The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation
The treatment of agarose embedded nuclear or cellular preparations with protein denaturing agents resulted in ordered cleavage of intact nuclear DNA into high molecular weight fragments with the pattern of fragmentation being unityped for various eukaryotic representatives. We snowed that the set of...
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| Zitieren: | The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation / V.T. Solovyan, I.О. Andreev, T.Y. Kolotova, P.V. Pogrebnoy, D.V. Tarnavsky // Биополимеры и клетка. — 1996. — Т. 12, № 3. — С. 67-76. — Бібліогр.: 51 назв. — англ. |
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irk-123456789-1539152019-06-15T01:29:05Z The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation Solovyan, V.T. Andreev, I.O. Kolotova, T.Y. Pogrebnoy, P.V. Tarnavsky, D.V. The treatment of agarose embedded nuclear or cellular preparations with protein denaturing agents resulted in ordered cleavage of intact nuclear DNA into high molecular weight fragments with the pattern of fragmentation being unityped for various eukaryotic representatives. We snowed that the set of DNA fragments represents the pre-existing DNA structural domains attributed to the higher levels of chromatin folding, and presented evidence allowing to interpret the nuclear DNA domain organization as a constituent component of topoisomerase III DNA complex with its ability to mediate the cleavage/religation reactions. We demonstrated that changes in the integrity of nuclear DNA, recognizable as an altered pattern of SDS-dependent cleavage of nuclear DNA into high molecular weight DNA fragments, took place at the early stage of apoptosis, upon number of stress challenges and in cells showing various proliferative status. The changes in the integrity of nuclear DNA affected by various influences were shown to be prompt and seem to be of transient nature. The results obtained allow to conclude that changes in the integrity of nuclear DNA revealed as an altered pattern of SDS-dependent high molecular weight DNA cleavage may present the specific genome reaction accompanying the physiological changes in the cells during apoptosis, stress response and differentiation. Уданій роботі показано, що при дії на «заплавлені» в агарозу препарати клітин та ядер білкових денатурантів відбувається упорядковане крупноблочне розщеплення інтактної ядерної ДНК. Фрагменти, що утворюються при цьому, являють собою передіснуючі структурні домени ядерної ДНК, які відповідають виищм рівням упакування хроматину. їх можна розглядати як конститутивний компонент ядерного комплексу ДНК/топоізомераза II, спроможного здійснювати реакцію розщеплення – воз'єднання ДНК Встановлено, що зміна нативності, або цілісності, ядерної ДНК, яка виявляється у зміні характеру DS-Na-золежного крупноблочного розщеплення, ядерної ДНК, відбувається на ранніх етапах апоптозу під дією різноманітних стресових факторів, а також у клітинах з різним рівнем проліферативної активності. Отримані результати дозволяють припустити, що спостережені зміни нативності ядерної ДНК можуть бути специфічною геномною реакцією, яка супроводжує фізіологічні процеси в клітині приаіюптозі, відповіді на стрес або діференціювання. В данной работе показано, что воздействие на препараты «заплавленных» в агарозу клеток или ядер белковых денатурантов приводит к упорядоченному крупноблочному расщеплению интактной ядерной ДНК. Фрагменты, образующиеся при этом, являют собой предсуществующие структурные домены ядерной ДНК, соответствующие высшим уровням упаковки хроматина. Их можно рассматривать как конститутивный компонент ядерного комплекса ДНК/ топоизомераза II, способного осуществлять реакцию расщепления/воссоединения ДНK Установлено, что изменение нативности, или целостности, ядерной ДНК, проявляющееся в изменении характера DS-Na-зависимого крупноблочного расщепления ядерной ДНК, происходит на ранних этапах апоптоза под влиянием всевозможных стрессовых факторов, а также в клетках с различным уровнем пролиферативной активности. Эти изменения происходят быстро и могут иметь обратимый характер. Полученные результаты позволяют предположить, что наблюдаемые изменения нативности ядерной ДНК могут представлять собой специфическую геномную реакцию, сопровождающую физиологические процессы в клетке при апоптозе, ответе на стресс или дифференцировке. 1996 Article The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation / V.T. Solovyan, I.О. Andreev, T.Y. Kolotova, P.V. Pogrebnoy, D.V. Tarnavsky // Биополимеры и клетка. — 1996. — Т. 12, № 3. — С. 67-76. — Бібліогр.: 51 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000430 http://dspace.nbuv.gov.ua/handle/123456789/153915 575.113/577.21 en Биополимеры и клетка Інститут молекулярної біології і генетики НАН України |
