p53-dependent cell cycle arrest induced by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts.

Abstract

Proteases are known to play important roles in cell growth control, although the underlying mechanisms are still poorly understood. Here we show that the protease inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal induced cell cycle arrest in platelet-derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome. Inhibition of the proteasome resulted in accumulation of the tumor suppressor p53, which was followed by an increase in the amount of the cyclindependent kinase-inhibitor p21. As a consequence, both phosphorylation and activity of the cyclin-dependent kinase 2/cyclin E complex were inhibited. We further observed that the retinoblastoma gene product, pRb, remained in the hypophosphorylated state, thus preventing cells from progression into the S-phase. These studies strongly support the hypothesis that the proteasome is a key regulator in the Gl-phase of cell cycle progression.There is evidence that intracellular proteases play a dominant role in cell cycle control. Accordingly, antiproliferative effects are shown by some cell-permeable protease inhibitors. For example, the lipid soluble derivative of E-64, E-64d, an inhibitor of cathepsins and calpains, has been shown to arrest squamous cell carcinoma cells in metaphase (1). Cathepsins are known as acid cysteine proteases in contrast to the neutral, Ca2+-requiring cysteine proteases, calpains (calpain 1 and calpain 2). Mellgren and coworkers (2) also suggested that calpains participate in cell growth regulation since benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone, which is thought to inhibit calpains, reduced proliferation of human TE2 (simian virus 40-transformed human esophageal epithelial cells) and C-33A cells (human cervical carcinoma cells). In vascular smooth muscle cells, N-acetyl-L-leucinyl-L-leucinyl-Lnorleucinal (LLnL) (calpain inhibitor 1) caused a repression of platelet-derived growth factor-(PDGF), as well as seruminduced cell cycle progression before G1/S transition (3). Unfortunately, these commonly used thiol protease inhibitors are not highly selective, making it difficult to precisely define the intracellular targets. LLnL, for example, is not selective for calpains, but inhibits also cathepsin L (4), as well as the proteasome complex (5). Consequently, the assumption that calpains function in cell cycle control (2, 3), remains to be verified. Furthermore, it is not known which distinct step within the cell cycle is affected by calpain inhibitors resulting in G1 arrest. Interestingly, cyclin B degradation, which requires ubiquitin-conjugation (6), can be inhibited by LLnL (7). In previous studies in which we addressed the question if proteolytic activity is required to allow confluent cultures to mitogenically respond to PDGF, we noticed that LLnL strongly inhibited PDGF-induced proliferation. We therefore studied the effect of LLnL in PDGF-AB-stimulated human fibroblasts to determine (i) the protease(s) that is/are inhibited by LLnL, (ii) the target of the proteas...

Preferential induction of apoptosis for primary human leukemic stem cells

“…This finding indicates that other effects of the drug treatment must be contributing to AML cell death. In seeking to characterize LSC apoptosis further, we hypothesized that proteasome inhibition might be stabilizing proteins relevant to apoptosis induction such as p53 (38)(39)(40). Moreover, the activity of IDR was expected to activate p53-mediated apoptosis mechanisms.…”

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 mitotic arrest induced by LLnL is associated with the inhibition of cyclin B degradation. In platelet-derived growth factorstimulated human ®broblasts, LLnL induces p53-dependent cell cycle arrest at the G 1 /S boundary by inhibiting the proteasome (Dietrich et al, 1996). To examine the role of JNK in cell cycle checkpoint control, LLnL was used to induce cell cycle arrest in the p53-null lung carcinoma cells transfected with a dominant-negative mutant of JNK1.…”

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

“…Genetic evidence is consistent with an essential role for the ubiquitin (Ub)-proteasome system to maintain cell viability, and yeast whose proteasome activity is ablated are not viable (10,11). Exposure of mammalian cells to pharmacological agents that interfere with proteasome function generally leads to cell cycle arrest (12) and death (13). Proteasome inhibitors include the natural compounds lactacystin and epoxomycin (14,15) as well as C-terminally modified peptide derivatives (1,16), which all bind to the N-terminal threonine in the catalytic site of active ␤ subunits (17).…”