REEVALUATION OF THE ROLE OF DE-NOVO PROTEIN-SYNTHESIS IN RAT THYMOCYTE APOPTOSIS

In this study, the role of de novo protein synthesis in rat thymocytes undergoing apoptosis after treatment with methylprednisolone (MPS), i onomycin, or thapsigargin was evaluated using several inhibitors of pr otein synthesis (cycloheximide, emetine, and puromycin). Cycloheximide (1 mu g/ml) inhibited DNA cleavage in rat thymocytes treated with tha psigargin, MPS, and ionomycin by 91, 94, and 96%, respectively, and re duced [H-3]leucine incorporation into cellular proteins by 87, 85, and 84%, respectively, Emetine (300 nM) inhibited protein synthesis in th ymocytes to an equivalent level but reduced DNA cleavage by only 49, 4 3, and 57% in cells treated with thapsigargin, MPS, or ionomycin, resp ectively. More than threefold;higher concentrations of emetine (1 mu M ) were required to suppress DNA fragmentation to a similar extent as o bserved with cycleheximide. Puromycin at a concentration (5 mu g/ml) t hat reduced [H-3]leucine incorporation by >80% enhanced DNA cleavage i n thymocytes treated with thapsigargin, MPS, or ionomycin. By itself, puromycin (0.1-5 mu g/ml), but not cycloheximide or emetine, induced D NA fragmentation in thymocytes with the concomitant inhibition of prot ein synthesis. An analogue of puromycin, puromycin aminonucleoside, wh ich has no effect on protein synthesis, did not induce DNA fragmentati on in thymocytes and did not prevent thymocyte apoptosis triggered by other agents. Both cycloheximide and emetine dose-dependently reduced thymocyte DNA cleavage induced by puromycin despite marked inhibition of protein synthesis by puromycin itself. At high concentration, purom ycin (50 mu g/ml) was less efficient in causing DNA cleavage when adde d alone and markedly inhibited chromatin degradation induced by thapsi gargin, MPS, or ionomycin. Prolonged treatment (24 h) of thymocytes wi th any one of the different translational inhibitors resulted in exten sive DNA fragmentation. Similarly, the protective effect of these inhi bitors on DNA degradation in thymocytes induced by thapsigargin, MPS, or ionomycin diminished after 24 h. The present study demonstrates a l ack of correlation between inhibition of protein synthesis and prevent ion of DNA fragmentation in thymocyte apoptosis and suggests that the effects of translational inhibitors on thymocyte apoptosis are nonspec ific, and that they may delay the onset of apoptosis rather than preve nt it. (C) 1995 Academic Press, Inc.