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Department of Oncology, Albert Einstein College of Medicine, Bronx, New York 10461 [Y-H. L., R. P-S.]; Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016 [L. L., B. N., M. B., and F. M. M.]; T. J. Martell Laboratory for Leukemia, Cancer and AIDS Research, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029 [J-D. J.]; and Millennium Pharmaceuticals Inc., Cambridge, Massachusetts 02139 [P. J. E., J. A.]

Treatment with the proteasome inhibitor, PS-341 resulted in concentration- and time-dependent effects on Bcl-2 phosphorylation and cleavage in H460 cells that coincided with the PS-341-induced G₂-M phase arrest. The observed Bcl-2 cleavage paralleled the degree of PS-341-induced apoptosis but was detected to a similar extent with comparable concentrations of two other proteasome inhibitors (MG-132 and PSI). Calpain inhibitors, ALLM and ALLN, and the caspase inhibitors, Z-VAD and AC-YVAD did not induce Bcl-2 phosphorylation and cleavage. Exposure to PS-341 resulted in an additional M_r 25,000 cleavage fragment of Bcl-2, whereas only a M_r 23,000 fragment was observed with other anticancer agents. The formation of the M_r 25,000 fragment was not prevented by caspase inhibitors unlike the M_r 23,000 fragment, which suggests mediation by a caspase-independent pathway. Cell fractionation studies revealed that the Bcl-2 cleaved fragments localize within membrane structures and was an early event (at 12 h, posttreatment), and before the observed cleavage of poly(ADP-ribose) polymerase (PARP), ß-catenin, and DNA fragmentation (at 36 h posttreatment). The M_r 23,000 Bcl-2 cleavage product was inhibited by the pan-caspase inhibitor and the inhibitors of capase-3, -8, -9; but the PARP cleavage was prevented only by the pan-caspase and caspase-3 inhibitors, which suggests that the M_r 23,000 Bcl-2 cleavage occurred at both the initiation and execution stages of apoptosis. The inhibition of the ubiquitin/proteasome pathway by PS-341 leads, at an early stage of apoptosis, to Bcl-2 phosphorylation and a unique proteolytic cleavage product, which are associated with G₂-M phase arrest and the induction of apoptosis.

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