Changes of Cyclins/CDKs in Fas-mediated apoptosis and the mechansim

Abstract

Objective To study the changes of Cyclins/CDKs expression in Fas-mediated apoptosis, and the action mechanism in leukaemia cell lines and peripheral blood lymphocytes (PBL) in vitro.Methods The target cells--leukaemia cell lines and activated PBL stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, and the changes of Cyclins expression that related to the duration of inducing apoptosis was detected by Cyclin/DNA bi-parameter flow cytometry. Rb protein and the phosphorylation site of CDK1 and CDK2 were checked by Western blotting. Results Cyclin D3 expression was increased and Cyclin E expression decreased evidently, and the expression of Cyclins A and B1 had no significant change before the induction of Fas-mediated apoptosis in Jurkat, Molt-4 cell lines and PBL which entering cell cycle progression, till the late stage of inducing apoptosis in these cells the expression of Cyclin D3 was decreased, while Cyclin E increased. Meanwhile, apoptosis couldn' t be induced by rhFas ligand in G0-phase PBL. The level of CDK2 Thr-160 phosphorylation was decreased through the induction of Fas-mediated apoptosis evidently. The phosphorylation of CDK1 had no significant changes. Conclusion Fas-mediated apoptosis was located at late G1-phase and determined by whether the target cells had passed through the restriction point to cell division cycle or not. And the cell cycle specificity of Fas-mediated apoptosis was the result of the cells with DNA damage being blocked at late G1-phase,due to the decrease of Cyclin E expression and hypophosphorylation of CDK2 and under the surveillance of G1-phase check point. The changes of Cyclins expression at the late stage of Fas-mediated apoptosis is the result of cell arrest at G1-stage. Key words: Apoptosis; Flow cytometry