosphatase and expression of transcription factor Oct4, and all this characterizes embryonic stem cells of different origin. Expression of hES specific cell surface antigens (SSEA-3, SSEA-4, TRA-1-81 and TRA-11-81 and TRA-1-60) was confirmed by immunofluorescence analysis with the corresponding monoclonal antibodies. An additional prove for species specificity of HESC lines is the lack of expression of mouse specific surface antigen SSEA1. The cell cycle of HESC-1 undifferentiated cells and embryoid bodies was analysed cytofluorimetrically.

Alkaline Phosphatase↗
PubMed2002

[Effect of an vacuolar proton pump inhibitor bafilomycin A1 on the intracellular processing of markers for receptor-mediated and liquid phase endocytosis].

By means of subcellular fractionation in density Percoll gradients, immunoblotting and immunofluorescense, the effect of BafA1 on endocytosis of EGF-receptor complexes and horse radish peroxidase (HRP) in A431, HER14 and HC11 cell lines was studied. It was shown that the pretreatment of all used cell lines with BafA1 completely inhibited EGF degradation, but did not interfere with the delivery of significant portion of EGF-receptor complexes to late endosomes and lysosomes and transition of the receptor to juxtranuclear region. At the same time, BafA1 was found to dramatically inhibit the delivery of fluid phase marker HRP to late endosomes of A431 cells. The BafA1 effect on endocytosis of high concentrations of EGF was similar to that on HRP endocytosis. Regulatory mechanisms of early-to-late endosomal compartment transition are discussed.

3T3 Cells↗
PubMed2001

[Effect of wortmannin on endocytosis of epidermal growth factor receptors].

Using subcellular fractionation of human carcinoma A431 cells in Percoll gradient it was shown that P13-kinase (P13-K) inhibitor wortmannin blocked the transition of the EGF-receptor complexes from the early to the late endosomes. Under conditions when the receptor TK-dependent sorting system is mainly involved, i.e. at low EGF concentrations, the efficiency of sorting was seen to fall 5-10-folls in the presence of wortmannin compared to the control. At high EGF concentrations of the toxin inhibitory effect was no more than 30%. Immunofluorescent analysis has demonstrated that wortmannin treatment led to a juxtranuclear localization of EGF-receptors, which is presumably characteristic of the late endosomes. However, this localization became obvious even in 15 min following endocytosis stimulation, when EGF-receptors, according to the Percoll data, were associated mainly with the early endosomes. A possible role of phosphatidylinositol metabolism products in endocytosis regulation is discussed in addition to the structural and functional organization of the early endosomal compartments. A conclusion is made that P13-K may be a component of the EGF receptor-specific sorting system.

Androstadienes↗
PubMed2001

[Dynamics of the interaction of Stat1 transcription factor with the internalized EGF receptor and karyopherins alpha in response to stimulation of cells with epidermal growth factor].

The present paper deals with two aspects of EGF-induced signal transduction via transcriptional factor STAT1. Utilizing vesicle fractionation in Percoll density gradient followed by co-immunoprecipitation, we observed the association of STAT1 with EGF receptor internalized in early endosomes. Co-immunoprecipitation studies with antikaryopherin alpha antibody showed the binding of activated STAT1 to nuclear import factors--karyopherins alpha. Both complexes demonstrate similar dynamics upon EGF treatment: they are formed at the early times, cannot be detected within 15 min, and reappear in 20 min or later. The complex of STAT1 and karyopherin alpha is localized in the cytoskeletal fraction.

Animals↗
PubMed2001

[The role of SRC kinase in activation of transcription factor STAT1].

Transcription factor STAT1 (Signal Transducers and Activators of Transcription) takes part in signal transduction from receptors of growth factors and many cytokines, including interferons. In this paper, the role of tyrosinkinases Src and JAK2 was estimated in activation of STAT1 by epidermal growth factor (EGF) and hyperosmotic shock. Using a pharmacological inhibitor of Src kinases CGP77675 and cells with knockout c-src, it was shown that Src activated STAT1 upon stimulation by both epidermal growth factor and hypersomatic shock. In contrast, JAK2 activity exerted no influence on these processes.

Animals↗
PubMed2001

[The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. I. Dynamics of EGF internalization, recycling, sorting, and degradation during inhibition of Src-kinase activity].

In the present work, the role of Src-kinase in regulation of different stages of EGF-receptor endocytosis was studied. We used murine fibroblasts with knockout of Src gene and CGP77675, and the inhibitor of Src-family kinases. The absence of Src protein in the cells did not lead to any changes in the rates of 125I-EGF internalization or recycling and caused only slight decrease in the rate of its degradation. At the same time, treatment of the wild type cells with the inhibitor resulted in a small decrease in internalization rate and an increase in recycling. The influence of the inhibitor on 125I-EGF degradation was also more pronounced. But even in this case, the effects were no more than 30% of control values. CGP77675 extended the same effect upon cells of HER14 and HC11 lines. Subcellular fractionation of these cells in Percoll gradient has also demonstrated a slight inhibition of 125I-EGF sorting from early to late endosomes. The more pronounced effect of the Src-family kinase inhibitor on the EGF endocytosis, compared to that of the absence of a single Src protein, suggests a compensating mechanism of the Src-family kinases. A conclusion is made that in spite of a slight influence on practically all stages of intracellular pathway of EGF-receptor complexes, Src-kinases are obviously not the key regulators of their endocytosis.

Animals↗
PubMed2001

[The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. Distribution of clathrin after stimulation of EGR endocytosis in various cell lines during inhibition of Src-kinase activity].

A distribution of EGF receptor and clathrin during EGF endocytosis in A431, HER14, WT and PURO cell lines was studied by indirect immunofluorescence. Though the initial distribution of EGF-receptors on A431 and HER14 cells was somewhat different, the late stages of endocytosis proceeded equally and were marked by formation of bright spots in the juxtanuclear region characteristic of the late endosomes. The Src-family kinase inhibitor CGP77675 had no influence on the dynamics of receptor endocytosis at the immunofluorescent level in both cell lines. Stimulation of EGF-receptor endocytosis in A431 cells did not also result in any redistribution of clathrin in the areas where the majority of EGF-receptors are localized, i.e. in the lateral plasma membrane both in the control cells and under CGP77675 treatment. Clathrin in A431, WT and PURO cells demonstrated even a punctuated pattern throughout the cytoplasm with some accumulation in the juxtanuclear region. This distribution depended neither on the absence or presence of Src activity nor on EGF addition. The data obtained indicate that 1) EGF-receptors do not serve as the initiation sites during clathrin coated pit assembly; 2) Src-kinase activation does not result in significant clathrin redistribution in the plasma membrane, and its influence on EGF endocytosis can be considered as a secondary effect.

Animals↗