Taxol-induced Cytosolic Accumulation of Cytochrome and Phosphorylation of Bcl-2 as Well as for Inhibiting ''Loop'' Domain Is Necessary for Taxol-induced Mobility Shift

Taxol, 1-ß-D-arabinofuranosy Icytosine (ara-C), and etoposide induce apoptosis in HL-60 cells that is blocked by overexpression of Bcl-2 or Ikl-\,, A ~60-amino acid "loop"domain of Bcl-2 and Bcl-xLthat contains phosphorylation sites is known to negatively regulate their antiapoptotic function. In the present studies, Taxol-, ara-C-, or etoposide-induced apoptosis was examined in HL-60/Bcl-2A and HL-60/Bcl-xLA cells that express the loop-deletional mutant cDNA constructs pl9Bcl-2A32-80 and pl8Bcl-xLA26-83, respectively. This was compared with control HL-60/ neo cells as well as HL-60/Bcl-2 and HL-60/Bcl-xLcells. The latter two cell lines overexpress full-length Bcl-2 and IH-l-x,, respectively. Immunoblot analyses showed that HL-60/neo and HL-60/Bcl-2A cells express similar levels of p26Bcl-2. In contrast, as compared with HL-60/neo, HL-60/BcIx, A cells expressed significantly lower levels of p26Bcl-2. p29Bcl-xLand p21Bax levels were similar in all cell types. Exposure to etoposide (50 fiM) or ara-C (100 /j\n for 4 h induced apoptosis in HL-60/neo cells, but not in HL-60/Bcl-2, HL-60/Bcl-xL, HL-60/Bcl-2A, or HL-60/Bcl-xLA cells. In contrast, Taxol treatment (500 IIMfor 24 h) triggered the molecular cascade of apoptosis, represented by the cytosolic increase of cytochrome c and poly(ADP-ribose) polymerase or the DNA fragmentation factor cleavage activity of caspase-3 in HL-60/neo cells as well as in HL-60/BclxLAand HL-60/Bcl-2A cells, but not in their counterparts overexpressing full-length Bcl-2 and Bcl-x,. Equal amounts of p26Bcl-2 were coimmunoprecipitated with apoptosis protease-activating factor 1 (APAF-1) in HL60/neo and HL-60/Bcl-2A cells, whereas a markedly higher level of p26Bcl-2 coimmunoprecipitated with APAF-1 in HL-60/Bcl-2 cells. In association with Taxol-induced apoptosis, the levels of Bcl-2 that were coimmunoprecipitated with APAF-1 declined in HL-60/neo and HL-60/ Bcl-2A cells. This was not observed in 111.-o<)/Bcl-2cells, in which Taxolinduced apoptosis was blocked. Previous studies have demonstrated that Taxol induces phosphorylation of Bcl-2 in association with Taxol-induced apoptosis of HL-60/neo cells. Immunoblot analysis demonstrated a Taxolinduced mobility shift of Bcl-2 but not pl9Bcl-2A. Taxol also increased [32P]P, incorporation in p26Bcl-2, but not in pl9Bcl-2A or plKBcl-x,.. These findings indicate that the loop domain is necessary for the Taxolinduced mobility shift and phosphorylation of Bcl-2. Loop domain also seems to be necessary for the antiapoptotic effect of Bcl-2 against Taxolinduced apoptosis but not ara-Cor etoposide-induced apoptosis.