IN VITRO DETERMINATION OF THE APOPTOTIC EFFECT OF HEPARIN ON LYMPHOBLASTS USING DNA ANALYSIS AND MEASUREMENTS OF Fas AND Bcl-2 PROTEINS BY FLOW CYTOMETRY

Abstract

Heparin has an apoptotic effect beside its anticoagulant, anti-inflammatory, antihypertensive, and antiproliferative effects. In this study, the authors detected the percentages of apoptotic lymphoblasts and the expressions of apoptotic Fas protein and antiapoptotic Bcl-2 protein with flow cytometry in vitro after the incubation of lymphoblasts with heparin. Eleven newly diagnosed acute lymphoblastic leukemia (ALL) children were included in the study. Lymphoblasts were incubated in all different levels of heparin concentrations (0, 10, and 20 U/mL) and the percentages of apoptotic lymphoblasts and the percentages of Fas protein and Bcl-2 proteins were simultaneously measured by flow cytometry at 0, 1, and 2 h. At 0, 1, and 2 h, apoptosis was determined when heparin was added in 10- and 20-U/mL concentrations (p <. 05). The apoptotic effect of heparin on lymphoblasts was higher at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <. 01). The highest apoptosis was detected in the 20-U/mL heparin concentration at the first hour. The expression levels of Fas protein on lymphoblasts were higher at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <. 001). The highest expression of Fas protein was observed in the 20-U/mL heparin concentration at the first hour. The expression levels of Bcl-2 protein on lymphoblasts were lower at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <. 001). The lowest expression of Bcl-2 protein was detected in the 20-U/mL heparin concentration at the first hour. Increased concentrations of heparin had an increasing effect on the percentages of apoptotic lymphoblasts. The expression percentages of Fas protein on lymphoblasts also increased, whereas the expression percentages of Bcl-2 protein on lymphoblasts decreased (p <. 05). These results suggest that low-dose heparin may cause significant apoptosis of lymphoblasts in newly diagnosed ALL patients.