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The treatment of agarose embedded nuclear or cellular preparations with protein denaturing agents resulted in ordered cleavage of intact nuclear DNA into high molecular weight fragments with the pattern of fragmentation being unityped for various eukaryotic representatives. We snowed that the set of DNA fragments represents the pre-existing DNA structural domains attributed to the higher levels of chromatin folding, and presented evidence allowing to interpret the nuclear DNA domain organization as a constituent component of topoisomerase III DNA complex with its ability to mediate the cleavage/religation reactions. We demonstrated that changes in the integrity of nuclear DNA, recognizable as an altered pattern of SDS-dependent cleavage of nuclear DNA into high molecular weight DNA fragments, took place at the early stage of apoptosis, upon number of stress challenges and in cells showing various proliferative status. The changes in the integrity of nuclear DNA affected by various influences were shown to be prompt and seem to be of transient nature. The results obtained allow to conclude that changes in the integrity of nuclear DNA revealed as an altered pattern of SDS-dependent high molecular weight DNA cleavage may present the specific genome reaction accompanying the physiological changes in the cells during apoptosis, stress response and differentiation. |
| format |
Article |
| author |
Solovyan, V.T. Andreev, I.O. Kolotova, T.Y. Pogrebnoy, P.V. Tarnavsky, D.V. |
| spellingShingle |
Solovyan, V.T. Andreev, I.O. Kolotova, T.Y. Pogrebnoy, P.V. Tarnavsky, D.V. The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation Биополимеры и клетка |
| author_facet |
Solovyan, V.T. Andreev, I.O. Kolotova, T.Y. Pogrebnoy, P.V. Tarnavsky, D.V. |
| author_sort |
Solovyan, V.T. |
| title |
The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation |
| title_short |
The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation |
| title_full |
The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation |
| title_fullStr |
The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation |
| title_full_unstemmed |
The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation |
| title_sort |
ordered disintegration of nuclear dna as a specific genome reaction accompanying apoptosis, stress response and differentiation |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
1996 |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/153915 |
| citation_txt |
The ordered disintegration of nuclear DNA as a specific genome reaction accompanying apoptosis, stress response and differentiation / V.T. Solovyan, I.О. Andreev, T.Y. Kolotova, P.V. Pogrebnoy, D.V. Tarnavsky // Биополимеры и клетка. — 1996. — Т. 12, № 3. — С. 67-76. — Бібліогр.: 51 назв. — англ. |
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Биополимеры и клетка |
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ISSN 0233-7657. Биополимеры и клетка. 1996. Т. 12. № 3
The ordered disintegration of nuclear DNA as a specific
genome reaction accompanying apoptosis, stress response
and differentiation
V. T. Solovyan* I . О . Andreev, T. Yu. Kolotova
P. V. Pogrebnoy , D. V. Tarnavsky2
Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine
150 Zabolotnogo str., Kyiv 252143, Ukraine
1 L I . Mechnikov Institute of Microbiology and Immunology
Kharkiv 131057, Ukraine
2R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of the National
Academy of Sciences of Ukraine
45 Vasylkivska str., Kyiv 252022, Ukraine
The treatment of agarose embedded nucleaf or cellular preparations with protein
denaturing agents resulted in ordered cleavage of intact nuclear DNA into high molecular
weight fragments with the pattern of fragmentation being unityped for various eukaryotic
representatives. We showed that the set of DNA fragments represents the pre-existing
DNA structural domains attributed to the higher levels of chromatin folding, and
presented evidence allowing to interpret the nuclear DNA domain organization as a
constituent component of topoisomerase II/DNA complex with its ability to mediate the
cleavage/ religation reactions. We demonstrated that changes in the integrity of nuclear
D N A , recognizable as an altered pattern of SDS-dependent cleavage of nuclear DNA into
high molecular weight DNA fragments, took place at the early stage of apoptosis, upon
number of stress challenges and in cells showing various proliferative status. The changes
in the integrity of nuclear DNA affected by various influences were shown to be prompt and
seem to be of transient nature. The results obtained allow to conclude that changes in the
integrity of nuclear DNA revealed as an altered pattern of SDS-dependent high molecular
weight DNA cleavage may present the specific genome reaction accompanying the
physiological changes in the cells during apoptosis, stress response and differentiation
Introduction. Genomic DNA within eukaryotic cell nucleus appears to be
organized into loop domains sized about 40—100 kb, which are fixed on the
protein backbone structure referred to as nuclear matrix or chromosome scaffold
[1—11]. Topoisomerase II have been shown to be a major component of the
nuclear matrix and chromosome scaffold fraction and play important role in
chromosome structure and condensation and in genome expression [12—14].
