V. Т. Solov'yan, I. О. Andreev THE FORMATION OF HIGH MOLECULAR WEIGHT DNA FRAGMENTS: THE HALLMARK OF APOPTOSIS OR EARLY GENOMIC EVENTS IN STRESS RESPONSE?

We studied the peculiarities of nuclear DNA fragmentation in primary culture of mu­ rine thymocytes and in human lymphoblastoid cell culture (line СЕМ) induced to apoptosis by various influences. We showed that in apoptotic cells the ordered high molecular weight DNA cleavage, recognizable as DNA fragment release of 50—100 and 250—300 kb in length, preceded to typical nuclear DNA fragmentation at the internucleosomal re­ gions. Comparative study of the high molecular weight and internucleosomal DNA fragmentation demonstrated that these show different sensitivity to Zn ions, actinomycin D and cycloheximide. In addition, the formation of large molecular weight frag­ ments in contrast to oligonucleosomal ones may be reverted in conditions promoting to topoisomerase 11 mediated rejoining of cleaved DNA. These finding suggest that the high molecular weight and internucleosomal fragmentation may follow two different program­ mes of DNA cleavage during apoptosis mediated, presumably, by diverse enzumes. We showed that similar processes of ordered high molecular weight DNA cleavage occur in apoptotic cells and in those subjected to the stress challenges. The formation of high molecular weight DNA fragments was demonstrated to proceed promptly, show no correlation with cell death and occassionally may be of transient nature. The data obtained allow to interpret the formation of high molecular weight fragments as a component of cell response to stress rather that an early step of DNA fragmentation du­ ring apoptosis.

We studied the peculiarities of nuclear DNA fragmentation in primary culture of mu rine thymocytes and in human lymphoblastoid cell culture (line СЕМ) induced to apoptosis by various influences.We showed that in apoptotic cells the ordered high molecular weight DNA cleavage, recognizable as DNA fragment release of 50-100 and 250-300 kb in length, preceded to typical nuclear DNA fragmentation at the internucleosomal re gions.Comparative study of the high molecular weight and internucleosomal DNA fragmentation demonstrated that these show different sensitivity to Zn 2+ ions, actinomycin D and cycloheximide.In addition, the formation of large molecular weight frag ments in contrast to oligonucleosomal ones may be reverted in conditions promoting to topoisomerase 11 mediated rejoining of cleaved DNA.These finding suggest that the high molecular weight and internucleosomal fragmentation may follow two different program mes of DNA cleavage during apoptosis mediated, presumably, by diverse enzumes.
We showed that similar processes of ordered high molecular weight DNA cleavage occur in apoptotic cells and in those subjected to the stress challenges.The formation of high molecular weight DNA fragments was demonstrated to proceed promptly, show no correlation with cell death and occassionally may be of transient nature.The data obtained allow to interpret the formation of high molecular weight fragments as a component of cell response to stress rather that an early step of DNA fragmentation du ring apoptosis.I ntroduction.Apoptosis (a programmed cell death) provides a widely spread physiological process, commonly occurring during embryogenesis in established tissues and upon various pathological conditions [1][2][3].It possesses distinctive morphological features.One of the most documen ted biochemical characteristic of apoptosis is the internucleosomal DNA fragmentation to yield oligonucleosomal fragments which upon resolu tion by conventional gel electrophoresis develop typical «ladder» [4][5][6].Lately there is growing evidence to suggest another type of ordered DNA cleavage during apoptosis resulting in appearance of high molecular weight (HMW) fragments of about 30-500 kb, which either precede to or are observed in the absence of internucleosomal fragmentation [7][8][9].This finding provided evidence to suggest that the HMW-DNA fragmen tation rather then internucleosomal one may be a key characteristic of apoptosis [9,10].
In this report we present evidence to show that the ordered nuclear DNA cleavage into the HMW fragments seems to differ by its characte ristics from internucleosomal fragmentation and occur not only during apoptosis, but in the cell subjected to the stress challenges.
