Proteolytic loss of bcl-x(L) in FL5.12 cells undergoing apoptosis induced by MK886

Apoptosis induced in the IL3-dependent murine pro-B lymphocytic (FL5.12) ce ll line by the 5-lipoxygenase activating protein inhibitor MK886 is accompa nied by the rapid loss of the antiapoptotic bcl-X-L and bcl-2, but not the proapoptotic bax proteins (Datta et al., J. Biol. Chem. 273, 28163-28169, 1 998). Since several reports indicate important roles for noncaspase proteas es mi apoptosis, the participation of lysosomes, as well as serine, cystein e, or aspartic acid proteases, in the effects of MK886 were investigated. C onsistent with the involvement of various proteases, lysosomal degranulatio n was evident, as observed by a decrease in acridine orange fluorescence at 2 h and an increase in cytosolic beta -hexosaminidase activity at 4 h afte r treating FL5.12 cells with 10 muM MK886. The disappearance of bcl-X-L fro m FL5.12 cells upon MK886 treatment was prevented in a dose-dependent manne r by pretreatment with leupeptin, pepstatin, phenylmethylsulfonyl fluoride, or the broad-spectrum caspase inhibitor Boc-D-FMK. Each of the noncaspase protease inhibitors partially inhibited MK886-induced apoptosis as measured by phosphatidylserine externalization and DNA fragmentation. The noncaspas e inhibitors also blocked about half of the increase in caspase-3-like acti vity. Boc-D-FMK completely inhibited this enzyme and prevented apoptosis. N one of the inhibitors were able to directly inhibit activated caspase-3 in cell lysates, suggesting their effects were upstream of caspase activation. These observations suggest the involvement of various proteases, possibly originating from lysosomes, upstream of active caspase-3, in the loss of bc l-X-L, protein and in the signaling pathway of MK886-induced apoptosis in F L5.12 cells. This pathway may be unique to MK886 since these same protease inhibitors had only minimal effects on etoposide-induced apoptosis and the accompanying moderate loss of bcl-X-L in FL5.12 cells. (C) 2001 Academic Pr ess.