benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and calpain-inhibitor-2

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.

Topics: Acetylcysteine; Acrylates; Amino Acid Chloromethyl Ketones; Apoptosis; Boronic Acids; Bortezomib; Calpain; Cathepsins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Drug Synergism; Genes, p53; Humans; Jurkat Cells; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Neoplasms; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Tumor Cells, Cultured; Tumor Suppressor Protein p53; U937 Cells

The caspase-mediated cleavage of a limited number of cellular proteins is a common feature of apoptotic cell death. This cleavage usually inhibits the function of the target protein or generates peptides that actively contribute to the death process. In the present study, we demonstrate that the cyclin-dependent kinase inhibitor p27Kip1 is cleaved by caspases in human leukemic cells exposed to apoptotic stimuli. We have shown recently that p27Kip1 overexpression delayed leukemic cell death in response to cytotoxic drugs. In transient transfection experiments, the p23 and the p15 N-terminal peptides generated by p27Kip1 proteolysis demonstrate an anti-apoptotic effect similar to that induced by the wild-type protein, whereas cleavage-resistant mutants have lost their protective effect. Moreover, stable transfection of a cleavage-resistant mutant of p27Kip1 sensitizes leukemic cells to drug-induced cell death. Altogether, these results indicate that proteolysis of p27Kip1 triggered by caspases mediates the anti-apoptotic activity of the protein.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Base Sequence; Calpain; Caspase 3; Caspase 6; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cysteine Proteinase Inhibitors; Etoposide; Humans; Leukemia; Leupeptins; Microtubule-Associated Proteins; Molecular Sequence Data; Mutation; Nucleic Acid Synthesis Inhibitors; Oligopeptides; Protein Serine-Threonine Kinases; Thimerosal; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins