An inducible lymphocyte nuclear Ca2+/Mg(2+)-dependent endonuclease associated with apoptosis.

Abstract

Apoptotic cell death is typically accompanied by internucleosomal chromatin fragmentation. Although a number of candidate enzymes have been proposed, there is as yet no direct evidence for the involvement of any particular endonuclease in this process. Here we demonstrate the existence of an endonuclease(s) that is up-regulated during apoptotic T cell death. The endonuclease(s) is located in the nucleus, and its activity is increased up to eightfold by a variety of stimuli or conditions that induce apoptosis in T cell hybridomas and thymocytes. Treatments that prevent TCR-mediated apoptosis, such as cyclosporin A or concomitant administration of glucocorticoids, also prevent the induction of enzyme activity. The endonuclease activity is associated with three molecular forms, designated A, B, and C, with apparent M(r) of 49K, 47K, and 45K, respectively, and constitutes the major endonuclease activity in T hybridoma cells. From A exists in resting cells, and its activity is increased threefold after the induction of apoptosis. Forms B and C are absent in resting cells and are induced up to 20-fold after stimuli that lead to apoptosis. All three forms are Ca2+/Mg2+ dependent and are inhibited by Zn2+. This enzyme(s) introduces double strand breaks and single strand nicks into supercoiled plasmid DNA, demonstrating the mode of DNA fragmentation characteristic of products of apoptotic chromatin degradation. The enzyme(s) produces DNA fragments with 5'-P and 3'-OH terminals, also consistent with apoptotic chromatin degradation. Finally, enzyme solubilized from cells activated to die cleaves chromatin in nuclei isolated from unstimulated T hybridoma cells, yielding the classic DNA ladder. Because of its biologic properties, we named this enzyme(s) inducible lymphocyte Ca2+/Mg(2+)-dependent endonuclease, or ILCME. Because inducible lymphocyte Ca2+/Mg(2+)-dependent endonuclease possesses the key features predicted for an apoptosis-specific enzyme, it is a new candidate for an enzyme(s) that participates in DNA cleavage in apoptotic T cells.