Cryobiology xxx (2010) xxx–xxx

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Cryobiology

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Proteolytic events in cryonecrotic cell death: Proteolytic activationof endonuclease P23 q

Nevena Grdovic *, Melita Vidakovic, Mirjana Mihailovic, Svetlana Dinic, Aleksandra Uskokovic,Jelena Arambašic, Goran PoznanovicDepartment of Molecular Biology, Institute for Biological Research ‘‘Siniša Stankovic”, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 July 2009Accepted 26 January 2010Available online xxxx

Keywords:ProteolysisEndonucleaseNecrosisApoptosisCathepsin D

0011-2240/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.cryobiol.2010.01.005

q This work was funded by the Ministry of Science oNo. 143002B.

* Corresponding author. Fax: +381 11 2761433.E-mail address: nevenag@ibiss.bg.ac.rs (N. Grdovic

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Although cryosurgery is attaining increasing clinical acceptance, our understanding of the mechanisms ofcryogenic cell destruction remains incomplete. While it is generally accepted that cryoinjured cells die bynecrosis, the involvement of apoptosis was recently shown. Our studies of liver cell death by cryogenictemperature revealed the activation of endonuclease p23 and its de novo association with the nuclearmatrix. This finding is strongly suggestive of a programmed-type of cell death process. The presumedorder underlying cryonecrotic cell death is addressed here by examining the mechanism of p23 activa-tion. To that end, nuclear proteins that were prepared from fresh liver, which is devoid of p23 activity,were incubated with protein fractions isolated from liver exposed to freezing/thawing that possessed apresumed p23 activation factor. We observed that the activation of p23 was the result of a proteolyticevent in which cathepsin D played a major role. Different patterns of proteolytic cleavage of nuclear pro-teins after in vitro incubation of nuclei and in samples isolated from frozen/thawed liver were observed.Although both processes induced p23 activation, the incubation experiments generated proteolytic hall-marks of apoptosis, while freezing/thawing of whole liver resulted in typical necrotic PARP-1 cleavageproducts and intact lamin B. As an explanation we offer a hypothesis that after freezing, cells possessthe potential to die through necrotic as well as apoptotic mechanisms, based on our finding that the cyto-sol of cells exposed to cryogenic temperatures contains both necrotic and apoptotic executors of celldeath.

� 2010 Elsevier Inc. All rights reserved.

Introduction

Cryogenic temperatures have a dual effect with respect to bio-logical material – they can preserve as well as destroy. The abilityto preserve is the direct consequence of the complete slowingdown of all biochemical and physiological processes at the temper-ature of liquid nitrogen to the extent that nothing, including dam-age can take place [53]. However, the processes of cooling tocryogenic temperatures and subsequent warming induce damageto cells [19,52]. The dual nature of cryogenic temperatures wereutilized during the last 50 years in refining the procedures usedfor preserving cells and tissues (cryopreservation), as well as tech-niques used for cell destruction by cryosurgery. Cryosurgery is asurgical technique used to destroy tumors and other abnormal tis-sues, based on cell injury induced by freezing. Cryosurgical tech-niques have been improved with technological advances that

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f the Republic of Serbia, Grant

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al., Proteolytic events in cryon

allow for the monitoring and better control of the freezing process[4]. As a result, a better understanding of the mechanisms of celldestruction by cryogenic temperature is warranted.

The three key factors involved in cryoinjury are (1) mechanicaland (2) chemical stress that occur during the freezing process [38]and (3) cellular hypoxia during thawing [28]. Mechanical stress is aconsequence of the formation of ice crystals which disrupt cellmembranes and organelles [49]. As ice is formed from pure waterby the exclusion of electrolytes and organic chemicals, the cells areexposed to a hyperosmotic environment and high concentrationsof electrolytes that also damage cells (solute effect) [37]. Asidefrom the cellular effects, cells in tissues die as a result of vascularinjury and the absence of a blood supply during the post-thawperiod [19]. All of these processes lead to cell death that can bedescribed as necrotic. However, during the past few years, non-ne-crotic mechanisms of freezing-induced injury have been proposed[1,25,54]. Thus, it seems that freezing can also induce an apoptoticresponse, primarily in the peripheral zone of the cryogenic lesionwhere the temperature is not sufficiently low to provoke immedi-ate cell death. The molecular mechanisms of necrotic and apoptoticcell death that are induced by freezing have not been examined in

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detail, although identification of the key players would allow forthe manipulation of cell death pathways and improve the efficacyof cryosurgical treatments.

