Lactacystin, a specific inhibitor of the proteasome, induces apoptosis andactivates caspase-3 in cultured cerebellar granule cells

The multicatalytic protease complex or proteasome is a fundamental nonlysos omal tool that the cell uses to process or degrade proteins at a fast rate through the ubiquitin and ATP-dependent proteolytic pathway. Examples of th ese important proteins include the tumor suppressor protein p53, various cy clins, the cyclin-dependent kinase inhibitor p27, NF kappa B, I kappa B, c- fos, and c-jun. The activation of proteolytic enzymes, including certain cy stein-proteases of the ced-3/ICE (interleukin-1 beta-converting enzyme) fam ily, is a characteristic feature of the apoptotic program. However, the rol e of the multicatalytic protease complex in apoptosis is not well known. In order to obtain further information regarding the participation of the ubi quitin-mediated pathway in the decision of the cell to execute the cell dea th program, we have used a specific inhibitor of the multicatalytic proteas e complex, lactacystin, in cultured cerebellar granule cells. Cells were ob tained from the cerebellum of 6- to 8-day-old Wistar rats and cultured in N eurobasal medium supplemented with B-27, Addition of lactacystin to the cul tures induced apoptosis of the granule cells in a time-dependent fashion. T he morphological changes produced by the proteasome inhibitor included nucl ear condensation and DNA fragmentation measured by the diphenylamine test, as well as a positive labeling by the TUNEL (terminal deoxynucleotidyltrans ferase mediated-dUTP nick end labeling) assay, all of them typical features of apoptosis. Concomitant with apoptosis, there were changes in the expres sion of the ubiquitin mRNA, a progressive depletion in the free ubiquitin p ool, and an increase in the high molecular weight ubiquitin-protein conjuga tes. Caspase-3, a member of the ced-3/ICE family of cystein-proteases, show ed a marked increase in activity in the lactacystin-treated cells. In flow cytometry studies, the amount of cells in the S phase of the cell cycle was smaller in the lactacystin-treated cells than in controls, suggesting that apoptosis could be due, in part, to an alteration of the cell cycle. J. Ne urosci, Res. 59:601-611, 2000, (C) 2000 Wiley-Liss, Inc.