P4.09 - Role of Caspase-3 during TNF-alpha induced membrane molecule shedding from K-562 cells

Abstract

ABSTRACT Caspases are a large family of evolutionary conserved proteases. Although caspases act mainly within the cell, their activity is triggered and modulated by external signals, including cytokines. Cytokine and caspase networks are closely inter-connected in various ways at different levels including multiple biological effects. The eritroleukemia K-562 cell line was used as target, after treatment with diverse dose of TNF-alpha. The correlation between Caspase-3 activation, degree of apoptosis and necrosis with changes of CD30 and CD45 expression on cell surface and their appearances in supernatant was estimated in kinetics study. Apoptosis, necrosis and cell surface expression of CD30 and CD45 where analyzed using Flow cytometry (FACS Calibur using Cell Quest Pro Software). The intra-cellular Caspase-3 activity, intracellular CD30, CD45 in thecell lysate as well as rheir soluble forms of CD45 and CD30 in supernatante of cultures cells where analyzed in our study by Western blotting methods. The quantification was performed by densitometry analysis at GS-710 Imaging Densitometer, using Image J software. Resuts indicated that Caspase-3 activities was increased in dose and time depending manner, which correlates with apoptosis rate. However, decrease of cell surface membrane molecule (CD30 and CD45) and their appearances of soluble forms in supernatants after TNF treated K-562 cells, are not uniform. Soluble CD30 is rapidly increased in supernatant of treated cells with maximal effects after 4h, without strong caspase-3 activation, while soluble CD45 in supernatant, correlates with caspase-3 activity and rate of apoptosis and showing maximal effects at 24h. Our data indicate that TNF-alpha and Caspase-3 inter reaction have been associated in regulation of diverse cellular effect's, including shedding of cell surface molecules in our model and K-562 tumor cells, during apoptosis and necrosis induction.