DETAILED ANALYSIS OF CELL-CYCLE KINETICS UPON PROTEASOME INHIBITION

We have studied specific effects of proteasome inhibition on cell cycl e progression, To this end, the protease inhibitors MG115, calpain inh ibitor I, and calpain inhibitor II, which display differential inhibit ory effects on proteasomes, were used, Cell kinetic studies using brom odeoxyuridine pulse labeling revealed a complete block of G1/S and met aphase transitions and a delayed progression through S phase Ln cell c ultures treated with 54 mu M of MG115, Calpain inhibitor I in similar concentrations displayed a fivefold lower effect on cell cycle kinetic s. Calpain inhibitor II and MG2M, which is a structural analogue of MG 115, had no effect on the cell cycle, The inhibitory effect of MG115 t reatment was reversible, because the cell cycle was immediately resume d when the MG115-containing culture medium was replaced by fresh cultu re medium. Because ubiquitinated proteins accumulated after MG115 trea tment, it was confirmed that ubiquitin-dependent protein degradation, and thus proteasomal activity were blocked, By comparison of biochemic al and in vitro proteasome inhibition experiments, it was hypothesized that chymotrypsin-like activity of proteasomes may play an important role in cell cycle kinetics. (C) 1997 Wiley-Liss, Inc.