Interakcija Bcl-2 i Bax proteina u limfocitima B iz periferne krvi bolesnika sa hroničnom limfocitnom leukemijom.

Background. Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterized by the accumulation of morphologically mature monoclonal CD5+ B cells in the early phase (G0/G1) of the cell cycle. The accumulation of neoplastically transformed B-lymphocytes (CLL cells) is primarily the consequence of apoptosis blocking in these cells. Bcl-2 proteins are well-known modulators of this process. Some of these proteins are anti-apoptotic while the others are pro-apoptotic. All contain at least one of the four conserved regions called the Bcl-2 homologous domains (BH1-BH4). Evidence indicates that Bcl-2 and Bax form homo- and heterodimers. The anti-apoptotic effect of Bcl-2 protein is based on its ability to bind Bax protein in the heterodimer form, and thus to block the forming of Bax/Bax proapoptotic homodimers. The ratio of Bcl-2/Bax represents the cell autonomous rheostat which determinates the type of the cell reaction to an apoptotic stimulus. Methods. The aim of this study was to determine the level of interaction between these two proteins in CLL cells using the co-immunoprecipition method. The study included the analysis of 20 peripheral blood specimens from 20 patients with CLL, and 20 peripheral blood specimens from healthy persons, who were in the control group. Specimens were precipitated with the monoclonal antibody for Bcl-2 protein, and immunoblotted with the palyclonal antibady for Bax protein (IP: Bcl-2/WB:Bax). At the same time, specimens were precipitated with the polyclonal antibody for Bax protein, and immunoblotted with the monoclonal antibody for Bcl-2 protein (IP: Bax/WB:Bcl-2). The intensity of Bcl-2 and Bax protein's binding compared to the control samples of the peripheral blood from healthy persons, was increased in CLL cells. Results. IP: Bax/WB: Bcl-2 showed a high level of "free" Bcl-2 protein which was not bound in the heterodimer form to Bax protein. Simultaneously IP: Bcl-2/ WB: Bax showed that a higher... [ABSTRACT FROM AUTHOR]

Uvod. Hronična limfocitna leukemija (HLL) je maligna bolest koju karakteriše progresivna akumulacija zrelih, monoklonskih, CD5+ B ćelija u ranoj fazi (G0/G1) ćelijskog ciklusa. Akumulacija neoplastički transformisanih limfocita (ćelija HLL) B primarno je posledica blokade procesa apoptoze ovih ćelija. Proteini Bcl-2 familije su poznati modulatori programirane ćelijske smrti po tipu apoptoze. Svi članovi Bcl-2 familije proteina sadrže najmanje jedan odčetiri visokokonzervisana Bcl-2 homologa domena koji su odgovorni za procese homodimerizacije i heterodimerizacije. Heterodimere formiraju i proteini sa suprotnim dejstvom na proces apoptoze. Antiapoptozno dejstvo Bcl-2 proteina zasniva se na mogućnosti da veže Bax protein (u formi heterodimera) i tako onemogu ći stvaranje proapoptoznih homodimera Bax proteina. Pretpostavlja se da odnos Bcl-2 i Bax proteina predstavlja autonomni ćelijski reostat koji opredeljuje vrstu ćelijskog odgovora na dejstvo apoptotskog stimulusa. Cilj ovog rada bio je da se korišćenjem metode koimunoprecipitacije utvrdi stepen interakcije Bcl-2 i Bax proteina u uzorcima mononukleusnih ćelija iz periferne krvi bolesnika sa HLL. Metode. U našem radu analizovano je 20 uzoraka periferne krvi bolesnika sa HLL i 20 uzoraka periferne krvi zdravih osoba koji sučinili kontrolnu grupu. Uzorci su precipitovani monoklonskim antitelom za Bcl-2 protein i imunoblotovani poliklonskim antitelom za Bax protein (IP: Bcl-2/WB: Bax). Istovremeno su uzorci precipitovani poliklonskim antitelom za Bax protein i imunoblotovani monoklonskim antitelom za Bcl-2 protein (IP: Bax/WB: Bcl-2). Rezultati. Intenzitet vezivanja Bcl-2 i Bax proteina bio je povišen u ćelijama HLL u odnosu na kontrolne uzorke. Rezultati IP: Bax/WB: Bcl-2 pokazali su postojanje slobodnog Bcl-2 proteina koji nije veza... [ABSTRACT FROM AUTHOR]