Internucleosomal deoxyribonucleic acid cleavage activity in apoptotic thymocytes: detection and endocrine regulation.
Apoptosis is a programmed form of cell death that occurs under numerous physiological conditions, including endocrine regulation of specific cell populations. We have investigated the biochemical mechanisms involved in glucocorticoid-induced apoptosis in rat thymocytes. Internucleosomal cleavage of chromatin into oligonucleosomal fragments is common to all forms of apoptosis and precedes the onset of cell death. To identify the endonuclease that is responsible for the specific pattern of DNA degradation in glucocorticoid-induced apoptosis, we have developed an assay to measure internucleosomal cleavage activity in thymocyte nuclear extracts. This assay uses nuclei from cells resistant to hormone-induced DNA fragmentation (HeLa cells) as a substrate for nuclear extracts prepared from thymocytes of adrenalectomized rats treated with either dexamethasone (dex) or vehicle (control). After incubation at room temperature for 90 min, the HeLa DNA is purified, and its integrity is analyzed by agarose gel electrophoresis. The appearance of internucleosomal fragments of HeLa DNA is indicative of nuclease activity in the thymocyte nuclear extract. Nuclear extracts prepared from thymocytes of rats treated with dex for 5 h caused internucleosomal cleavage of HeLa DNA, whereas extracts from control rats did not result in any DNA fragmentation. Regulation of nuclease activity by dex was time dependent. Internucleosomal cleavage activity in thymocyte extract from dex-treated animals was detected as early as 2 h after hormone treatment and occurred before any detectable change in cell viability. Maximal extractable nuclease activity was coincident with decreased thymocyte viability and thymic involution. In contrast, extracts from medullary thymocytes, which are the only thymocytes that survive 72 h of glucocorticoid treatment, did not contain nuclease activity by this assay. Regulation of internucleosomal cleavage activity by dex was dose dependent and was specific for the glucocorticoid class of steroid hormones. Furthermore, the dex-induced response was inhibited by pretreating rats with the glucocorticoid receptor antagonist RU486, indicating that receptor-mediated processes are involved in the regulation of nuclease activity. The similarities between the regulation of internucleosomal cleavage activity reported here and the previously described degradation of thymocyte DNA in vivo makes this nuclease a likely constituent of the apoptotic process.
Schwartzman
R A
RA
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Cidlowski
J A
JA
D000315:Adrenalectomy; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology / Q000502:physiology; D002470:Cell Survival; D004247:DNA / Q000378:metabolism / Q000502:physiology*; D003907:Dexamethasone / Q000494:pharmacology; D004702:Endocrine Glands / Q000502:physiology*; D012739:Gonadal Steroid Hormones / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects / Q000502:physiology; D015735:Mifepristone / Q000494:pharmacology; D009707:Nucleosomes / Q000502:physiology*; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
D012739:Gonadal Steroid Hormones; D009707:Nucleosomes; D015735:Mifepristone; D003907:Dexamethasone; D004247:DNA