PS-341, a novel proteasome inhibitor, induces Bcl-2 phosphorylation and cleavage in association with G2-M phase arrest and apoptosis.
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 BcI-2 phosphorylation and cleavage. Exposure to PS-341 resulted in an additional Mr 25,000 cleavage fragment of Bcl-2, whereas only a Mr 23,000 fragment was observed with other anticancer agents. The formation of the Mr 25,000 fragment was not prevented by caspase inhibitors unlike the Mr 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 approximately 12 h, posttreatment), and before the observed cleavage of poly(ADP-ribose) polymerase (PARP), beta-catenin, and DNA fragmentation (at approximately 36 h posttreatment). The Mr 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 Mr 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.
Ling
Yi-He
YH
Department of Oncology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Liebes
Leonard
L
Ng
Bruce
B
Buckley
Michael
M
Elliott
Peter J
PJ
Adams
Julian
J
Jiang
Jian-Dong
JD
Muggia
Franco M
FM
Perez-Soler
Roman
R
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D002460:Cell Line; D003546:Cysteine Endopeptidases; D053938:DNA Fragmentation; D005434:Flow Cytometry; D016195:G2 Phase; D006801:Humans; D008938:Mitosis; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000031:analogs & derivatives* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D001897:Boronic Acids; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D011719:Pyrazines; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C425776:tyrosyl-valyl-alanyl-aspartic acid fluoromethyl ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D000069286:Bortezomib; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex