©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Large-scale Fragmentation of Mammalian DNA in the Course of Apoptosis Proceeds via Excision of Chromosomal DNA Loops and Their Oligomers (*)

(Received for publication, June 2, 1995)

Maria A. Lagarkova (1) Olga V. Iarovaia (1) (2) Sergey V. Razin (1) (2)(§)

From the  (1)Institute of Gene Biology RAS, Vavilov Str. 34/5, 117334 Moscow, Russia and the (2)International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

It has been shown recently that apoptotic degradation of genomic DNA in mammalian cells starts by excision of large DNA fragments ranging in size from 50 kilobases to more then 300 kilobases. Although it was suggested that the above fragments could represent chromosomal DNA loops, the supposition was not supported by direct experimental evidence. In present work, we have studied the specificity of nucleolar and euchromatic gene long-range fragmentation in mouse and human cells triggered to undergo apoptosis either by tumor necrosis factor or by serum deprivation. Separation of the excised large DNA fragments by pulsed field gel electrophoresis followed by Southern analysis has demonstrated that in all cases studied the above fragmentation proceeds in a specific way. Furthermore, the patterns of DNA long-range fragmentation in the cells undergoing apoptosis were indistinguishable from the patterns of DNA cleavage into chromosomal loops by the high salt-insoluble topoisomerase II of the nuclear matrix. These results suggest the conclusion that apoptotic degradation of chromosomal DNA starts by excision of DNA loops and their oligomers.


INTRODUCTION

Extensive digestion of cellular DNA by endogenous nucleases is considered to be a hallmark of programmed cell death (apoptosis) (1, 2, 3) . Recent evidence suggests that degradation of genomic DNA in the course of apoptosis starts by excision of 50-300-kb (^1)DNA fragments(4, 5, 6, 7, 8, 9, 10) which are then further digested to oligonucleosomal ladders. It has been suggested that the pattern of large scale fragmentation of chromosomal DNA reflects a periodicity of 30 nm chromatin fibril organization into loops attached to the nuclear matrix(4, 5, 10) . The only reason for this supposition was, however, a certain similarity in sizes. Although it was easy to analyze the specificity of the long-range DNA fragmentation by nucleases within any characterized genomic area, the pattern of this fragmentation could not be compared directly with the partitioning of DNA into loops because of the absence of a reliable procedure for mapping the DNA loop anchorage sites. However, this kind of comparison became possible with the elaboration of a general approach for DNA loop excision from the genome by topoisomerase II-mediated DNA cleavage at matrix attachment sites(11, 12) . Using this approach, we have demonstrated that in mammalian cells the nucleolar genes are organized into uniform loops equal in size to the size of the rDNA repeat and separated by the matrix attachment areas in nontranscribed spacers(11) . In the present study, the patterns of nucleolar and euchromatic gene long-range fragmentation in the course of apoptosis induced either by tumor necrosis factor (TNF-alpha) or by serum deprivation were compared with these generated by the high salt-insoluble topoisomerase II. They were found to be essentially similar if not identical.


EXPERIMENTAL PROCEDURES

Cultivation of Cells and Induction of Apoptosis

Mouse L929 cells were grown in Dulbecco's modified medium supplemented with 10% fetal calf serum. For induction of apoptosis, the recombinant human TNF-alpha was added up to a concentration of 5 units/ml. Human lymphoblastoid B-cells (line RPMI-6410t) were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum. For induction of apoptosis, the cells were first incubated for 2 h in the presence of actinomycin D (1 µM). Then they were transferred to a fresh medium (RPMI 1640) supplemented with TNF-alpha (5 or 10 units/ml). Human erythroleukemia K562 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. Apoptosis was triggered by replacement of this medium with the RPMI medium without serum.

To determine the cell viability, the cells were stained with trypan blue and counted under the microscope.

Excision of chromosomal DNA loops by DNA cleavage at matrix attachment sites with the high salt-insoluble topoisomerase II was carried out exactly as described(11, 12) .

Pulsed Field Gel Electrophoresis and Southern Analysis

DNA samples for pulsed field gel electrophoresis (PFGE) were prepared by lysis of cells embedded in agarose blocks followed by digestion of proteins with proteinase K (1 mg/ml final concentration) in solution containing 400 mM Na-EDTA and 1% SDS, as has been described previously(11, 12) . PFGE was carried out in a Bio-Rad CHEF III system in TBE buffer at 14 °C for 22 h at a voltage gradient of 6 V/cm with the switch time ramped linearly from 10-90 s. After electrophoresis, the separated DNA fragments were transferred to Hybond N membranes (Amersham) and were hybridized to the P-labeled cloned DNA fragment representing coding sequences for the 28 S rRNA (rDNA probe) or to the P-labeled cloned DNA fragment derived from the third exon of the human c-myc gene (c-myc probe). Prehybridization (5-10 h) and hybridization (12-16 h) were carried out at 65 °C in the following solution: 5 SSPE, 5 Denhardt's solution, 0.5% (w/v) SDS (Bio-Rad), 200 µg/ml denatured salmon sperm DNA (Sigma). After hybridization, the filters were washed twice (30 min each) at 65 °C in 2 SSPE, 0.5% SDS solution and once (30 min) at 65 °C in 0.1 SSPE, 0.5% SDS solution. The filters were then exposed to Fuji RX film at -75 °C with an intensifying screen (DuPont).


