In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal.

Abstract

The cytotoxic neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN) inhibits cell-cycle progression in CHO cells, affecting the G1/S and metaphaseanaphase 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 Hi kinase activity remains elevated during mitotic arrest. Duration of mitotic arrest depends on ALLN concentration; high concentrations (>50 ,ug/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. Cydin B degradation fails to occur, and histone Hi kinase remains activated for the duration of mitotic arrest at all ALLN concentrations.Proteolysis of cyclin proteins is crucial in the regulation of cell-cycle progression in a wide variety of cell types (1). Cyclin B degradation appears to involve ubiquitination (2-4), and although mutant cyclin B protein is conjugated with ubiquitin, it is not degraded (2, 5). The failure of cyclin B degradation blocks cell-cycle progression at the metaphaseanaphase transition in vitro (2, 5). In developing clam embryo cells, normal cell-cycle progression requires that cycin B degradation occur within a narrow time window (6), and thus both the timing and extent of cyclin degradation are important in regulating progression through mitosis. In vitro studies with clam oocyte extracts show that a variety of agents, including some protease inhibitors, can alter the rate ofcyclin B proteolysis (7). Although important aspects of cyclin B proteolysis are known, many details of the process in vivo remain largely undescribed (8).The cell-permeant tripeptide neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN) significantly reduces the in vivo rate of degradation of the enzyme hydroxymethylglutaryl-CoA reductase in Chinese hamster ovary (CHO) cells (9). ALLN is cytotoxic to CHO cells, and selection with ALLN leads to the development of drug resistance, resulting from the amplification of the MDR 1 gene (10) as well as overexpression of an aldo-keto reductase (21). However, the cellular effects of ALLN responsible for its cytotoxicity have not been defined. In this report we show that ALLN is a strong inhibitor of cell-cycle progression in CHO cells. It is active at two cell-cycle transition points, G1/S and mitosis, and also inhibits S-phase progression under some circumstances. In mitosis, ALLN inhibits the metaphase-anaphase transiti...

Role of c-Jun N-terminal kinase 1 (JNK1) in cell cycle checkpoint activated by the protease inhibitor N-acetyl-leucinyl-leucinyl-norleucinal

“…LLnL has been demonstrated to induce cell cycle arrest in CHO cells in a dose-dependent manner (Sherwood et al, 1993) and in PDGF-stimulated human fibroblasts (Dietrich et al, 1996). To examine the e ects of LLnL on lung cancer cells, Calu-1 lung carcinoma cells were treated with various doses of LLnL and viable cells were counted by trypan blue exclusion.…”

“…The peptide aldehyde N-acetyl-leucinylleucinyl-norleucinal (LLnL), an inhibitor of the chymotryptic site on the 20S proteasome, can inhibit the proteolytic activity of the proteasome (Visnitsky et al, 1992) and block the degradation of proteins and the processing of NFkB precursors into active proteins (Rock et al, 1994;Palombella et al, 1994). LLnL inhibits cell cycle progression in Chinese hamster ovary (CHO) cells, a ecting the G 1 /S and metaphaseanaphase transition points, as well as S phase progression (Sherwood et al, 1993). The mitotic arrest induced by LLnL is associated with the inhibition of cyclin B degradation.…”

Androgens Negatively Regulate Forkhead Transcription Factor FKHR (FOXO1) through a Proteolytic Mechanism in Prostate Cancer Cells

“…7, E and F, left panel). ALLnL was functional since cyclin B, a calpain target protein (57), was stabilized in the cells treated with ALLnL (data not shown). Likewise, no effect of EGTA on the androgen-induced appearance of the 60-kDa FLAG-tagged FKHR band was detected (Fig.…”

The role of the ubiquitin-proteasome pathway in apoptosis

“…Early studies evaluating the consequences of inhibition of this function revealed mutations in a ubiquitinactivating enzyme in Chinese hamster ovary cells led to an arrest at G 2 /M, 80 as did yeast mutations which prevented the assembling of ubiquitin chains. 81 Inhibition of the proteasome resulted in cell-cycle arrest at the G 1 /S boundary in Chinese hamster ovary cells 82 as well as in Neuro 2A and MG-63 osteosarcoma cells, 83 while HeLa cells arrested at G 2 /M, 84 and other studies noted cellular arrest at both G 1 /S and G 2 / M. 85 This is by no means a complete review of the effects of proteasome inhibition on progression through the cell cycle, however, which is beyond the scope of this work. The reader is referred to several recent reviews.…”