"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 or Bcl-xL.A 60-amino acid "loop" domain of Bcl-2 and Bcl-xL 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-2delta and HL-60/Bcl-xLdelta cells that express the loop-deletional mutant cDNA constructs p19Bcl-2delta32-80 and p18Bcl-xLdelta26-83, respectively. This was compared with control HL-60/neo cells as well as HL-60/Bcl-2 and HL-60/Bcl-xL cells. The latter two cell lines overexpress full-length Bcl-2 and Bcl-xL, respectively. Immunoblot analyses showed that HL-60/neo and HL-60/Bcl-2delta cells express similar levels of p26Bcl-2. In contrast, as compared with HL-60/neo, HL-60/Bcl-xLdelta cells expressed significantly lower levels of p26Bcl-2. p29Bcl-xL and p21Bax levels were similar in all cell types. Exposure to etoposide (50 microM) or ara-C (100 microM) for 4 h induced apoptosis in HL-60/neo cells, but not in HL-60/Bcl-2, HL-60/Bcl-xL, HL-60/Bcl-2delta, or HL-60/Bcl-xLdelta 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-xLdelta and HL-60/Bcl-2delta cells, but not in their counterparts overexpressing full-length Bcl-2 and Bcl-xL. Equal amounts of p26Bcl-2 were coimmunoprecipitated with apoptosis protease-activating factor 1 (APAF-1) in HL-60/neo and HL-60/Bcl-2delta 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-2delta 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-2delta. Taxol also increased [32P]Pi incorporation in p26Bcl-2, but not in p19Bcl-2delta or p18Bcl-xL. 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.
Department of Medicine, Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
G
G
Fang
B S
BS
Chang
C N
CN
Kim
C
C
Perkins
C B
CB
Thompson
K N
KN
Bhalla
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053477:Apoptotic Protease-Activating Factor 1; D001665:Binding Sites; D015153:Blotting, Western; D003561:Cytarabine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D005047:Etoposide / Q000037:antagonists & inhibitors / Q000494:pharmacology; D018922:HL-60 Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D009154:Mutation; D017239:Paclitaxel / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C506592:APAF1 protein, human; D000964:Antimetabolites, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D053477:Apoptotic Protease-Activating Factor 1; C494811:BCL2L1 protein, human; D003574:Cytochrome c Group; D004273:DNA, Neoplasm; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D003561:Cytarabine; D005047:Etoposide; D011065:Poly(ADP-ribose) Polymerases; D017239:Paclitaxel