50-kb Chromatin Fragmentation in the Absence of Apoptosis

Treatment with ionic detergents of nuclei isolated from various continuously growing cell lines generally yields chromatin samples of high viscosity. Extensive treatment with nuclease-free proteinase K or pronase solubilized the viscous lysates with >90% of the DNA migrating at 50 kb. Freshly prepared human peripheral blood T cells also yield a substantial fraction of their DNA in an 50- to 100-kb band. The cleavage sites may coincide with a class of DNase I-hypersensitive regions, since digestion of chromatin by DNase I at 10 U/ml, without protease, also yields fragments of preferentially 50-kb size. Occasionally, the oligonucleosomal ladder was also detected together with high molecular weight degradation products. Remarkably, all of these fragmentation patterns were seen in healthy, resting or proliferating cells, i.e., in the absence of apoptosis. Tritiated thymidine incorporation could be readily detected in the 50-kb DNA fragments. The effect of an apoptotic intracellular milieu on the integrity of isolated chromatin is apparently imitated by the extensive protease treatment used in our DNA isolation protocol.