Materials and Methods.Cell lines and culture conditi ons.In this work we used the human lymphoblastoma cultured cells (line СЕМ) and primary cell culture of murine thymocytes obtained from the thymus of a 4-5 week old mice (line BALB/c).СЕМ cultured cells were routinely incubated in RPMI 1640 medium supplemented with 10 % fetal calf serum (FCS) in an atmosphere of 95 % air, 5% CO2 to give a final suspension of 210 cell/ml.Thymocyte primary culture was pre» pared from intact thymocytes to final suspension of 2-Ю 7 cells in RPMI ® V. T. SOLOV'yAN, I. O. ANDREEV, 1993 1640/10% FCS and incubated under conditions indicated above for at least 6 h either with or without apoptotic inducers.Apoptosis was indu ced by incubation of cells either with 1 цМ.dexamethasone or 7.5 цМ teniposide (VM 26).The cell viability was estimated by Trypan blue exclusion.
Sample preparation.200 ці of cell suspension were placed into the well of cell culture plate followed by addition of equal volume of 1 % low-melting agarose prepared on TEN-buffer (10 mM Tris-HCl, pH 1.5, 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 fractio nated either conventional or field inversion gel electrophoresis (FIGE) to detect the pattern of nuclear DNA cleavage.Conventional.gelelectro phoresis was carried out in 1.4 % agarose at 50 V for 4-5 h using 0.5X XTBE buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA, pH 8-8.5).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 [11].In some cases FIGE was carried out for 5-6 h al lowing to resolve both low-and HMW DNA.After electrophoresis the gel was stained with 1 mg/ml ethidium bromide for 10 min, viewed using a UV transilluminator and photographed using Mikrat 300 film.
Results and Discussion.Previously we showed that the treatment of agarose-embedded nuclear DNA preparations with the protein denaturants resulted in appearance of two main types of nuclear DNA fragments sized about 50-100 and 250-300 kb, which proved to be unityped for various eukaryote representatives [12].The ordered cleavage of nuclear DNA into the HMW fragments (HMW-fragmentation) seems to be of physiological value since the formation of HMW DNA fragments may be involved in the early events accompanying the programmed cell death.This is evidenced from the data presented in Fig. 1, which show that the dexamethasone induced apoptosis in primary culture of thymocytes is associated with extensive nuclear HMW-DNA cleavage, which precedes to typical for apoptosis internucleosomal DNA fragmentation and is de tectable immediately after cell treatment with dexamethasone.In the ab sence of dexamethasone certain proportion of cells seems to undergo the spontaneous apoptosis accompanied with the HMW-and internucleoso mal fragmentation (Fig. 1).
Incubation of apoptotic thymocytes with Zn 2+ ions, which are known to inhibit the apoptosis specific endonucleases, demonstrates that these completely counteract to the internucleosomal DNA fragmentation but fail to prevent the accumulation of HMW DNA fragments (Fig. 1).Mo reover, Zn 2 + ions seem to increase the dexamethasone induced HMW-DNA cleavage at least at the early stage of apoptosis (Fig. 1).
The fact that the internucleosomal and HMW-DNA fragmentation show various response to Zn 2 + ions suggests that they may follow two different, with regards to their properties, programmes of DNA cleavage during apoptosis.It is supported by the observation that the internucleo somal DNA fragmentation in contrast to the HMW-DNA cleavage is completely prevented by actinomyein D and cycloheximide (results not shown).
In addition, the formation of HMW DNA fragments but not the internucleosomal DNA fragmentation may be reversible.This, however, is to be the case only at the early steps of apoptosis JFig.2).
Similarly to the primary culture of thymocytes, in cultured human lymphoblastoma cells (line СЕМ) the apoptosis to be provoked either by dexamethasone or teniposide is accompanied with progressive DNA cleavage into HMW-DNA fragments of about 250-300 and 50-100 kb with the intensity of fragmentation being correlated with cell death (Fig. 3).However, in control cells (cells non stimulated to apoptosis) the HMW-DNA cleavage is also observed without any relationship with changes in cell viability (Fig. 3).The data presented suggest that the formation of HMW-DNA fragments does not correlates with the Cell death directly and may be observed in cells non stimulated to apoptosis.
As evidenced from the data presented in Fig. 4 the recovery of cells after heat shock is accompanied with the transient increase in HMW- (Fig. 4).Similarly, cell incubation in serum deficient medium is also accompanied with the increase in HMW-DNA fragmentation which ra pidly declines following serum addition and after 24 h incubation with fresh serum disappears (Fig. 5).These data suggest that the formation of HMW-DNA fragments takes place in cells subjected to the stress challenges and is reversible after removal of stress influences.
