Spontaneous and drug-induced apoptosis is mediated by conformational changes of Bax and Bak in B-cell chronic lymphocytic leukemia.
The role of Bax and Bak, 2 proapoptotic proteins of the Bcl-2 family, was analyzed in primary B-cell chronic lymphocytic leukemia (CLL) cells following in vitro treatment with fludarabine, dexamethasone, or the combination of fludarabine with cyclophosphamide and mitoxantrone (FCM). A strong correlation was found between the number of apoptotic cells and the percentage of cells stained with antibodies recognizing conformational changes of Bax (n = 33; r = 0.836; P <.001) or Bak (n = 10; r = 0.948; P <.001). Preincubation of CLL cells with Z-VAD.fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), a broad caspase inhibitor, abolished caspase-3 activation, exposure of phosphatidylserine residues, and reactive oxygen species generation; partially reversed the loss of transmembrane mitochondrial potential (DeltaPsim); but did not affect Bax or Bak conformational changes. These results indicate that the conformational changes of Bax and Bak occur upstream of caspase activation or are caspase independent. Following drug-induced apoptosis, Bax integrates into mitochondria, as demonstrated by fluorescence microscopy and Western blot, without changes in the total amount of Bax or Bak protein. Fludarabine and FCM induce p53 stabilization, but do not seem to be essential in inducing Bax and Bak conformational changes, as they are also observed in dexamethasone-treated CLL cells. These results demonstrate that, in CLL cells, the change in the intracellular localization of Bax from cytosol to mitochondria and the conformational changes of Bax and Bak are among the early steps in the induction of cell death.
Bellosillo
Beatriz
B
Department of Hematology, Institute of Hematology and Oncology from Hospital Clínic, Postgraduate School of Hematology Farreras-Valentí, Barcelona, Spain.
Villamor
Neus
N
López-Guillermo
Armando
A
Marcé
Silvia
S
Bosch
Francesc
F
Campo
Elias
E
Montserrat
Emili
E
Colomer
Dolors
D
D000368:Aged; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002452:Cell Count; D003520:Cyclophosphamide / Q000494:pharmacology / Q000627:therapeutic use; D003907:Dexamethasone / Q000494:pharmacology / Q000627:therapeutic use; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008875:Middle Aged; D008942:Mitoxantrone / Q000494:pharmacology / Q000627:therapeutic use; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D014740:Vidarabine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
C494772:BAK1 protein, human; C494826:BAX protein, human; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D003907:Dexamethasone; D003520:Cyclophosphamide; D008942:Mitoxantrone; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D014740:Vidarabine; C024352:fludarabine