leupeptins and Lymphoma--T-Cell

Failure to perform the Fas-related apoptosis pathway can account for tumor resistance both to chemotherapeutic agents and to immunological effectors. We studied the role of NK-kappaB in Fas-resistance, employing the Fas-sensitive human T-lymphoma HuT78 cell line and its Fas-resistant variants HuT78B1 and HuT78G9. All these cell lines expressed high levels of constitutively activated NF-kappaB. Pretreatment of cells with NF-kappaB inhibitors (PDTC, MG132, or SN50) strongly enhanced CH11-induced apoptosis in HuT78 and Hut78G9 cells, while only MG132 showed a similar potentiating effect in HuT78B1. The described synergism was significantly inhibited by pretreatment with the anti-Fas-blocking antibody ZB4 or with the pancapsase inhibitor Z-VAD-FMK, but not by capsase-8 or -9 inhibitors. Overall, these data suggest that NF-kappaB inhibition may restore the Fas-pathway in Fas-resistant NF-kappaB-overexpressing tumors.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; fas Receptor; Humans; Leupeptins; Lymphoma, T-Cell; NF-kappa B; Peptides; Proline; Thiocarbamates

The proteasome is the primary protease used by cells for degrading proteins and generating peptide ligands for class I molecules of the major histocompatibility complex. Based on the properties of cells adapted to grow in the presence of the proteasome inhibitor 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone (NLVS), it was proposed that proteasomes can be replaced by alternative proteolytic systems, particularly a large proteolytic complex with a tripeptidyl peptidase II activity. Here we show that NLVS-adapted cells retain sensitivity to a number of highly specific proteasome inhibitors with regard to antigenic peptide generation, accumulation of polyubiquitinated proteins, degradation of p53, and cell viability. In addition, we show that in the same assays (with a single minor exception), NLVS-adapted cells are about as sensitive as nonselected cells to Ala-Ala-Phe-chloromethylketone, a specific inhibitor of tripeptidyl peptidase II activity. Based on these findings, we conclude that proteasomes still have essential proteolytic functions in adapted cells that are not replaced by Ala-Ala-Phe-chloromethylketone-sensitive proteases.

Topics: Amino Acid Chloromethyl Ketones; Aminopeptidases; Animals; Antigen Presentation; Antigens; Boronic Acids; Bortezomib; CD8-Positive T-Lymphocytes; Cell Survival; Cysteine Endopeptidases; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Drug Resistance; Endopeptidases; Enzyme Activation; H-2 Antigens; Leupeptins; Lymphoma, T-Cell; Mice; Multienzyme Complexes; Neoplasm Proteins; Oligopeptides; Peptide Fragments; Phenols; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Pyrazines; Selection, Genetic; Serine Endopeptidases; Sulfones; Thymus Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tyramine; Ubiquitins

Proteases play an important role in regulation of apoptosis. To elucidate the role of proteasome in apoptosis, we examined the effects of the proteasome inhibitors, carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal and carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal on RVC lymphoma cells. Cells exposed to the proteasome inhibitors arrested at G2/M phase followed by internucleosomal DNA cleavage, chromatin condensation, and formation of apoptotic bodies dose- and time-dependently. Ubiquitinated histone H2A levels decreased in the exposed cells, suggesting a relationship between deubiquitination of histone H2A and the chromatin disarray seen in apoptosis. Northern blots showed an increase in expression of polyubiquitin genes early in the incubation. These findings suggest that the ubiquitin-mediated proteasome-proteolytic system is involved in regulating the cell cycle and apoptosis in RVC cells.

Topics: Animals; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; G2 Phase; Histones; Leupeptins; Lymphoma, T-Cell; Mice; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; RNA, Messenger; Tumor Cells, Cultured; Ubiquitins