Thus the data presented indicate that the formation of HMW-DNA fragments occurs at the early stages of apoptosis and seems to differ from the internucleosomal fragmentation with regards to its properties.The HMW-DNA cleavage may be detected immediately after apoptosis induction takes place in normal cells and in those subjected to stress challenges may not correlate with cell viability and seems to be rever sible This allows to interpret the formation of HMW-DNA fragments Fig. 2. The rejoining of clea ved high molecular weight DNA fragments in apoplotic thymocy tesT Cells were treated will 1 u.M dexamelhasone and allo wed to incubate for the time de signatcd at the top of the Fign re (h).After incubation eel's were embedded into agarose (_) or incubated at 55 °С fo 10 min ( + )' followed by agaro se embedding.After gelation cells were treated with SDS and fractionated by FIGE undci conditions allowing to separate both large-and low moleculn weight fragments; C -contro' cells (not treated with dexamethasone), ЛІ-lambda DNA as being not a hallmark of apoptosis but rather as a reaction of cell ge nome to stress challenges.
Our results as well as the data reported earlier [7][8][9][10] demonstra te that the process of programmed cell death is accompanied with the ordered cleavage of nuclear DNA into HMW fragments, which precedes to the internucleosomal DNA fragmentation.At the same time our tindings reveal that the HMW-DNA cleavage and internucleosomal DNA fragmentation seem to differ in regard to their characteristics and may present diverse processes of DNA cleavage during apoptosis.The fact that formation of HMW fragments and internucleosomal DNA cleavage exhibit distinct sensitivity to Zn 2 + ions, actinomycin D and cycloheximide suggests that different enzymes may be involved into these processes.The observation that topoisomerase II specific poison teniposide may modulate the heat-shock mediated turnover of HMW fragments (Fig. 4) suggests the topoisomerase II involvement in HMW-DNA cleavage.This is further confirmed by the fact (Fig. 2) that the HMW-DNA fragmen tation may be reversible in conditions that initiate topoisomerase II me diated DNA cleavage toward religation of cleaved DNA [13][14][15].Our data show that the formation of HMW-DNA fragments takes place both in apoptotic cells and those subjected to stress challenges.
Since any stress influences may interfere with cellular viabili ty one should believe that the increased HMW-DNA cleava ge may reflect the fact that cells become predisposed to the apoptosis.However, our data show that formation of HMW-DNA fragments fails to correlate directly with the changes in cell viability, takes place in normal cells and is reversib le after removal of stress influences.In addition, in apoptotic cells the increased HMW-DNA cleavage may be detected immediately after cell treatment with dexamethasone or Zn 2 + ions (Fig. 1).These findings add credence to the idea that the formation of HMW-DNA fragments repre sents more likely the cellular response to stress than the early step of apoptotic nuclear DNA fragmentation.In this context it is of interest the observation that the loss of capacity to religation of the HMW-DNA fragments at elevated temperature is coincident with oligonucleosomal ladder development during apoptosis (Fig. 2).This can be explained by the loss of topoisomerase II activity at this step of apoptosis due to non specific proteolysis which may occur in apoptotic cells [16,17].Alter native explanation may be that at the early steps of apoptosis the for mation of HMW-DNA fragments, mediated presumably by topoisomera se II, represents the cellular response to apoptotic inductors and may not be directly implicated in «apoptosis-specific» DNA fragmentation, which takes place at the advanced stages of apoptosis coinciding with the development of oligonucleosomal ladder.
Our interpretation of HMW-DNA cleavage is based on previously obtained data showing that fractionation of nonapoptotic lysed nuclear preparation by pulsed field gel electrophoresis results in appearance of subset of discrete DNA fragments correlating with the higher levels of chromatin structural organization [12].In accordance with our early data as well as with those obtained by other workers the fragments of about 50-100 kb may be ascribed to nuclear DNA loop domains [12,18,19].We provided evidence that nuclear DNA structural domains are involved in functioning DNA/topoisomerase complex with its ability to mediate the cleavage/religation equilibrium reactions [20'].