Several lines of evidence suggest that type II enzyme appears to be concentrated
in a number of discrete anchoring complexes, which probably form the basis of
the chromatin loop domains [15, 16]. The organization of nuclear DNA into
ioop domains provides reasons to believe that each structural domain may
correspond to individual functional genome unit, which is compartmentalized
*Corespondence address.
г
© Y T SOLOVYAN, I O ANDREEV, T Yu KOLOTOVA, P V POGREBNOY, D Y TAR.NA'VSKV, 1Q96
67
SOLOVYAN V. Т. ET AL.
relative to regulatory elements by DNA sequences that are bound to the nuclear
matrix [17—22] (for review see [19]). Several investigators reported that
changes in DNAse I sensitivity that can extend over several dozens of kilobases
accompany changes in expression of specific genes [23, 24] (for review see
[3 ]), with the boundaries of transcriptionally active nuclease-sensitive domains
being commonly coincided with matrix attachment regions [25—28 ]. In a
number of studies using a variety of fractionation techniques both replicating
DNA and transcriptionally active sequences were shown to exhibit an altered
interaction with nuclear matrix [11, 29—35] (for review see [19, 33—35]).
These data indicate that some functionally significant structural rearrangements
of chromatin may occur during DNA replication and transcription.
Recently it was shown that the formation of discrete set of high molecular
weight (HMW) DNA fragments proceeds at the early stages of apoptosis [36,
37 ], which may be interpreted as a key event involved in the programmed cell
death [38, 39]. This finding suggests that the ordered disintegration of nuclear
DNA into HMW-DNA fragments may represent an early event in specific cell
programme resulting in cell self-demise.
In the present investigation we report data to show that the changes in the
integrity of nuclear DNA accompany various cell programmes including
apoptosis, stress response, proliferation and may be interpreted as a specific
genome reaction being of physiological value.
Material and Methods. Cell lines and culture conditions. The human
lymphoblastoma cultured cells (line СЕМ) and primary cell culture of murine
thymocytes obtained from the thymus of 4—5 week old mouse (line BALB/c)
were used for investigation of apoptosis and stress response. Human cultured
cells were routinely incubated in RPMI 1640 medium supplemented with 10 %
fetal calf serum (FCS) in atmosphere of 95 % air, 5 % CO2 to give a final
suspension of 5-Ю6 cells/ml. Thymocyte primary culture was prepared from
intact thymocytes to final suspension of 2-Ю7 cells in RPMI 1640/10 % FCS
and incubated under conditions indicated above for at least 6 h either with or
without apoptotic inducers. Apoptosis was induced by incubation of cells either
with 1 μ M dexamethasone or 7.5 μΜ teniposide (VM-26). The cell survival
fraction was estimated by Trypan blue exclusion.
Preparation of cellular samples to FIGE fractionation. 200 μ\ of cell
suspension were placed into the well of cell culture plate followed by addition
of equal volume of 1 % low-melting point agarose prepared on TEN-buffer
(10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl). After gelation the
equal volume of lysing buffer (TEN + 1 % SDS) was layered followed by
incubation for 1 h at 37 °С. Agarose plugs containing the lysed cells were used
for analysis by agarose gel electrophoresis.
Gel electrophoresis. Lysed cell preparations were fractionated either by
conventional or field inversion gel electrophoresis (FIGE) to detect the
pattern of nuclear DNA cleavage. Conventional gel electrophoresis was carried
out in 1.4 % agarose at 50 V for 4—5 h using 0,5xTBE buffer (0.089 M Tris,
0.089 M boric acid, 0.002 M EDTA, pH 8.5). FIGE was performed in 1 %
agarose at 85 V for 18 h in 0,5xTBE buffer under constant pulses of electric
field (24 s «forward» and 8 s «backward») allowing to monotonous resolution of
DNA molecules sized up to 500 kb [50]. In some cases FIGE was carried out
for 5—6 h allowing to resolve both low- and high molecular weight DNA. After
electrophoresis the gel was stained with 1 μ%/πή ethidium bromide for 10 min,
viewed using UV transilluminator and photographed using Mikrat 300 film.