RESULTS AND DISCUSSION

Analysis of the Specificity of Nucleolar DNA Long-range Fragmentation in the Course of Apoptosis Induced by Tumor Necrosis Factor

The tumor necrosis factor (TNF-alpha) can trigger apoptosis in a variety of cultured cells(13, 14, 15) . In some cases, exposure of cells to the TNF-alpha alone is sufficient for triggering apoptosis(15, 16) , whereas in other cases the synergetic treatment with TNF-alpha and actinomycin D is necessary(15, 16, 17) . In our experiments, both types of the TNF-alpha-sensitive cells were studied. Human lymphoblastoid RPMI-6410t cells were triggered to undergo apoptosis by treatment with TNF-alpha and actinomycin D. Mouse L929 cells, known to be sensitive to the TNF-alpha independently on transcription inhibition(18) , were treated with TNF-alpha alone. As follows from the data represented in Table 1, more than 50% of TNF-alpha/actinomycin D-treated RPMI-6410t cells became trypan blue-positive after 48 h of cultivation. A similar cytotoxic effect was observed after 12 h of cultivation of TNF-alpha-treated L929 cells. In both cases, a significant portion of cells in the population possessed typical ``apoptotic'' morphology (not shown). When DNA from these cells was analyzed by PFGE, a substantial amount of material was found in the 50-600-kb size area (Fig. 1A). In contrast to most of the other authors(4, 5, 6) , we failed to observe a discontinuity in size distribution of the excised large DNA fragments. The reason for this discrepancy is not clear at the moment. We cannot exclude that the 300-kb band observed previously by several authors (4, 5, 6) is a compression artifact of the field inversion gel electrophoresis used for separation of the excised DNA fragments. Indeed, the sharpness of the above band varied significantly in the experiments of different authors (one may, for example, compare the data presented in Refs. 4 and 5). Furthermore, in no case did the authors cited above show proportional separation of the phage -DNA concatemers within the size area ranging from 50 to 300 kb (i.e. where the discontinuous distribution of genomic DNA fragments was observed). Finally, when other researchers have used PFGE for separation of the excised large DNA fragments, no discontinuity in the size distribution of these fragments has been observed(7) . In our conditions of PFGE, the compression artifact was observed close to the top of the gel (a relatively sharp band of about 700 kb). This compression band accumulated fragments ranging in size from 700 kb to more then 1000 kb. No compression band was seen in the lane loaded with DNA from control cells because in this case the DNA was virtually nondegraded, and hence it remained within the block loaded at the start of the gel. The start area is not shown in Fig. 1A.




Figure 1: Analysis of the specificity of nucleolar gene long-range cleavage in cells undergoing apoptosis triggered by TNF-alpha. A, separation of the released DNA fragments by PFGE (staining with ethidium bromide). B, hybridization of the separated DNA fragments (after Southern transfer) with the P-labeled rDNA probe (autoradiograph). Lane 1, fragmentation of DNA by the high salt-insoluble topoisomerase II of the nuclear matrix (cleavage of DNA at the basement of the loops(11, 12) ). Lanes 2-4, fragmentation of DNA in RPMI-6410t cells pretreated with actinomycin D (1 µg/ml) and cultivated for 48 h in the absence of TNF-alpha (lane 2) or in the presence of 5 units/ml (lane 3) and 10 units/ml (lane 4) TNF-alpha. Lanes 5-7, fragmentation of DNA in mouse L929 cells cultivated for 0 h (lane 5), 12 h (lane 6), and 18 h (lane 7) in the medium containing 5 units/ml TNF-alpha. All lanes were loaded with DNA from the same amount of cells. The start area (i.e. where the agarose blocks with embedded DNA were inserted in the gel) is not shown. Arrows at the left side of the A indicate positions of the molecular weight markers.



The results of Southern hybridization with the rDNA probe are shown in Fig. 1B. It is clear that in both cases of TNF-alpha-induced apoptosis the long-range fragmentation of nucleolar genes proceeds in a specific fashion. Hybridization of Southern filters with a probe representing coding sequences for the 28 S rRNA revealed a regular pattern of bands with the sizes divisible by the size of rDNA repeat (Fig. 1B, lanes 3 and 4 and lanes 6 and 7). Importantly, exactly the same pattern of the nucleolar DNA fragmentation was generated when the high salt-extracted cells were treated with an inhibitor of topoisomerase II (lane 1). As has been discussed previously, the pattern of cellular DNA fragmentation by the high salt-insoluble topoisomerase II reflects directly the mode of DNA organization into loops(11, 12) . The similarity of this pattern to the pattern of long-range DNA fragmentation in the course of apoptosis suggests that this fragmentation proceeds via excision of DNA loops and their oligomers.

