Although Ara-C is a highly active drug against AML, resistance to high-dose Ara-C (HIDAC) is common. HIDAC is known to induce apoptosis of AML cells. Recently, apoptotic stimuli have been shown to cause mitochondrial perturbations including the release of cytochrome c into the cytosol, resulting in the cleavage and activity of caspase-3 (CPP32, Yama or apopain) and other cysteine proteases of the ICE/ced-3 family which are the executors of apoptosis. Premature activity of p34cdc-2 kinase (CDK1) has also been shown to mediate the characteristic morphologic and biochemical features of apoptosis. In addition, recently, some of the other antileukemic drugs have been shown to induce Fas ligand and engage the Fas (CD95) receptor-triggered pathway to apoptosis. Bc1-2 and Bcl-xL are important anti-apoptotic membrane channel proteins that heterodimerize with the pro-apoptotic Bax. This reduces the levels of free Bax that promote apoptosis. Although overexpression of Bcl-xL or Bcl-2 in human AML cells has been demonstrated to confer a distal mechanism of resistance against HIDAC, the activity of the effector molecules or the precise step(s) in the pathway to HIDAC-induced apoptosis that are blocked have not been comprehensively examined. By utilizing the human AML HL-60 (p53 null) and/or ML-1 cells (p53wt), this application will investigate the effect of the enforced overexpression of the anti-apoptotic (Bcl-2 or Bcl-xL) or pro-apoptotic (Bax or Bcl-x-S) on the potential involvement and ordering of the molecular events during HIDAC-induced apoptosis. These include the determinations of 1) whether HIDAC treatment alters the levels of free (unbound) Bax or Bak relative to those bound to Bc1-2 and Bcl-xL; 2) whether HIDAC induces mitochondrial perturbations including the release of cytochrome c into the cytosol, loss of membrane potential or increase in the reactive oxygen species; 3) the role of CDK1 activity in mediating HIDAC-induced apoptosis; and 4)whether Fas receptor-signaling is involved in HIDAC-induced apoptosis. These in vitro studies may define molecular targets or elucidate mechanisms that could be manipulated to promote apoptosis and overcome HIDAC resistance in AML.