Results and Discussion. We showed in our previous works * that the
fractionation of agarose embedded nuclei samples, treated with SDS, by field
inversion gel electrophoresis (FIGE) results in appearence of two main types of
discrete DNA fragments sized about 50—100 kb and 250—300 kb, with the
68
THE ORDERED DISINTEGRATION OF NUCLEAR4 DNA
pattern being comparable for various eukaryotic representatives [40]. The
treatment of nuclei preparations with protein denaturing agents represents the
decisive prerequisite for ordered HMW-DNA cleavage. No DNA fragment is
released into gel provided these agents lacking even if nuclei were destroyed
with fiigh concentration of EDTA [40].
Re-fractionation of nuclear preparations by FIGE with various pulsed field
switching intervals or by orthogonal field alteration gel electrophoresis
(OFAGE) showed that the group of DNA fragments of about 250—300 kb
contributes to the limited mobility zone under FIGE employed while the another
group of DNA represents the set of DNA fragments with the real size of about
50—100 kb (results not shown).
As evidenced from the data presented in Fig. 1, the pattern of SDS-depen-
Fig. 1. The pattern of lymphocytes nuclear DNA
fragmentation in cells ( / ) , nuclei (2) and nucleoids
(3) preparations. Human lymphocytes were collected
by centrifugation, resuspended in TE-buffer
containing 0.15 M NaCl and embedded into low-
melting agarose. After gelation samples were treated
with 1 % SDS followed by HGE fractionation. For
preparation of nuclei, cells were. resuspended in
buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM
MgCl2, 1 % triton X-100, 0.32 M sucrose. After
centrifugation nuclear pellet was resuspended in the
same buffer without triton followed by addition of
equal volume of 1 % agarose. Nucleoids (histone-
depleted nuclei) were prepared by extensive nuclei
treatment with 2 M NaCl followed by embedding into
agarose. After gelation nuclei and nucleoid
preparations were treated with 1 % SDS and
fractionated by FIGE. M — molecular weight stan-
dards, lambda DNA oligomers
dent HMW-DNA cleavage is similar to that found in fractionated cell-, nuclei-
and «nucleoid» preparations. The only difference is that in the nucleoids DNA
fragments of about 50—100 kb represent the major DNA cleaved product. The
maintaining of an ordered nuclear DNA fragmentation in nucleoids (histone-
depleted nuclei) suggests that the HMW-DNA fragments revealed by FIGE may
be relevant to the higher-level DNA structural organization. Based on the data
of structural organization of histone-depleted nuclei, it is conceivable to ascribe
the observed 50—100 kb DNA fragments to DNA loop domains the average
length of which varies between 40—100 kb [10, 41 ].
Data presented in Fig. 2 show that nuclear DNA structural domains seem
to be involved in functioning topoisomerase II/DNA complex with the main
ability to carry out cleavage/rejoining reactions. Thus, the specific modulator of
topoisomerase II activity, teniposide (VM-26), enhances the ordered HMW-
DNA cleavage which results in the increased amount of DNA released into gel
and in breaking down of the large DNA fragments into 50—100 kb ones. (Fig.
2, B, line I, 2). On the other hand, the conditions provoked by the
Fig. 2. Modulation of topoisomerase II-mediated
cleavage/religation reactions in cultured human
cells: A — cultured lymphoblastoma cells (line
СЕМ) were incubated at 37 °С (1), subjected at
4 °С for 30 min (2) followed by incubation at 37
°С for 30 min (J); B — the same cells were
incubated at 37 °С without ( / ) or with 7.5 μΜ
teniposide (2) for 20 min followed teniposide
treatment the cells were iftcubated at 55 0C for 10
min (J) (A, B — after incubation cells were
embedded into agarose, treated with SDS and
fractionated by FIGE)
69
SOLOVYAN V. Т. ET AL.
topoisomerase ΙΙ-mediated reverse reaction lead to the rejoiiling of cleaved DNA
domains (Fig. 2).
