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

Division of Hematology/Oncology, Department of Medicine, Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322 [G. F., C. N. K., C. P., K. N. B.], and Departments of Molecular Genetics and Cell Biology and Medicine, Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637 [B. S. C., C. B. T.]

Taxol, 1-ß-D-arabinofuranosylcytosine (ara-C), and etoposide induce apoptosis in HL-60 cells that is blocked by overexpression of Bcl-2 or Bcl-x_L. A 60-amino acid "loop" domain of Bcl-2 and Bcl-x_L that 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-2 and HL-60/Bcl-x_L cells that express the loop-deletional mutant cDNA constructs p19Bcl-232-80 and p18Bcl-x_L26–83, respectively. This was compared with control HL-60/neo cells as well as HL-60/Bcl-2 and HL-60/Bcl-x_L cells. The latter two cell lines overexpress full-length Bcl-2 and Bcl-x_L, respectively. Immunoblot analyses showed that HL-60/neo and HL-60/Bcl-2 cells express similar levels of p26Bcl-2. In contrast, as compared with HL-60/neo, HL-60/Bcl-x_L cells expressed significantly lower levels of p26Bcl-2. p29Bcl-x_L and p21Bax levels were similar in all cell types. Exposure to etoposide (50 µM) or ara-C (100 µM) for 4 h induced apoptosis in HL-60/neo cells, but not in HL-60/Bcl-2, HL-60/Bcl-x_L, HL-60/Bcl-2, or HL-60/Bcl-x_L cells. In contrast, Taxol treatment (500 nM for 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/Bcl-x_L and HL-60/Bcl-2 cells, but not in their counterparts overexpressing full-length Bcl-2 and Bcl-x_L. Equal amounts of p26Bcl-2 were coimmunoprecipitated with apoptosis protease-activating factor 1 (APAF-1) in HL-60/neo and HL-60/Bcl-2 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-2 cells. This was not observed in HL-60/Bcl-2 cells, in which Taxol-induced 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 Taxol-induced mobility shift of Bcl-2 but not p19Bcl-2. Taxol also increased [³²P]P_i incorporation in p26Bcl-2, but not in p19Bcl-2 or p18Bcl-x_L. These findings indicate that the loop domain is necessary for the Taxol-induced mobility shift and phosphorylation of Bcl-2. Loop domain also seems to be necessary for the antiapoptotic effect of Bcl-2 against Taxol-induced apoptosis but not ara-C- or etoposide-induced apoptosis.

¹ To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Department of Medicine, Winship Cancer Center, Emory University School of Medicine, 1365-B Clifton Road N.E., Atlanta, GA 30322. Phone: (404) 778-3992; Fax (404) 778-3965.

Received 1/19/98. Accepted 6/ 9/98.

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