benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Pituitary-Neoplasms

Proteasome inhibitors induce apoptosis in some malignant cells, and we show here that these inhibitors induce apoptosis in rat pituitary MMQ and GH3 tumor cells but not in normal pituitary cells. Three proteasome inhibitors, PSI, MG-132, and lactacystin, but not the calpain inhibitor, ALLM, dose- and time-dependently caused apoptosis in these cells, and 10 microM PSI caused apoptosis in 70% of MMQ cells and in 25% of GH3 cells within 24 h. A lower PSI dose (10 nM) inhibited GH3 cell growth without causing significant apoptosis or affecting prolactin secretion. Primary rat pituitary cells were resistant to both PSI and MG-132 and did not undergo apoptosis. In MMQ cells, DNA synthesis was slowed (approximately 30%) after 6 h of 10 microM PSI treatment and a partial cell cycle block at G2/M was evident after 8 h. Colorimetric caspase substrate assay and Western blotting of caspase substrates showed that caspases 2 and 3 are activated by PSI while caspases 6 and 8 remained inactive. A broad-range caspase inhibitor, caspase inhibitor III, prevented apoptosis induced by PSI. The results show that proteasome inhibitors induce apoptosis in rat pituitary tumor cells by specific caspase activation. This novel group of drugs may potentially be used in treatment of aggressive pituitary tumors, especially as their action appears relative for tumor cells.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Caspases; Cells, Cultured; Cysteine Endopeptidases; Enzyme Activation; Enzyme Inhibitors; Female; Growth Hormone; In Situ Nick-End Labeling; Leupeptins; Multienzyme Complexes; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactinoma; Proteasome Endopeptidase Complex; Rats; Tumor Cells, Cultured

The goal of these studies was to define the rate-limiting steps in the inactivation of type 2 iodothyronine deiodinase (D2). We examined the effects of ATP depletion, a lysosomal protease inhibitor, and an inhibitor of actin polymerization on D2 activity in the presence or absence of cycloheximide or 3,3', 5'-triiodothyronine (reverse T3, rT3) in rat pituitary tumor cells (GH4C1). We also analyzed the effects of the proteasomal proteolysis inhibitor carbobenzoxy- L-leucyl-L-leucyl-L-leucinal (MG132). The half-life of D2 activity in hypothyroid cells was 47 min after cycloheximide and 60 min with rT3 (3 nM). rT3 and cycloheximide were additive, reducing D2 half-life to 20 min. D2 degradation was partially inhibited by ATP depletion, but not by cytochalasin B or chloroquine. Incubation with MG132 alone increased D2 activity by 30-40% for several hours, and completely blocked the cycloheximide- or rT3-induced decrease in D2 activity. These results suggest that D2 is inactivated by proteasomal uptake and that substrate reduces D2 activity by accelerating degradation through this pathway. This is the first demonstration of a critical role for proteasomes in the post-translational regulation of D2 activity.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chloroquine; Cycloheximide; Cysteine Endopeptidases; Cytochalasin B; Iodide Peroxidase; Iodothyronine Deiodinase Type II; Isoenzymes; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Pituitary Neoplasms; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Rats; Thyroxine; Triiodothyronine, Reverse; Tumor Cells, Cultured; Ubiquitins