Bcl-xL 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-xL (HL-60/neo cells), paclitaxel-induced phosphorylation of Bcl-2 is followed by increased intracellular free Bax levels. This, in turn, is followed by the cleavage and activation of the key cysteine protease, CPP32beta/Yama, and cleavage of poly(ADP-ribose) polymerase, resulting in the DNA fragmentation of apoptosis. Cotreatment with the benzoquinone ansamycin Geldanamycin depleted Raf-1 but did not decrease Bcl-2 levels or impair paclitaxel-induced Bcl-2 phosphorylation in HL-60/neo cells. Also, Geldanamycin did not affect paclitaxel-induced apoptosis of HL-60/neo cells. As compared to the control HL-60/neo, HL-60/Bcl-xL cells contain Bcl-2 as well as an enforced overexpression of Bcl-xL. Immunoprecipitation studies with anti-Bcl-2 and/or anti-Bcl-x antibodies demonstrated that HL-60/Bcl-xL cells possess lower free Bax but higher levels of Bax heterodimerized to Bcl-2 and Bcl-xL. Following treatment of HL-60/Bcl-xL cells with paclitaxel, although Bcl-2 phosphorylation was observed, it was not followed by increased free Bax levels, cleavage of CPP32beta/Yama and poly(ADP-ribose) polymerase, or induction of the DNA fragmentation of apoptosis. These findings indicate the order of molecular events leading to paclitaxel-induced apoptosis and show that Raf-1 may not be involved in paclitaxel-induced phosphorylation of Bcl-2 or apoptosis of HL-60 cells.
Ibrado
A M
AM
Department of Medicine, Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Liu
L
L
Bhalla
K
K
D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones; D053148:Caspase 3; D020169:Caspases; D002453:Cell Cycle / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000378:metabolism; D018922:HL-60 Cells / Q000187:drug effects*; D006801:Humans; D047029:Lactams, Macrocyclic; D009363:Neoplasm Proteins / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D011809:Quinones / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011809:Quinones; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D017239:Paclitaxel; C001277:geldanamycin