Abstract
The mechanism of glucocorticoid-induced internucleosomal DNA cleavage and cytolysis of lymphatic cells is not known. Recent data (Compton, M.M., and Cidlowski, J.A. (1987) J. Biol. Chem. 262, 8288-8292) suggested that in vivo treatment of rat thymocytes with glucocorticoids induces a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis. In this paper, the possibility that lymphocytolysis may result from glucocorticoid-induced nuclease(s) was examined. Using the rat thymocytes as a model system, we have shown by electrophoretic, enzymatic, and amino acid sequence analysis that the putative glucocorticoid-induced nucleases identified recently by Compton and Cidlowski are in fact H1, H1(0), and core histones, and their gross appearance is not the result of new histone protein synthesis, but a result of the release of histone-containing nucleosomes during chromatin breakdown. Evidence presented here shows that the putative induced nuclease activity is an artifact of the assay system employed. Because our data do not support induction of a glucocorticoid-induced nuclease(s), we examined the possibility that DNA cleavage might be induced by activation of a constitutive endogenous endonuclease. We have shown that it is possible to produce characteristic internucleosomal DNA cleavage of rat thymocytes, merely by incubating intact nuclei from untreated adrenalectomized rat thymocytes with Ca2+ and Mg2+ for a short period of time. However, in glucocorticoid-sensitive human CEM-C7 lymphocytes activation of internucleosomal DNA cleavage was independent of calcium uptake. We conclude that glucocorticoid induction of internucleosomal DNA fragmentation does not necessarily require expression of a new nuclease(s), but is the result of the activation of a constitutive endogenous endonuclease(s). Also, our data suggest that the mechanism which controls activation of internucleosomal DNA cleavage in rat thymocytes differs from that which operates in CEM-C7 lymphocytes.
Highlights
8292) suggested that in vivo treatment of rat thymo- bition and cessation of proliferation, which is followed cytes with glucocorticoids induces a nucleolytic “lysis gene”product(s)responsible for lymphocytolysis
In cells that respond by cytolysis, clease(s), we examined the possibilitthyat DNA cleav- these alterations arefollowed by chromatin condensation and age might be induced by activation of a constitutive cleavage of DNA which is believed to beinduced by activation endogenousendonuclease
Thymocyte DNA isolated from Me2SO-treated control rats was used as a control ( l a w duce a nucleolytic“lysis gene” product(s) responsiblefor 3)
Summary
8292) suggested that in vivo treatment of rat thymo- bition and cessation of proliferation, which is followed cytes with glucocorticoids induces a nucleolytic “lysis gene”product(s)responsible for lymphocytolysis. The N-terminal aminoacid treated rat thymocytes or human lymphocytes was assayed sequence of one of these proteins with an apparemntolecular on SDS-polyacrylamide gels containing DNA as described mass of 18 kDa was as follows: PEPAKSAPAPKKGSK- above, apparent false nuclease activities were observed asso-
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