BCR-ABL EXERTS ITS ANTIAPOPTOTIC EFFECT AGAINST DIVERSE APOPTOTIC STIMULI THROUGH BLOCKAGE OF MITOCHONDRIAL RELEASE OF CYTOCHROME-C AND ACTIVATION OF CASPASE-3

Bcr-Abl expression in leukemic cells is known to exert a potent effect against apoptosis due to antileukemic drugs, but its mechanism has no t been elucidated. Recent reports have indicated that a variety of apo ptotic stimuli cause the preapoptotic mitochondrial release of cytochr ome c (cyt c) into cytosol, which mediates the cleavage and activity o f caspase-3 involved in the execution of apoptosis. Whether Bcr-Abl ex erts its antiapoptotic effect upstream to the cleavage and activation of caspase-3 or acts downstream by blocking the ensuing degradation of substrates resulting in apoptosis, has been the focus of the present studies. In these, we used (I)the human acute myelogenous leukemia (AM L) HL-60 cells that are stably transfected with the bcr-abl gene (HL-6 0/Bcr-Abl) and express p185 Bcr-Abl; and (2) the chronic myelogenous l eukemia (CML)-blast crisis K562 cells, which have endogenous expressio n of p210 Bcr-Abl. Exposure of the control AML HL-60 cells to high-dos e Ara-C (HIDAC), etoposide, or sphingoid bases (including C-2 Ceramide , sphingosine, or sphinganine) caused the accumulation of cyt c in the cytosol, loss of mitochondrial membrane potential (MMP), and increase in the reactive oxygen species (ROS). These preapoptotic events were associated with the cleavage and activity of caspase-3, resulting in t he degradation of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) and DNA fragmentation factor (DFF), internucleosomal DNA fragm entation, and morphologic features of apoptosis. In contrast, in HL-60 /Bcr-Abl and K562 cells, these apoptotic stimuli failed to cause the c ytosolic accumulation of cyt c and other associated mitochondrial pert urbations, as well as the failure to induce the activation of caspase- 3 and apoptosis. While the control HL-60 cells showed high levels of B cl-2 and barely detectable Bcl-x(L), HL-60/Bcr-Abl cells expressed hig h levels of Bcl-x(L) and undetectable levels of Bcl-2, a pattern of ex pression similar to the one in K562 cells. Bar and caspase-3 expressio ns were not significantly different between HL-60/Bcr-Abl or K562 vers us HL-60 cells. These findings indicate that Bcr-Abl expression blocks apoptosis due to diverse apoptotic stimuli upstream by preventing the cytosolic accumulation of cyt c and other preapoptotic mitochondrial perturbations, thereby inhibiting the activation of caspase-3 and exec ution of apoptosis. (C) 1998 by The American Society of Hematology.