Apoptosis is a highly organized, physiological process of celldeath regulated by homeostatic mechanisms that provides the safeelimination and disposal of cells. It is triggered and executedthrough an elaborate network of biochemical events and is energydependent [16]. While normal developmental signals and cyto-toxic stimuli primarily elicit apoptosis, acute cell damage resultingfrom accidental exposure of cells to extreme physical damage,toxic insults and severe changes of physiological conditions in-duces a non-apoptotic type of cell death referred to as necrosis.Necrosis does not require the expenditure of cellular energy andis traditionally considered to be a disorganized process. Accumu-lating evidence obtained from studies of cell death occurring underdifferent conditions has brought about a change in this view andnecrosis is increasingly seen as a process that is executed by a con-served program which is supported by a limited set of biochemicalevents [17,55]. Based on our previous results we proposed thateven acute cell death, which is induced by cryogenic temperatures,is accompanied by an ordered sequence of molecular events [22].This assumption was supported by the finding that exposure of li-ver tissue to cryogenic temperatures was accompanied by the acti-vation of an enzyme with an endonucleolytic activity that wasdesignated as endonucleases p23. Endonuclease p23 was charac-terized as a 23 kDa nuclear, Mg2+-dependent, sequence non-spe-cific endonuclease which could participate in DNA degradationduring cell death [23]. It was shown that during in vivo andex vivo cryonecrosis, respectively, induced by repeated freezingand thawing of liver tissue in an anesthetized rat or after thaw-ing of liver frozen in liquid nitrogen, the enzyme became activeand established a stable association with the proteinaceousfoundation of the nucleus, termed the nuclear matrix [22]. Inview of the observed activation of p23 during cryonecrosis, weproposed that rat liver cell death in response to acute cell dam-age exhibits an inherent, programmed response in which p23plays an important role. What was missing from our work thatwould further support this assumption was the mechanism ofp23 activation.

As p23 activity was completely inhibited in the presence of cat-ion concentrations that resembled the in vivo state in nuclei, wesuggested that the enzyme is maintained in an inactive state underphysiological conditions [23]. Changes in cellular organelle andplasma membranes that occur during necrosis cause alterationsin cellular ion concentrations and pH [40,48]. Thus, it is possiblethat alterations of the intracellular milieu affect p23 activity. How-ever, it does not seem likely that the activity of an enzyme that ispotentially dangerous to the cell is solely regulated at this level.Proteolysis represents the main mechanism of regulation of activ-ities of most proteins that play specific roles in the apoptotic pro-cess and initiation of the proteolytic cascade during apoptosisserves to either activate or inactivate key proteins, depending ontheir role in cell death [10]. Given that proteolysis is also impli-cated in necrotic cell death, although its exact role, the proteasesinvolved and the target proteins remain unclear [3], we assumedthat activation of p23 during cryo-induced injury was mediatedby a proteolysis.

To obtain an insight into the upstream molecular events thatcould be responsible for the activation of p23, in the ex vivo modelsystem of liver cell necrosis induced by exposure of liver to cryo-genic temperatures, we performed a series of experiments inwhich different cellular fractions were examined for the presenceof a proteolytic activity capable of activating p23. Identificationof a protease involved and the molecular events that result in acti-vation of p23 would contribute to better understanding of the pro-cess that underlies cryo-induced cell death.

Please cite this article in press as: N. Grdovic et al., Proteolytic events in cryon(2010), doi:10.1016/j.cryobiol.2010.01.005

Materials and methods

Animals

All animal procedures were approved by the Committee for Eth-ical Animal Care and Use of the Institute for Biological Research,Belgrade, which acts in accordance with the Guide for the Careand Use of Laboratory Animals published by the US National Insti-tute of Health (NIH Publication No. 85/23, revised 1986). Male 30-day-old albino rats of the Wistar strain (Rattus norvegicus) wereused. The animals were kept at constant temperature, humidityand dark/light intervals. The rats were starved for 24 h before tothe experiments. They were killed by decapitation, the liver wasremoved and processed as indicated below.

