title: Identification and characterization of glucocorticoid-regulated nuclease(s) in lymphoid cells undergoing apoptosis.
abstract: Apoptosis is a physiological process by which selected cells are deleted from a population in response to specific regulatory signals. A hallmark of apoptosis is the internucleosomal degradation of DNA prior to cell death. We are studying glucocorticoid-induced lymphocytolysis as a model system for apoptosis within the immune system. In rat thymocytes, the internucleosomal DNA cleavage which occurs following glucocorticoid treatment is both time- and dose-dependent, and is blocked by the glucocorticoid antagonist RU 486, indicating that this effect is mediated by the glucocorticoid receptor. Similar experiments using glucocorticoid-responsive (wt) and glucocorticoid-resistant (nt-) S49.1 lymphoma cell lines confirm that internucleosomal DNA degradation and cell death are glucocorticoid receptor-mediated events and thus reflect the direct effects of glucocorticoids on lymphocytes. In an effort to identify the nuclease(s) responsible for the DNA degradation, we have developed two assays to detect nucleases whose activity is altered by glucocorticoid treatment. The first assay involves electrophoresing extracts of nuclear protein from control and glucocorticoid-treated lymphoid cells into SDS-polyacrylamide gels containing [32P]DNA within the gel matrix. This assay is used to estimate the molecular mass of the nuclease, based on the observed in situ nuclease activity. The second assay uses HeLa nuclei as a substrate to detect internucleosomal cleavage activity present in nuclear extracts of control and glucocorticoid-treated lymphoid cells. Using these assays we have identified a novel Ca2+, Mg(2+)-dependent nuclease with an apparent molecular weight of 18 kDa in both S49 wt cells and rat thymocytes treated with glucocorticoids. Furthermore, nuclear extracts of glucocorticoid-treated, but not control, rat thymocytes and S49 wt cells were capable of cleaving HeLa chromatin at internucleosomal sites. In an effort to determine the identity of the nuclease capable of internucleosomal cleavage of DNA, nuclear extracts from dex-treated rat thymocytes were fractionated by gel filtration chromatography under non-denaturing conditions, and the fractions were analyzed using the [32P]DNA SDS-PAGE and HeLa nuclei assays. When analyzed under native conditions, the 18 kDa nuclease described previously appears to exist as a congruent to 25 kDa protein which may be part of a high molecular weight complex.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
authors.forename: L M
authors.initials: LM
authors.lastname: Caron-Leslie
authors.forename: R A
authors.initials: RA
authors.lastname: Schwartzman
authors.forename: M L
authors.initials: ML
authors.lastname: Gaido
authors.forename: M M
authors.initials: MM
authors.lastname: Compton
authors.forename: J A
authors.initials: JA
authors.lastname: Cidlowski
mesh_terms: D000818:Animals; D016923:Cell Death; D002474:Cell-Free System; D002478:Cells, Cultured; D004249:DNA Damage; D003851:Deoxyribonucleases / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000201:enzymology*; D051379:Mice; D051381:Rats
chemical_list: D005938:Glucocorticoids; D003907:Dexamethasone; D003851:Deoxyribonucleases