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

Taxol, 1-beta-D-arabinofuranosylcytosine (ara-C), and etoposide induce apoptosis in HL-60 cells that is blocked by overexpression of Bcl-2 o r Bcl-x(L). A similar to 60-amino acid ''loop'' domain of Bcl-2 and Bc l-x(L) that contains phosphorylation sites is known to negatively regu late their antiapoptotic function. In the present studies, Taxol-, ara -C-, or etoposide-induced apoptosis was examined in HL-60/Bcl-2 Delta and HL-60/Bcl-x(L)Delta cells that express the loop-deletional mutant cDNA constructs p19Bcl-2 Delta 32-80 and p18Bcl-x(L)Delta 26-83, respe ctively. This was compared with control HL-60/neo neo tells as well as HL-60/Bcl-2 and HL-60/Bcl-x(L) cells. The Latter two cell lines overe xpress full-length Bcl-2 and Bcl-x(L), respectively, Immunoblot analys es showed that HL-60/neo and HL-60/Bcl-2 Delta cells express similar l evels of p26Bcl-2, In contrast, as compared with HL-60/neo, HL-60/Bcl- x(L)Delta cells expressed significantly lower levels of p26Bcl-2. p29B cl-x(L) and p21Bax levels were similar in all cell types. Exposure to etoposide (50 mu M) or ara-C (100 mu M) for 4 h induced apoptosis in H L-60/neo cells, but not in HL-60/Bcl-2, HL-60/Bcl-x(L), HL-60/Bcl-2 De lta, or HL-60/Bcl-x(L)Delta cells. In contrast, Tar;ol treatment (500 nM for 24 h) triggered the molecular cascade of apoptosis, represented by the cytosolic increase of cytochrome c and poly(ADP-ribose) polyme rase or the DNA fragmentation factor cleavage activity of caspase-3 in HL-60/neo cells as wed as in HL-60/Bcl-x(L)Delta and HL-60/Bcl-2 Delt a cells, but nut in their counterparts overexpressing full-length Bcl- 2 and Bcl-x(L). Equal amounts of p26Bcl-2 were coimmunoprecipitated wi th apoptosis protease-activating factor 1 (APAF-1) in HL-60/neo and HL -60/Bcl-2 Delta cells, whereas a markedly higher level of p26Bcl-2 coi mmunoprecipitated with APF-1 in HL-60/Bcl-2 cells. In association with Taxol-induced apoptosis, the levels of Bcl-2 that were coimmunoprecip itated with APAF-1 declined in HL-60/neo and HL-60/Bcl-2 Delta cells. This was not observed in HL-60/Bcl-2 cells, in which Taxol-induced apo ptosis was blocked. Previous studies have demonstrated that Taxol indu ces phosphorylation of Bcl-2 in association with Taxol-induced apoptos is of HL-60/neo cells. Immunoblot analysis demonstrated a Taxol-induce d mobility shift of Bcl-2 but not p19Bcl-2 Delta. Taxol also increased [P-32]P-i incorporation in p26Bcl-2, but not in p19Bcl-2 Delta or p18 Bcl-x(L). These findings indicate that the loop domain is necessary fo r the Taxol-induced mobility shift and phosphorylation of Bcl-2, Loop domain also seems to be necessary fur the antiapoptotic effect of Bcl- 2 against Taxol-induced apoptosis but not ara-C- or etoposide-induced apoptosis.