The ordered disintegration of nuclear DNA into HMW-DNA domains
seems to be of physiological value since the formation of HMW DNA fragments
may be involved in the early events accompanying programmed cell death. This
is evidenced from the data presented in Fig. 3, which show that the
Fig. 3. Gel electrophoretic analysis of nuclear DNA cleavage during dexamethasone induced apoptosis
in murine thymocytes. Cells were treated with 1 μΜ dexamethasone and allowed to incubate for the
time indicated at the Юр of the Figure. After incubation cells were embedded into low-melting point
agarose, treated with SDS and fractionated either by FIGE (top panels) or conventional gel
electrophoresis (bottom panels). C — control cells (not treated with dexamethasone)
dexamethasone induced apoptosis in primary culture of thymocytes is associated
with enhanced nuclear DNA cleavage into fragments of about 50—100 kb,
which is detectable at the early stage of apoptosis and precedes typical for
apoptosis internucleosomal DNA fragmentation. It seems likely, however, that
the ordered HMW-DNA cleavage is not restricted to apoptotic cell death. The
data presented in Fig. 4 show that in vitro incubation of primary culture of
thymocytes without apoptotic inducers is accompanied with the rapid increase
of nuclear HMW-DNA cleavage to be kept «frozen» without typical for apoptosis
oligonucleosomal «ladder» development. These data suggest that ordered
Fig. 4. Gel electrophoretic analysis of nuclear DNA
cleavage in primary culture of thymocytes non-
stimulated to apoptosis. Intact murine thymocytes
were resuspended in RPMI medium supplemented
with 10 % FCS and allowed to incubate for the time
indicated at the top of the Figure (h). After
incubation cells were embedded into low-melting
point agarose, treated with SDS and fractionated b?
FIGE
70
THE ORDERED DISINTEGRATION OF NUCLEAR4 DNA
disintegration of nuclear DNA revealed as an enhanced HMW-DNA cleavage
contribute not only to the apoptosis but may be implicated in cell response to
stress challenges induced by thymocyte introduction into in vitro culture. As a
confirmation to this may be data presented in Fig. 5, indicating that both
Fig. 5. The pattern of ordered DNA cleavage in cultured CEM cells in response to cold shock. Cells
were incubated for 10 min at 4 °С (A) or 1 h at 15 °С (B) followed by incubation at 37 °С for the
time indicated at the top of figure (h). After incubation cells were embedded into low-melting point
agarose, treated with SDS and fractionated by FIGE. C — control cells not subjected to shock
short-time acute and prolong gentle cold shock, appear to induce in cultured
human lymphoblastoma cells (line СЕМ) rapid changes in the pattern of
HMW-DNA cleavage which is invariably persistent for at least 2 h recovering
after cold shock.
As evidenced from the data presented in Fig. 6 the ordered disintegration
of nuclear DNA into HMW-DNA fragments may be of transient nature. Thus,
cultured CEM cell incubation in serum-free medium is accompanied by the
increased formation of HMW-DNA fragments to be rapidly declined following
serum addition and practically disappeared after 24 h incubation with fresh
medium with serum (Fig. 6). Similarly, the dilution of monolayer HeLa cells
with fresh medium is also associated with rapid changes in the pattern of
SDS-dependent HMW-DNA cleavage (Fig. 7). The generation of HM W-DNA
fragments is progressively decreased during cell incubation in fresh medium
and completely disappeared after 24 h of incubation. However, after 2 /3 of
new monolayer has been established, the formation of HMW-DNA is resumed
(Fig. 7).
These data indicate that changes in the pattern of SDS-dependent
HMW-DNA cleavage, seem to be of transient nature, and may be implicated
not only in cell response to stress challenges but also to accompany the changes
in the proliferation status of the cell. It allows to interpret the changes in the
integrity of nuclear DNA structural domains as a specific genome reaction being
of physiological value.
Our data show that the treatment of agarose-embedded nuclear or cellular
preparations with SDS results in ordered fragmentation of nuclear DNA into
HMW-DNA fragments with the pattern of fragmentation being comparable for
various eukaryotic representatives. The maintaining of an ordered nuclear DNA
fragmentation in histone-depleted nuclei (Fig. 1) suggests that the formation of
HMW-DNA fragments appears to be independent on the presence of histones
and may be due to the periodicity of DNA folding in the cell nucleus. This
provides reasons to believe that HMW-DNA fragments revealed by FIGE
fractionation of SDS treated nuclear preparations represent pre-existing
structural domains to reflect the higher levels of chromatin organization.
Based on the data of structural organization of histone-depleted nuclei, it
71
SOLOVYAN V. Т. ET AL.
Fig. 6. The transient nature of ordered DNA cleavage in cultured CEM cells during serum starvation.
