Ubiquitin and regulation of apoptosis in Jurkat cells

Abstract

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.