benzyloxycarbonyl-isoleucyl-glutamyl(o-tert-butyl)-alanyl-leucinal and carbobenzoxy-leucyl-leucyl-norvalinal

The 26S proteasome is a ubiquitin-dependent proteolytic system that has been implicated in the regulation of cell cycle progression and apoptosis. We investigated the effects of the proteasome inhibitors MG115 and PSI alone or in combination with different concentrations of the antiandrogen hydroxyflutamide on the cellular proliferation, apoptosis and viability of 10 prostatic adenocarcinoma cell cultures. Treatment with both proteasome inhibitors resulted in apoptosis induction, whereas the combinations with hydroxyflutamide generally did not, with the exception of MG115 combined with 10(-7) M hydroxyflutamide. MG115 caused a significant decrease in cellular proliferation, as did the combinations of both proteasome inhibitors with hydroxyflutamide, whereas hydroxyflutamide alone was only effective at a concentration of 10(-5) M. Cellular viability was significantly reduced when both proteasome inhibitors were combined with 10(-5) M hydroxyflutamide. Although the results varied among different cell lines, we conclude that proteasome inhibitors are able to induce apoptosis and reduce cellular proliferation. They might prove effective as antineoplastic substances in prostatic adenocarcinoma alone or in combination with antiandrogens.

Topics: Adenocarcinoma; Androgen Antagonists; Apoptosis; Cell Division; Cell Survival; Cysteine Proteinase Inhibitors; Drug Interactions; Flutamide; Humans; Leupeptins; Male; Oligopeptides; Prostatic Neoplasms; Protease Inhibitors; Tumor Cells, Cultured

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

The rat gastric GATA DNA-binding protein, GATA-6 (GATA-GT1), was stably expressed in CHO-K1 cells. The GATA-6 protein was localized in the nucleus but not in the cytoplasm. Interestingly, when cells were treated with dibutyryl cAMP, the GATA-6 protein was specifically degraded. Such a phenomenon was not observed in the presence of 5'-AMP or dibutyryl cGMP. The cellular level of the GATA-6 protein was restored upon removal of dibutyryl cAMP. Degradation was also induced by cholera toxin, which increased the cellular cAMP concentration, and was inhibited by a protein kinase A inhibitor. However, activators of protein kinase C did not have any effect. The degradation was inhibited by proteasome inhibitors (PSI (benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinal) and MG115 (benzyloxycarbonyl-Leu-Leu-norvalinal)) but not by those of lysosomes and serine proteases. These results suggest that a kinase-mediated protein phosphorylation is the cellular signal for degradation of the GATA-6 protein. This finding constitutes a novel aspect of regulation by GATA DNA-binding proteins, which are essential for developmental processes and tissue-specific transcription.

Topics: Amino Acid Sequence; Animals; Bucladesine; CHO Cells; Cholera Toxin; Conserved Sequence; Cricetinae; Cyclic AMP; Dibutyryl Cyclic GMP; Diglycerides; DNA-Binding Proteins; Fluorescent Antibody Technique, Indirect; Gastric Mucosa; GATA6 Transcription Factor; Inositol 1,4,5-Trisphosphate; Kinetics; Leupeptins; Molecular Sequence Data; Oligopeptides; Proadifen; Protease Inhibitors; Rats; Recombinant Fusion Proteins; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Zinc Fingers