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), ionomycin, or thapsigargin was evaluated using several inhibitors of protein synthesis (cycloheximide, emetine, and puromycin). Cycloheximide (1 microgram/ml) inhibited DNA cleavage in rat thymocytes treated with thapsigargin, MPS, and ionomycin by 91, 94, and 96%, respectively, and reduced [3H]leucine incorporation into cellular proteins by 87, 85, and 84%, respectively. Emetine (300 nM) inhibited protein synthesis in thymocytes to an equivalent level but reduced DNA cleavage by only 49, 43, and 57% in cells treated with thapsigargin, MPS, or ionomycin, respectively. More than threefold higher concentrations of emetine (1 microM) were required to suppress DNA fragmentation to a similar extent as observed with cycloheximide. Puromycin at a concentration (5 micrograms/ml) that reduced [3H]leucine incorporation by > 80% enhanced DNA cleavage in thymocytes treated with thapsigargin, MPS, or ionomycin. By itself, puromycin (0.1-5 micrograms/ml), but not cycloheximide or emetine, induced DNA fragmentation in thymocytes with the concomitant inhibition of protein synthesis. An analogue of puromycin, puromycin aminonucleoside, which has no effect on protein synthesis, did not induce DNA fragmentation 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, puromycin (50 micrograms/ml) was less efficient in causing DNA cleavage when added alone and markedly inhibited chromatin degradation induced by thapsigargin, MPS, or ionomycin. Prolonged treatment (24 h) of thymocytes with any one of the different translational inhibitors resulted in extensive DNA fragmentation. Similarly, the protective effect of these inhibitors on DNA degradation in thymocytes induced by thapsigargin, MPS, or ionomycin diminished after 24 h. The present study demonstrates a lack of correlation between inhibition of protein synthesis and prevention of DNA fragmentation in thymocyte apoptosis and suggests that the effects of translational inhibitors on thymocyte apoptosis are nonspecific, and that they may delay the onset of apoptosis rather than prevent it.
Chow
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Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Peters
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Orrenius
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004640:Emetine / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D008775:Methylprednisolone / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D011692:Puromycin Aminonucleoside / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013950:Thymus Gland / Q000166:cytology*
D011500:Protein Synthesis Inhibitors; D013729:Terpenes; D011691:Puromycin; D015759:Ionomycin; D011692:Puromycin Aminonucleoside; D019284:Thapsigargin; D004247:DNA; D003513:Cycloheximide; D000252:Calcium-Transporting ATPases; D008775:Methylprednisolone; D004640:Emetine