(Received for publication, June 2, 1995)
From the
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.
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 ()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-
) 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.
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) .
Figure 1:
Analysis of the
specificity of nucleolar gene long-range cleavage in cells undergoing
apoptosis triggered by TNF-. 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-
(lane 2) or in the presence of 5 units/ml (lane 3) and 10 units/ml (lane 4) TNF-
. 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-
. 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--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.
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.
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.