Relevance. Impairment of apoptosis regulation in P19 cells is correlated with generation of oxidative stress. Under hypoxia, changes in mitochondrial functions occur, which may exacerbate oxidative stress in the tumor cell. The aim of the study was to evaluate the effects of N-ethylmaleimide and 1,4-dithioerythritol on implementation and regulation of apoptosis in P19 cells under hypoxia in vitro . Materials and methods . P19 cells (mouse teratocarcinoma) cultured under hypoxia served as the material for the study. For redox status modulation, 5mM N-ethylmaleimide and 1,4-dithioerythritol in the final concentrations of 5 mM were used. The intracellular concentration of calcium ions, the transmembrane potential and the number of Annexin V, CD95 and CD120 positive cells were determined by flow cytometry. The levels of reduced, oxidized and protein-bound glutathione, protein SH groups, hydroxyl radical and protein carbonyl derivatives were measured by spectrophotometry. Results. The alteration in the redox status of the glutathione system under hypoxia, accompanied by oxidative modification of proteins (glutathionylation and carbonylation), influences the metabolism in the tumor cell on the whole. Under the effects of 1,4-dithioerythritol, an SH group protector, this alteration promotes formation of additional mechanisms to escape apoptosis, whereas under the effects of N-ethylmaleimide, an SH group blocker, it, on the contrary, promotes apoptosis activation. Conclusions . The changes in the redox homeostasis of the tumor cell and modulation of oxidative modification of proteins (glutathionylation and carbonylation) under hypoxia are one of the promising approaches to targeted regulation of cell death.

Relevance. Impairment of apoptosis regulation in P19 cells is correlated with generation of oxidative stress. Under hypoxia, changes in mitochondrial functions occur, which may exacerbate oxidative stress in the tumor cell. The aim of the study was to evaluate the effects of N-ethylmaleimide and 1,4-dithioerythritol on implementation and regulation of apoptosis in P19 cells under hypoxia in vitro. Materials and methods. P19 cells (mouse teratocarcinoma) cultured under hypoxia served as the material for the study. For redox status modulation, 5mM N-ethylmaleimide and 1,4-dithioerythritol in the final concentrations of 5 mM were used. The intracellular concentration of calcium ions, the transmembrane potential and the number of Annexin V, CD95 and CD120 positive cells were determined by flow cytometry. The levels of reduced, oxidized and protein-bound glutathione, protein SH groups, hydroxyl radical and protein carbonyl derivatives were measured by spectrophotometry. Results. The alteration in the redox status of the glutathione system under hypoxia, accompanied by oxidative modification of proteins (glutathionylation and carbonylation), influences the metabolism in the tumor cell on the whole. Under the effects of 1,4-dithioerythritol, an SH group protector, this alteration promotes formation of additional mechanisms to escape apoptosis, whereas under the effects of N-ethylmaleimide, an SH group blocker, it, on the contrary, promotes apoptosis activation. Conclusions. The changes in the redox homeostasis of the tumor cell and modulation of oxidative modification of proteins (glutathionylation and carbonylation) under hypoxia are one of the promising approaches to targeted regulation of cell death.

Introduction. Hypoxia in tumor growth contributes to mitochondrial dysfunction and exacerbates oxidative stress in the immortalized cell. The objective of the study was to investigate the molecular mechanisms of the effects of N-acetylcysteine on redox regulation of tumor cell apoptosis under hypoxia.Material and Methods. P19 cells (mouse teratocarcinoma) cultured under hypoxia served as the material for the study. The redox status was modulated with N-acetylcysteine in the final concentration of 5 mM. The level of reactive oxygen species, concentration of calcium ions, transmembrane potential and the number of CD95-, CD120- and Annexin V-positive cells were determined by flow cytometry. The concentration of glutathione system components as well as the levels of protein SH groups and protein carbonyl derivatives were measured by spectrophotometry.Results. The use of N-acetylcysteine under hypoxic conditions was accompanied by the increased total glutathione concentration and protein SH groups levels, decreased levels of Са2+ ions, proteinbound glutathione and protein carbonyl derivatives, as well as the production of reactive oxygen species and more appropriate functioning of P19 cells mitochondria. N-acetylcysteine contributed to the development of additional resistance of P19 cells to apoptosis under hypoxia.Conclusion. The alteration in the state of the glutathione system under hypoxia influences the changes in tumor cell metabolism on the whole and promotes formation of additional mechanisms to escape apoptosis.