Cells were incubated in serum-free medium (-) for the time indicated at the top of figure (h).
Following starvation serum was added and cells were incubated in serum-containing medium (+) for
the time indicated at the top of figure (h). After incubation cells were embedded into agarose, treated
with SDS and fractionated by FIGE. C — control cells before serum starvation
Fig. 7. The transient nature of ordered DNA cleavage in cultured HeLa cells. Monolayer cells were
washed with cultural medium to remove dead cells and treated with tripsine to obtain cellular
suspension. Portion of cellular suspension was embedded into agarose to analyse nuclear DNA
cleavage (control cells — C), while the equal another portions were supplemented with fresh medium
and allowed to incubate for the time indicated at the top of figure (h). After incubation cells were
washed with cultural medium to remove dead cells, tripsinized, collected by centrifugation and
embedded into agarose followed by SDS treatment and FIGE fractionation
is conceivable to ascribe the 50—100 kb DNA fragments to DNA loop domains
the average length of which varies between 40—100 kb [10, 41 ]. Similar results
were obtained by other authors as well [42—44 ]. As far as 250—300 kb DNA
fragments are concerned these contribute to the limited mobility zone under
FIGE employed and may be interpreted as a heterogeneous population of DNA
fragments consisting of noncleaved loop domains. This is supported by the fact
that these fragments may be converted into 50—100 kb ones by a number of
influences including topoisomerase II specific poisons [40 ]. The sensitiveness of
SDS-dependent HMW-DNA cleavage to topoisomerase ΙΙ-specific poison
teniposide and rejoining of cleaved HMW-DNA domain under conditions
specifically stimulating the topoisomerase II-mediated religation reaction (Fig.
2) add credence to the idea that the nuclear DNA structural domains are
involved in functioning topoisomerase II/DNA complex the main property of
which is its ability to mediate the cleavage/rejoining reactions.
Our data evidence that the pattern of SDS-dependent HMW-DNA cleavage
may follow the physiological changes in the cells thus suggesting that changes
in the integrity of nuclear DNA may be of physiological value. Thus, the
enhanced HMW-DNA cleavage was shown to be the early event involved in
programmed cell death. Comparable data were obtained recently by other
investigators [36—39 ] to demonstrate that the formation of HMW-DNA
fragments may be considered as a biochemical hallmark of apoptosis.
Our results indicate that changes in the integrity of nuclear DNA revealed
as an altered pattern of SDS-dependent HMW-DNA cleavage occur not only
during apoptosis but upon cell challenging with stress stimuli (Fig. 3—6). Since
any stress influence may interfere with cellular viability one should believe that
the increased HMW-DNA fragmentation may reflect the fact that cells become
predisposed to apoptosis. The same reason may be for the monolayer HeLa
cells, which show the increased formation of HMW-DNA fragments (Fig. 7),
72
THE ORDERED DISINTEGRATION OF NUCLEAR4 DNA
However, our data show that the ordered disintegration of nuclear DNA in
apoptotic thymocytes seems to be uncoupled with apoptosis-specific
internucleosomal DNA cleavage and takes place in those thymocytes not
stimulated to apoptosis (Fig. 4). In addition, the quite different pattern of
HMW-DNA cjeavage may be observed even in the same plant tissues showing
various differentiation level (like shoots and roots of pea seedlings). As
evidenced from the results in all cases under study there is a tendency to
enhanced HMW-DNA fragmentation in quiescent or terminally differentiated
tissues, which is revealed as a changed proportion between the two types of
DNA fragments towards 50—100 kb fragments increase [51 ].
These results suggest that HMW-DNA cleavage seems to be unrelated with
apoptosis-specific nuclear DNA degradation, but more likely represent the
cellular response being of physiological value. This is further supported by the
observation that formation of HMW-DNA fragments seems may be of transient
nature (Fig. 6, 7). Taken together our results suggest that there may be a
specific cellular response resulting in structural rearrangements of nucleai\pNA
domains, which accompany the physiological changes in the cells during various
cellular programmes including apoptosis, stress response and differentiation.
