BCL-X(L) OVEREXPRESSION INHIBITS PROGRESSION OF MOLECULAR EVENTS LEADING TO PACLITAXEL-INDUCED APOPTOSIS OF HUMAN ACUTE MYELOID-LEUKEMIA HL-60 CELLS

Paclitaxel has been shown to activate Raf-1 and cause phosphorylation of Bcl-2, which has been correlated with paclitaxel-induced apoptosis of cancer cells. In the present studies, we demonstrate that in human AML HL-60 cells that express Bcl-2 but little Bcl-x(L) (HL-60/neo cell s), paclitaxel-induced phosphorylation of Bcl-2 is followed by increas ed intracellular free Bax levels. This, in turn, is followed by the cl eavage and activation of the key cysteine protease, CPP32 beta/Yama, a nd cleavage of poly(ADP-ribose) polymerase, resulting in the DNA fragm entation of apoptosis. Cotreatment with the benzoquinone ansamycin Gel danamycin depleted Raf-1 but did not decrease Bcl-2 Levels or impair p aclitaxel-induced Bcl-2 phosphorylation in HL-60/neo cells. Also, Geld anamycin did not affect paclitaxel-induced apoptosis of HL-60/neo cell s. As compared to the control HL-60/neo, HL-60/Bcl-x(L) cells contain Bcl-2 as well as an enforced overexpression of Bcl-x(L). Immunoprecipi tation studies with anti-Bcl-2 and/or anti-Bcl-x antibodies demonstrat ed that HL-60/Bcl-x(L) cells possess lower free Bar but higher levels of Bax heterodimerized to Bcl-2 and Bcl-x(L). Following treatment of H C60/Bcl-x(L) cells with paclitaxel, although Bcl-2 phosphorylation was observed, it was not followed by increased free Bax levels, cleavage of CPP32 beta/Yama and poly(ADP-ribose) polymerase, or induction of th e DNA fragmentation of apoptosis. These findings indicate the order of molecular events leading to paclitaxel-induced apoptosis and show tha t Raf-1 may not be involved in paclitaxel-induced phosphorylation of B cl-2 or apoptosis of HL-60 cells.