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Overexpression of Bcl-x_L Inhibits Ara-C-induced Mitochondrial Loss of Cytochrome c and Other Perturbations That Activate the Molecular Cascade of Apoptosis

Division of Hematology/Oncology, Department of Medicine, Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322 [C. N. K., Y. H., A. M. I., L. L., G. F., K. B.]; and University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 [X. W.]

High-dose Ara-C (HIDAC) induces the cleavage and activity of caspase-3 (CPP32ß/Yama/apopain), resulting in the morphological and biochemical features of apoptosis. High levels of the antiapoptotic Bcl-x_L or Bcl-2, relative to the proapoptotic Bax, have been shown to inhibit HIDAC-induced cleavage and activity of caspase-3 and apoptosis of the human acute myeloid leukemia HL-60 cells. In a previous report, we demonstrated this inhibition, using the control HL-60 (HL-60/neo) cells and their counterparts, HL-60/Bcl-x_L, which have enforced overexpression of Bcl-x_L and a significantly lower ratio of free to bound Bax. Results of the present studies demonstrate that, in the initiation phase of apoptosis of HL-60/neo cells due to HIDAC (10 or 100 µM for 4 h), cytochrome c is released from the mitochondria to the cytosol, followed by the loss of mitochondrial membrane potential (m) and an increase in the reactive oxygen species; these events precede and trigger the cleavage and activity of caspase-3. These HIDAC-induced early mitochondrial and cytosolic perturbations, which represent the initiation phase of HIDAC-induced apoptosis, were inhibited in HL-60/Bcl-x_L cells. HIDAC treatment for 4 h also modestly increased the intracellular levels of free Bax relative to Bax bound to Bcl-2 and Bcl-x_L in HL-60/neo but not in HL-60/Bcl-x_L cells. In HL-60/neo cells, HIDAC-induced progressive accumulation of cytochrome c in the cytosol, the decrease in m, and the increase in reactive oxygen species were not inhibited by coculture with the tetrapeptide inhibitors of caspases that have been previously shown to inhibit Ara-C-induced cleavage and activity of caspase-3 and apoptosis. These findings indicate that Bcl-x_L inhibits HIDAC-induced preapoptotic mitochondrial perturbations, which prevent the accumulation of cytochrome c in the cytosol, thereby preserving caspase-3 in the inactive zymogen state and checking the molecular cascade of apoptosis.

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

Received 5/ 6/97. Accepted 6/10/97.

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