Our interpretation of ordered HMW-DNA cleavage is based on previously
obtained data showing that nuclear DNA structural domains are involved in
functioning topoisomerase II/DNA complex [45]. We demonstrated that the
properties of this complex are comparable with those described for in vitro
established complex purified enzyme/plasmide DNA [46, 47]. The main
property of this complex was shown to be its ability to mediate the
cleavage/rejoining reactions of DNA structural domains ([45]; Fig. 2, present
communication). Based on the studies with purified topo II enzymes and DNA
the two-stage model for topoisomerase II-mediated cleavage/religation reactions
has been proposed [46, 47]. According to this model an enzyme/DNA cleavable
complex is the key covalent in- rmediate in the topoisomerase II mediated DNA
turnover, being in rapid equiiiorium with noncleavable complex [46—48 ]. The
exposure of the cleavable complex but not noncleavable one to protein
denaturants (such as SDS or alkali) results in cleaved DNA product involving
the covalent linking of topoisomerase II submnits to the 5'-ends of broken DNA
[46, 47 ]. Proceeding from the results that structural domains of nuclear DNA
contribute to the functioning topoisomerase II/DNA complex [45] it seems
appropriate to interpret the ordered disintegration of nuclear DNA into
HMW-DNA fragments as DNA structural domain turnover from «noncleavable»
to «cleavable» state, mediated by topo II enzymes. Thus, our results allow to
conclude that changes in the integrity of nuclear DNA revealed as an altered
pattern of SDS-dependent HMW DNA-cleavage may present the specific
genome reaction accompanying the physiological changes in the cells. This
reaction may be interpreted as the turnover of structural domains between
«cleavable» and «noncleavable» state whose physiological value remains to be
elucidated.
This study was supported in part by Grant of Fund «Fundamental
Research» of State Committee of Science and Technology, project 5.3/140.
В. Т. Солов'ян, I. О. Андреевf Т. Ю. Колотова, П. В. Погребной, Д В. Тарнавський
Упорядкована дезинтеграція ядерної ДНК як специфічна геномна реакція, що супроводжує
апоптоз, відповідь на стрес і диференціювання
Резюме
Уданій роботі показано, що при дії на «заплавлені» в агарозу препарати клітин та ядер білкових
денатурантів відбувається упорядковане крупноблочне розщеплення інтактної ядерної ДНК.
Фрагменти, що утворюються при цьому, являють собою передіснуючі структурні домени
73
SOLOVYAN V. Т. ET AL.
ядерної ДНК, які відповідають виищм рівням упакування хроматину. їх можна розглядати як
конститутивний компонент ядерного комплексу ДНК/топоізомераза II, спроможного
здійснювати реакцію розщеплення — воз'єднання ДНК Встановлено, що зміна нативності, або
цілісності, ядерної ДНК, яка виявляється у зміні характеру DS-Na-золежного крупноблочного
розщеплення, ядерної ДНК, відбувається на ранніх етапах апоптозу під дією різноманітних
стресових факторів, а також у клітинах з різним рівнем проліферативної активності.
Отримані результати дозволяють припустити, що спостережені зміни нативності ядерної
ДНК можуть бути специфічною геномною реакцією, яка супроводжує фізіологічні процеси в
клітині приаіюптозі, відповіді на стрес або діференціювання
B. T. Соловьян, И. О. Андреев, Т. Ю. Колотова, П. В. Погребной, Д. В. Тарнавский
Упорядоченная дезинтеграция ядерной ДНК как специфическая геномная реакция,
сопровождающая апоптоз, ответ на стресс и дифференцировку
Резюме
В данной работе показано, что воздействие на препараты «заплавленных» в агарозу клеток или
ядер белковых денатурантов приводит к упорядоченному крупноблочному расщеплению
интактной ядерной ДНК. Фрагменты, образующиеся при этом, являют собой
предсуществующие структурные домены ядерной ДНК, соответствующие высшим уровням
упаковки хроматина. Их можно рассматривать как конститутивный компонент ядерного
комплекса ДНК/ топоизомераза II, способного осуществлять реакцию
расщепления/воссоединения ДНK Установлено, что изменение нативности, или целостности,
ядерной ДНК, проявляющееся в изменении характера DS-Na-зависимого крупноблочного
расщепления ядерной ДНК, происходит на ранних этапах апоптоза под влиянием всевозможных
стрессовых факторов, а также в клетках с различным уровнем пролиферативной активности.
Эти изменения происходят быстро и могут иметь обратимый характер. Полученные
результаты позволяют предположить, что наблюдаемые изменения нативности ядерной ДНК
могут представлять собой специфическую геномную реакцию, сопровождающую
физиологические процессы в клетке при апоптозе, ответе на стресс или дифференцировке.
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UDC 575.113/577.21 Received 10.08.95
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