INVIVO INHIBITION OF CYCLIN-B DEGRADATION AND INDUCTION OF CELL-CYCLE ARREST IN MAMMALIAN-CELLS BY THE NEUTRAL CYSTEINE PROTEASE INHIBITOR N-ACETYLLEUCYLLEUCYLNORLEUCINAL

The cytotoxic neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN) inhibits cell-cycle progression in CHO cells, affecting the G1/S and metaphase-anaphase transition points, as well as S phase. Mitotic arrest induced by ALLN is associated with the inhibition of cyclin B degradation. At mitosis-inhibiting concentrations of ALLN, cells undergo nuclear-envelope breakdown, spindle formation, chromosome condensation, and congression to the metaphase plate. However, normal anaphase events do not occur, and cells arrest in a metaphase configuration for a prolonged period. Steady-state levels of cyclin B increase to greater than normal mitotic levels, and cyclin B is not degraded for an extended period. Histone H1 kinase activity remains elevated during mitotic arrest. Duration of mitotic arrest depends on ALLN concentration; high concentrations (>50 mug/ml) produce a prolonged mitotic arrest, whereas at lower concentrations, cells are transiently delayed through mitosis (up to 4-12 hr), after which they undergo aberrant cell division resulting in randomly sized daughter cells containing variable amounts of DNA. Cyclin B degradation fails to occur, and histone H1 kinase remains activated for the duration of mitotic arrest at all ALLN concentrations.