Overexpression of Apaf-1 promotes apoptosis of untreated and paclitaxel- or etoposide-treated HL-60 cells.
Recent studies have demonstrated that Apaf-1 is the adaptor molecule which in the presence of cytosolic cytochrome c (cyt c) and dATP interacts with procaspase-9, resulting in the sequential cleavage and activity of caspase-9 and caspase-3, followed by apoptosis. In the present studies, we determined the effect of enforced overexpression of Apaf-1 on the apoptotic threshold in the human myeloid leukemia HL-60 cells. Our findings demonstrate that both transient and stable transfections resulted in a 2.5-fold higher expression of Apaf-1, which was associated with approximately a 5-fold increase in the percentage of apoptosis in the transfectants (HL-60/Apaf-1) as compared with the control HL-60/neo cells. In cells overexpressing either Bcl-2 or Bcl-xL, transient overexpression of Apaf-1 did not induce apoptosis. Stably overexpressing Apaf-1 levels significantly sensitized HL-60/Apaf-1 cells to apoptosis induced by clinically achievable concentrations of paclitaxel or etoposide (P < 0.01). This increase in paclitaxel- or etoposide-induced apoptosis of HL-60/Apaf-1 cells was not associated with any significant alterations in Bcl-2, Bcl-xL, Bax, Fas, or Fas ligand expression. It was, however, clearly associated with caspase-9 cleavage, as well as the poly(ADP-ribose) polymerase and DFF45 cleavage activity of caspase-3. Coexpression of the catalytically inactive, dominant-negative, mutant caspase-9, XIAP, or treatment with the caspase inhibitor, zVAD, significantly inhibited the increase in apoptosis of HL-60/Apaf-1 cells (P < 0.01). These data indicate that the intracellular levels of Apaf-1 is an important molecular determinant of the threshold for apoptosis induced by paclitaxel and etoposide.
Perkins
C
C
Department of Medicine, Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Kim
C N
CN
Fang
G
G
Bhalla
K N
KN
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053477:Apoptotic Protease-Activating Factor 1; D005047:Etoposide / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D017239:Paclitaxel / Q000494:pharmacology*; D011506:Proteins / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D051028:bcl-2-Associated X Protein
C506592:APAF1 protein, human; D000972:Antineoplastic Agents, Phytogenic; D053477:Apoptotic Protease-Activating Factor 1; C494826:BAX protein, human; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D005047:Etoposide; D017239:Paclitaxel