Based on the studies with purified topo II enzymes and DNA the two-stage model has been proposed for topoisomerase II-mediated cle avage/religation reactions [13][14][15].According to this model the enzyme/DNA cleavable complex is the key covalent intermediate in the to poisomerase II mediated DNA turnover, which is in rapid equilibrium with noncleavable complex.The exposure of the cleavable complex but not noncleavable one to protein denaturants (such as SDS or alkali) re sults in cleaved DNA product involving the covalent linking of topoiso merase II subunits to the 5'-ends of broken DNA [13][14][15].
Our and other data reveal that the decisive prerequisite for the HMW-DNA cleavage seems to be the exposition of agarose embedded nuclear and cellular preparations either to protein denaturants (SDS) or to topoisomerase II specific poisons followed by SDS treatment [12,19,20].No DNA fragment releases into gel provided these agents lacking even if nuclei were destroyed with high concentrations of EDTA or Tri ton X-100 [12].Base on the SDS-dependent HMW-DNA cleavage as well as on the previous data suggesting the involvement of DNA structural domain in functioning DNA/topoisomerase complex it seems reasonable to note that the formation of HMW-DNA fragments at the early stages of apoptosis and upon stress challenge should be by no means associated with the real DNA fragmentation.It is appropriate to suggest that in these cases the changes in the integrity J9] of DNA structural domains takes place.In terms of functioning DNA/topoisomerase complex the changes in integrity of DNA structural domains may be interpreted as their transition from noncleavable to cleavable state mediated by topo isomerase II.Whether the specific subset of structural domains is invol ved in these conversions or changes in the integrity of random set of structural domains takes place remains to be elucidated.

Fig. 1 .
Fig. 1.Gel electrophoretic analysis of DNA degradation during dexamethasone induced apoptosis in thymocytes.Cells were treated with 1 u,M dexamethasone and allowed to incubate for the time designated at the top of the Figure (h) without (lanes /-5) or with 1 mM Zn 2 + (lanes 9-14).After incubation cells were embedded into !ow-meltingagarose, treated with SDS (see Materials and Methods) and fractionated either by FIGE (A) or conventional' gel electrophoresis (B).CO -control cells (not treated with dexamethasone) at the beginning of in vitro incubation, C6 -the same cells after 6 h in vitro incubation, M -lambda oligomers.Time of incubation designated on the top of the figure, numbers of lanes designated between the panels A and B, these designa tions are shared by both panere of the figure

Fig. 3 .
Fig. 3.The ordered DNA cleavage du ring apoptosis in cultured СЕМ cells: A -Cells were incubated for the time designated at the top of the Figure (h) without apoptosis inducers (Con) or with 1 uM dexamethasone (Dex) or with 7.5 |лМ teniposide (Ten) (Af ter incubation cells were embedded in to agarose, treated with SDS and fractionated by FIGE; M -lambda oligomers); B -The time course of cell viability estimated by Trypan blue exclusion

Fig. 4 .
Fig. 4. The transient nuclear DNA cleavage in cultured СЕМ cells during heat-shock recovering.Cells were heat-shocked at 42 °С for 10 min followed by incubation at 37 °С for the time designated at the top of Figure (h) without (-) or with 7.5 [iM tenipo side (-)-).Following incubation cells were «mbedded into agarose, treated with SDS and fractionated by FIGE; C--control cells; M -molecular weight standards: lambda ША+HindIII digest of lambda DNA

Fig. 5 .
Fig. 5.The transient nuclear DNA cleavage in cultured СЕМ cells during serum starvation.Cells were incuba ted in serum-free medium (-) for the time indicated at the top of Figure (h).Following starvation serum was added cells were incubated in serumcontaining medium (+) for the time indicated at the top of Figure (h).Af ter incubation cells were embedded into agarose, treated with SDS and fractionated by FIGE; C -control cells before serum starvation В. Т. Солов'ян, I. О. Андреев ФОРМУВАННЯ ВЕЛИКИХ ФРАГМЕНТІВ ДНК: КЛЮЧОВА ХАРАКТЕРИСТИКА АПОПТОЗУ ЧИ РАННЯ ГЕНОМНА ВІДПОВІДЬ НА СТРЕС?