EXPERIMENTAL CELL RESEARCH 235, 130–137 (1997) ARTICLE NO. EX973651 The Cleavage of Nuclear DNA into High Molecular Weight DNA Fragments Occurs Not Only during Apoptosis but Also Accompanies Changes in Functional Activity of the Nonapoptotic Cells V. T. Solov’yan, 1 I. O. Andreev, T. Yu. Kolotova, P. V. Pogribniy,* D. T. Tarnavsky,* and V. A. Kunakh Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Kyiv 252627, Ukraine; and *R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Science of Ukraine, Kyiv 252022, Ukraine clear matrix or chromosome scaffold [1 – 11]. Topoisom- In this paper we demonstrate that apoptosis in pri- erase II has been shown to be a major component of the mary culture of murine thymocytes and in continu- nuclear matrix and plays an important role in chromo- ously growing human cells is associated with the pro- some structure, chromosome condensation, and genome gressive disintegration of nuclear DNA into high mo- expression [12 – 14]. Several lines of evidence suggest lecular weight (HMW) – DNA fragments of about 50 – 150 that topoisomerase II is concentrated in a number of kb. We also show that the formation of similarly sized discrete anchoring complexes which probably form the HMW–DNA fragments takes place in the same cells in bases of the chromatin loop domains [11, 12, 15, 16]. the absence of apoptotic inducers. Unlike an apoptotic The organization of nuclear DNA into loop domains fragmentation of nuclear DNA, the formation of HMW – provides reasons to believe that each structural domain DNA fragments in nonapoptotic cells is rapidly in- may correspond to individual functional genome units, duced, has no correlation with the cell death, and is which are compartmentalized relative to regulatory el- not associated with the development of oligonucleoso- ements by DNA sequences that are bound to the nu- mal ‘‘ladder’’ or apoptotic changes in nuclear morphol- clear matrix [17, 18]; for review see [19–22]. Several ogy. The disintegration of DNA into HMW-fragments investigators have reported that changes in DNase I is also observed in nuclei isolated from healthy, non- sensitivity, which can extend over tens of kilobases, apoptosizing tissues of various eukaryotes. We show accompany changes in expression of specific genes [11, that the formation of HMW–DNA fragments in the ab- sence of apoptotic inducers is strongly dependent on 23, 24], with the boundaries of transcriptionally active the ionic detergents, is responsive to the topoisomer- nuclease-sensitive domains generally coinciding with ase II-specific poison, teniposide, and is completely re- matrix attachment regions [25–28]. In a number of versible under conditions that favor topoisomerase II- studies, both replicating DNA and transcriptionally ac- dependent rejoining reaction. Also, we demonstrate tive sequences were shown to exhibit an altered inter- that the formation of HMW–DNA fragments in contin- action with nuclear matrix [6, 29 – 32]; reviewed in [33 – uously growing cell lines caused either by serum dep- 36]. These data indicate that some functionally sig- rivation or monolayer establishment is of a transient nificant structural rearrangements of chromatin may nature and rapidly reverses to the control level follow- accompany various cellular programs associated with ing serum addition or dilution of monolayer. The re- the induction of cell proliferation or changes in tran- sults suggest that the cleavage of nuclear DNA into scription. HMW–DNA fragments is associated not only with Recently, it was demonstrated that DNA loop do- apoptosis but also accompanies changes in functional mains in animal cells may be excised by topoisomerase activity of nonapoptotic cells.  1997 Academic Press II-mediated DNA cleavage at scaffold attachment sites when either intact cells or nucleoids (high-salt- extracted nuclei) were treated with inhibitors of topo- INTRODUCTION isomerase II [37, 38]. The average size of cleaved DNA loop domains estimated by this approach ranged from Genomic DNA within the eukaryotic cell nucleus ap- 40 to 75 kb [37]. The fragmentation of nuclear DNA pears to be organized into loop domains which are fixed into similarly sized DNA fragments was observed in to a protein backbone structure referred to as the nu- apoptotic cells [39–43], which led to the conclusion that the release of DNA loops from the nuclear scaffold is a crucial intracellular event in apoptotic cells (see 1 To whom correspondence and reprint requests should be ad- dressed. Fax: /380-44-2660759. E-mail: kunakh@imbig.kiev.ua. review [44]). 130 0014-4827/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.