Introduction.One of the crucial tasks in medicine is studying the molecular mechanisms of selective management of tumor cell apoptosis following conformational changes in protein molecules (ubiquitination).The purpose of the study. The aim of the project is to establish the role of ubiquitin and ubiquitinligase in dexamethasone-induced apoptosis in Jurkat cells.Materials and methods.The study was carried out on the Jurkat tumor cell line (intact cells and cells cultured in the presence of an apoptosis inducer dexamethasone in the final concentration of 10 µmol. In intact and dexamethasone-affected Jurkat cells, implementation of apoptosis and the amount of FAS-, TNF Receptor 1 and cells with reduced mitochondrial membrane potential were assessed by flow cytometry using FITC-conjugated Annexin V and Propidium Iodide. The levels of NF-κB, Apaf-1, ubiquitin and ubiquitin ligase were determined by Western blot analysis. The activity of caspase-3 was measured by spectrofluorometry.Results.When adding the apoptosis inducer dexamethasone to the Jurkat cell culture, we registered a fall in the concentration of ubiquitin and a rise in the level of ubiquitinligase against the backdrop of activated receptor(an increase in the amount of Annexin V positive cells, FASand TNF Receptor 1) and mitochondrialmediated (an increase in the number of cells with reduced mitochondrial membrane potential and elevation of Apaf-1 level) pathways of apoptosis, as opposed to the intact cell culture. We estimated the completion of apoptosis by determining the activity of caspase-3 in the investigated tumor cells.Conclusion.The obtained findings allow the conclusion that ubiquitination of regulatory and effector proteins in programmed cell death is one of the molecular mechanisms that regulates and selectively controls apoptosis in Jurkat cells.

Introduction. Changes in the redox status of tumor cells can be used as one of the molecular mechanisms of apoptosis aimed at increasing the susceptibility of tumor cells to chemotherapeutic agents. Purpose: to study the mechanisms of dysregulation of apoptosis in P19 tumor cells under the conditions of redox status modulation. Material and methods. Apoptosis in P19 tumor cells was assessed by flow cytometry analysis. The number of annexin-positive cells, the expression of CD95 and CD120, as well as the intracellular calcium ion concentration and the percentage of cells with reduced mitochondrial transmembrane potential were measured. The protein-glutathione mixed-disulfide level and the GSH/GSSG ratio were determined by spectrophotometry. To modulate redox status of cells, the protector and blocker of SH-groups, or N-acetylcysteine were used. Results. Incubation of cultures in the presence of SH-group blocker resulted in the imbalance in the glutathione system with increased concentration of glutathionylated proteins. A decreased redox status led to an increased CD95 and CD120 expression levels on the membrane of P19 tumor cells, as well as to decreased mitochondrial potential and increased intracellular calcium ion concentration, thus contributing to the launch of a P19 tumor cells. The presence of SH-group blocker and N-acetylcysteine resulted in an increased number of annexinpositive cells. Conclusion. Along with the development of oxidative stress, the molecular redox-dependent mechanisms of apoptosis dysregulation through the mitochondrial and receptor-mediated pathways were identified in the P19 tumor cells.

Introduction. According to modern perceptions, tumor growth, along with oxidative stress formation, is accompanied by hypoxia. Nowadays studying the regulation of cellular molecular system functioning by conformational changes in proteins appears to be a topical issue.Research goal was to evaluate the state of the glutathione system and the level of protein glutathionylation in P19 embryonal carcinoma (EC) cells under hypoxic conditions.Material and methods. P19 EC cells (mouse embryonal carcinoma) cultured under normoxic and hypoxic conditions served the research material. The concentration of total, oxidized, reduced and protein-bound glutathione, the reduced to oxidized thiol ratio as well as glutathione peroxidase and glutathione reductase activity were determined by spectrophotometry.Results. Glutathione imbalance was accompanied by a decrease in P19 EC cell redox status under hypoxic conditions against the backdrop of a rise in protein-bound glutathione.Conclusions. As a result of the conducted study oxidative stress formation was identified when modeling hypoxia in P19 embryonal carcinoma cells. The rise in the concentration of protein-bound glutathione may indicate the role of protein glutathionylation in regulation of P19 cell metabolism and functions under hypoxia.

