mdl-201053 and acetylleucyl-leucyl-norleucinal

Caspases, members of the cysteine protease family, execute UVB-induced apoptosis in several cell lines and keratinocytes. Several researchers investigating UVB-induced apoptosis have demonstrated a dose-dependent protective effect of the synthetic peptide caspase inhibitor zVAD-fmk. However, zVAD-fmk displays a dose-dependent protective effect against UVB-induced apoptosis, even at doses higher than those required to block all known proapoptotic caspases. In addition, it is known that zVAD-fmk also inhibits other cysteine proteases including cathepsins and calpains, and these proteases have recently been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes. This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors zFA-fmk, CA-074-Me and ALLN all failed to prevent UVB-induced apoptosis in HeLa cells and keratinocytes. In conclusion, the protective effect of zVAD-fmk at high concentrations indicates that other proteases than caspases are active in the execution of UVB-induced apoptosis but further studies are needed to identify these proteases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calpain; Caspase Inhibitors; Cathepsins; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; HeLa Cells; Humans; Keratinocytes; Ketones; Leupeptins; Phospholipases A; Ultraviolet Rays

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Bucladesine; Calpain; Cyclin B; Cyclin B1; Cysteine Proteinase Inhibitors; Dipeptides; Fluorouracil; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ketones; Leupeptins; Lovastatin; Nuclear Proteins; Protein Processing, Post-Translational; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Tumor Cells, Cultured