Analysis of the Specificity of Nucleolar DNA Long-range Fragmentation in Cells Triggered to Undergo Apoptosis by Serum Deprivation

It has been reported previously (6) that fragmentation of DNA in the course of apoptosis triggered by serum deprivation also starts by excision of the ``loop-sized'' DNA fragments. Hence, in the next set of experiments, we have analyzed the specificity of nucleolar DNA long-range fragmentation in the course of apoptosis induced by serum deprivation. In these experiments, we have used human erythroleukemia K562 cells. After 68 h of cultivation in the absence of serum, the percentage of dead (trypan blue-positive) cells in the population exceeded 50% (Table 1), and, after 168 h of cultivation in the same medium, virtually all cells were dead. PFGE analysis of the corresponding DNA samples revealed a wide size distribution (50 to 600 kb) of the excised large fragments of the genomic DNA in a sample taken after 68 h of cultivation in the medium lacking serum (Fig. 2A). The loop-sized DNA fragments could still be seen after 168 h of cultivation without serum, although the average size of these fragments was significantly lower. Southern analysis with the rDNA probe revealed the same patterns of regular bands as in the samples from cells undergoing apoptosis triggered by TNF-alpha treatment (compare Fig. 1B and Fig. 2B). Hence, a conclusion may be drawn that cells of different lineages entering apoptosis in response to different treatments start to degrade the nucleolar genes by excision of chromosomal DNA loops and their oligomers.


Figure 2: Analysis of the specificity of nucleolar gene long-range cleavage in human K562 cells triggered to undergo apoptosis by serum deprivation. A, separation of the released DNA fragments by PFGE (staining with ethidium bromide). B, hybridization of the separated DNA fragments (after Southern transfer) with the P-labeled rDNA probe (autoradiograph). Lanes 1-3 were loaded with DNA samples from the same amount of cells cultivated in the medium lacking serum for 24, 96, and 168 h, respectively. Lane (A) represents distribution of the phage -DNA concatemers.



Analysis of Specificity of Non-nucleolar DNA Long-range Fragmentation in Human K562 Cells Triggered to Undergo Apoptosis by Serum Deprivation

The nucleolus constitutes a special (morphologically distinct) domain of the cell nucleus. One may thus argue that conclusions drawn in experiments with ribosomal genes may not necessarily be valid for the other parts of the genome. In order to clarify the situation, we have analyzed the specificity of long-range fragmentation of the genomic region bearing the c-myc gene in human K562 cells triggered to undergo apoptosis by serum deprivation. The experiments were carried out in the same way as those described above except that the c-myc probe was used for the Southern analysis. One may see (Fig. 3, lanes 2-5) that an 80-kb DNA fragment bearing the c-myc gene was accumulated in K562 cells undergoing apoptosis. A similar DNA fragment was excised from the genome by topoisomerase II-mediated cleavage at matrix attachment sites (compare lane 1 and lanes 2-5 in Fig. 3). Hence, it is likely that, as in the case of nucleolar genes, the degradation of the euchromatic part of the genome in cells undergoing apoptosis starts by excision of chromosomal DNA loops.


Figure 3: Analysis of specificity of the c-myc gene locus long-range cleavage in human K562 cells triggered to undergo apoptosis by serum deprivation. A, separation of the released DNA fragments by PFGE (staining with ethidium bromide). B, hybridization of the separated DNA fragments (after Southern transfer) with the P-labeled c-myc probe (autoradiograph). Lanes represent distribution of the length markers (phage -DNA concatemers). Lane 1 was loaded with DNA cleaved by topoisomerase II at matrix attachment sites. Lanes 2-6 were loaded with DNA from the same amount of cells cultivated in the medium without serum for 120 h, 96 h, 48 h, 24 h, and 0 h, respectively.




FOOTNOTES

*
This work was supported by Grant 93-04-21558 from Russian Foundation for Support of Fundamental Science, Grants MKF000 and MKF300 from the International Science Foundation, and International Centre for Genetic Engineering and Biotechnology Grant CRP/RUS93-06 (to S. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy. Tel.: 39-40-375-7352; Fax: 39-40-226-555.

(^1)
The abbreviations used are: kb, kilobase(s); TNF-alpha, tumor necrosis factor alpha; PFGE, pulsed field gel electrophoresis.


ACKNOWLEDGEMENTS

The plasmid containing Xenopus laevis rDNA sequences was a generous gift of Prof. T. Moss (Laval University, Quebec, Canada). Recombinant human TNF-alpha with the specific activity of 10^6 units/mg was a generous gift of Dr. Korobko (Institute of Bioorganic Chemistry RAS, Moscow).


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.