Reaction of the glutathione system of Jurkat tumor cells and blood lymphocytes was evaluated under conditions of culturing with 5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole (KRIBB3), a selective inhibitor of heat shock protein Hsp27. The results indicated the regulatory role of Hsp27 in the maintenance of the functional activities of glutathione reductase, glutathione peroxidase, and realization of apoptotic death of Jurkat cells and blood lymphocytes. Inhibition of Hsp27 in Jurkat tumor cells led to imbalance of the glutathione system and increase of the share of annexin-positive cells.

The research objective is to determine the role of the gluthatione system components in realization of the receptor pathway of Jukart tumor cell apoptosis.Apoptosis realization using FITC-labeled annexin V and propidium iodide as well as the amount of TNF R1- and Fas-presenting cells has been evaluated by flow cytofluorometry; activity caspase-3 registered a spektroflyuorimetrichesky method. The concentration of reduced and oxidated gluthatione has been determined by spectrophotometry.The material for the research was intact Jukart tumor cells and the ones incubated in the presence of a selective inhibitor of the key gluthatione synthesis enzyme – buthionine-sulfoximine.The research has shown that the gluthatione system plays an important regulatory role in activation of the receptor pathway of Jukart tumor cell apoptosis.The gluthatione system components are targets for activation of programmed cell death in tumor growth.

Aim. To experimentally select the optimum concentration of hydrogen peroxide capable of efficiently induce oxidative stress and launch the programmed death of the maximum number of lymphocytes, but not induce the necrosis. Methods. Jurkat tumor cell line (human T-lymphoblastic leukemia) lymphocytes isolated from the blood of healthy donors (15 males, 18 females) aged 18 to 25 years were the objects of the study. To confirm the object of study, blood cells typing for CD5 using flow cytometry was performed. To model the oxidative stress in vitro, blood lymphocytes were incubated in the presence of hydrogen peroxide at a final concentration of 0.3, 0.5, 1.0 and 2.0 mM. Reduced and oxidized glutathione levels estimation, the ratio between the fractions, and the level of reactive oxygen forms in lymphocytes for a relative assessment of the oxidative stress degree in cancer cells, were used. Results. An optimal final concentration of hydrogen peroxide — 0.5 mM — was established, causing an increase of active oxygen forms concentration in cells, comparable to levels in tumor cells, the formation of a maximum number of annexin positive cells and minimum number propidium-positive cells and the comparable ratio of the reduced and oxidized glutatione levels. Conclusion. The optimum concentration of hydrogen peroxide (0.5 mM) was selected for the oxidative stress formation in the peripheral blood lymphocytes to study the apoptosis dysregulation in oxidative stress in Jurkat line tumor cells (human T-lymphoblastic leukemia).

Aim. To experimentally select the optimum concentration of hydrogen peroxide capable of efficiently induce oxidative stress and launch the programmed death of the maximum number of lymphocytes, but not induce the necrosis. Methods. Jurkat tumor cell line (human T-lymphoblastic leukemia) lymphocytes isolated from the blood of healthy donors (15 males, 18 females) aged 18 to 25 years were the objects of the study. To confirm the object of study, blood cells typing for CD5 using flow cytometry was performed. To model the oxidative stress in vitro, blood lymphocytes were incubated in the presence of hydrogen peroxide at a final concentration of 0.3, 0.5, 1.0 and 2.0 mM. Reduced and oxidized glutathione levels estimation, the ratio between the fractions, and the level of reactive oxygen forms in lymphocytes for a relative assessment of the oxidative stress degree in cancer cells, were used. Results. An optimal final concentration of hydrogen peroxide - 0.5 mM - was established, causing an increase of active oxygen forms concentration in cells, comparable to levels in tumor cells, the formation of a maximum number of annexin positive cells and minimum number propidium-positive cells and the comparable ratio of the reduced and oxidized glutatione levels. Conclusion. The optimum concentration of hydrogen peroxide (0.5 mM) was selected for the oxidative stress formation in the peripheral blood lymphocytes to study the apoptosis dysregulation in oxidative stress in Jurkat line tumor cells (human T-lymphoblastic leukemia).