Abstract
Internucleosomal DNA cleavage is the key molecular event of the cytolytic phase of glucocorticoid-induced lymphocytolysis. We find that novobiocin, the topoisomerase II inhibitor, is a potent inducer of in vivo internucleosomal DNA cleavage in human CEM lymphocytes. This in vivo effect is very rapid, time- and dose-dependent, requires cellular integrity, and does not require de novo protein synthesis. Recently our data (Alnemri, E. S., and Litwack, G. (1989) J. Biol. Chem. 264, 4104-4111) suggested that activation of DNA cleavage in CEM-C7 lymphocytes by glucocorticoids is independent of calcium uptake. Similarly, the novobiocin effect is also independent of calcium uptake and does not occur in isolated CEM nuclei or in CEM cells treated previously with the divalent cation ionophore A23187. Internucleosomal DNA cleavage induced by novobiocin or glucocorticoid generates blunt-ended double-stranded DNA fragments possessing 3'-hydroxyls and 5'-phosphates. As demonstrated by gel retardation analysis and DNase I footprinting, novobiocin causes the disruption and unfolding of an in vitro reconstituted mononucleosome so that it becomes more susceptible to DNase I cleavage. Our data suggest that 1) novobiocin rapid activation of internucleosomal DNA cleavage and chromatin changes in CEM lymphocytes are molecular features of apoptosis or programmed cell death. 2) CEM lymphocytes apparently do not express a Ca2(+)-dependent endonuclease. 3) The mechanism(s) of glucocorticoid or novobiocin-induced DNA cleavage in CEM lymphocytes involves activation of a constitutive non Ca2(+)-dependent endonuclease. We propose that the majority of nuclear chromatin is maintained in a highly compact and charge-neutralized state and that disruption of this highly ordered structure, directly by novobiocin or indirectly by glucocorticoid, may lead to the exposure and unmasking of internucleosomal linker DNA regions which are substrates for a constitutive non-Ca2(+)-dependent endonuclease.
Highlights
Novobiocin Activates Internucleosomal DNA Cleavage of Human Lymphocyte Genome in Vivo-To examine the effect of novobiocin on chromatin structure of human lymphocytic cell lines, CEM-C7 cells were treated with O-1200 pg/ml novobiocin for 3 h
Fig. lA shows that novobiocin was able to activate internucleosomal DNA cleavage in CEM-C7 cell line in a dose-dependent manner
The novobiocin-induced DNA cleavage pattern is strongly similar to that obtained after glucocorticoid treatment of rat thymocytes or human CEMC7 lymphocytes [18]
Summary
Was isolated and analyzed as described under “Experimental Procedures.” Table II shows that regardless of the presence or absence of EGTA, novobiocin was able to cause internucleosomal DNA cleavage in CEM-C7 lymphocytes. DNA was isolated and analyzed as described under “Experimental Procedures.” Fig. 4, A and B, show that incubation of rat thymocyte nuclei with various Ca2+ concentrations resulted in a dose-dependent activation of internucleosomal DNA cleavage.
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