Mechanism of tissue-specific induction of internucleosomal deoxyribonucleic acid cleavage activity and apoptosis by glucocorticoids.
Cell death occurring by apoptosis has become widely recognized as an integral component of the life cycle of many cell types. Apoptosis can be induced in many tissues by a wide variety of endogenous signals, including glucocorticoids. However, even though there are glucocorticoid receptors present in almost all cells, only certain lymphoid cells are susceptible to glucocorticoid-induced apoptosis. The basis for this selective regulation of programmed cell death is unknown. Internucleosomal chromatin degradation is an integral characteristic of apoptosis, common to all cells undergoing this form of programmed cell death. Thus, we have developed an in vitro assay that recapitulates apoptotic DNA degradation in isolated nuclei and have identified a nuclease activity with internucleosomal specificity that is present in nuclear extracts of thymocytes undergoing glucocorticoid-induced apoptosis. We have now extended these studies to analyze the molecular properties of the crude enzyme and to further elucidate the mechanism of its regulation during the tissue-specific induction of apoptosis. In vitro, optimal internucleosomal cleavage activity was detected in the presence of millimolar concentrations of calcium and magnesium or manganese. Nuclease activity was inhibited by three agents previously shown to block apoptosis (zinc, aurintricarboxylic acid, and sodium), suggesting that the nuclease we detected in apoptotic thymocytes is responsible for the DNA degradation associated with apoptosis. Size-fractionation analysis of thymocyte nuclear extract under native conditions revealed a protein with an apparent molecular mass of 22.7 kilodaltons and a sedimentation coefficient of 3.5S. Interestingly, when extracts from control thymocytes, inactive in internucleosomal cleavage activity, were subjected to gel filtration or sucrose density gradient fractionation, internucleosomal cleavage activity was detected. The physical characteristics of this endonuclease activity were identical to those found in thymocyte extract from glucocorticoid-treated rats. Repressed or latent internucleosomal cleavage activity was also detected in hepatocyte nuclear extracts, but this activity was not activated by glucocorticoid treatment. These data suggest that glucocorticoid-induced apoptosis involves cell-specific activation of a latent endonuclease, rather than nuclease induction.
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
R A
RA
Schwartzman
J A
JA
Cidlowski
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D004720:Endonucleases / Q000378:metabolism; D006367:HeLa Cells / Q000648:ultrastructure; D006801:Humans; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D009707:Nucleosomes / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013950:Thymus Gland / Q000648:ultrastructure; D015032:Zinc / Q000494:pharmacology
D009707:Nucleosomes; D008345:Manganese; D001312:Aurintricarboxylic Acid; D003907:Dexamethasone; D004247:DNA; D004720:Endonucleases; D008274:Magnesium; D015032:Zinc; D002118:Calcium