Liver treatments

Whole excised liver was frozen in liquid nitrogen and subse-quently thawed for 5 min at 37 �C. Freezing and thawing cycleswere performed three times as will be indicated, in order to com-plete the process of cryonecrosis [22]. Control homogenates wereprepared from fresh liver immediately after excision.

Isolation of the rat liver nuclei and nuclear matrices

All buffers contained 1 mM phenylmethyl sulfonyl fluoride(PMSF), a serine protease inhibitor. The livers were homogenizedin 0.25 M sucrose, 50 mM Tris–HCl (pH 7.4), 5 mM MgSO4 andthe nuclei were isolated and purified by ultracentrifugation ofthe homogenate in sucrose as described previously [31]. The iso-lated nuclei were incubated with soluble proteins as will be indi-cated or processed for nuclear matrix isolation. Nuclear matriceswere isolated essentially as described previously [42]. Freshly iso-lated nuclei were stabilized by incubation at 42 �C for 20 min. Thenuclei were then treated with 2 mM Na-tetrathionate (1 h, 4 �C, in0.25 M sucrose, 50 mM Tris–HCl (pH 7.4), 5 mM MgSO4) andwashed twice with the same buffer without Na-tetrathionate. Nu-clei were incubated in 0.25 M sucrose, 50 mM Tris–HCl (pH 7.4),5 mM MgSO4 overnight at 4 �C to allow endogenous nucleasedigestion and subjected to consecutive extraction/centrifugationsteps: twice with high-salt buffer (2 M NaCl, 10 mM Tris–HCl (pH7.4), 0.2 mM MgSO4), once with freshly prepared 1% Triton X-100in the same buffer without NaCl, followed by two washes with10 mM Tris–HCl (pH 7.4), 0.2 mM MgSO4. The morphologicalintegrity of the isolated nuclear matrix structures was checkedby light microscopy.

Isolation of cytosol proteins

The livers were minced and homogenized in buffer containing2 M sucrose, 10 mM Hepes (pH 7.6), 25 mM KCl, 5 mM MgCl2,1 mM EDTA (pH 8.0), 1 mM spermidine, 1 mM PMSF, 1 mM DTT.After ultracentrifugation (72,000g, 30 min, 4 �C, Beckman SW-28rotor), the supernatant was collected and subjected to ultracentri-fugation (82,000g, 60 min, 4 �C, Beckman Ti-50 rotor). The cytosolsupernatant was collected, frozen in small aliquots and stored at�80 �C until use.

Isolation of total nuclear lysates

Purified nuclei were pelleted by centrifugation (1000g, 10 min,4 �C) and resuspended in lysis buffer that contained 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.2 mM PMSF,1 lg/ml aprotinin, 1 lg/ml leupeptin, 1 lg/ml pepstatin. After incu-bation (4 �C, 30 min), the lysate was centrifuged (10,000g, 15 min at

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4 �C). The obtained supernatant (nuclear lysate) was dialyzedagainst Tris–HCl (pH 7.4), frozen in small aliquots and stored at�80 �C until use.

Incubation of nuclear and nuclear matrix proteins with differentsoluble fractions (in vitro activation of p23)

One hundred micrograms of nuclear or nuclear matrix proteinswas incubated with 200 lg of either cytosol or nuclear lysate pro-teins for 30 min at 30 �C, in incubation buffer that contained50 mM Tris–HCl (pH 7.4), 5 mM CaCl2, 0.5 mM EDTA. After theincubation step, nuclear or nuclear matrix proteins were pelletedby centrifugation at 5000g, for 10 min at 4 �C and subjected toactivity gel analysis. In experiments in which the effect of proteaseinhibition was tested, preincubation of 200 lg of cytosol proteinswith a cocktail of protease inhibors (aprotinin 25 lg/ml, pepstatin10 lg/ml, PMSF 2 mM and leupeptin 25 lg/ml), for 15 min at roomtemperature in the incubation buffer was preceded the incubationwith nuclear proteins.

Activity gel analysis

Nuclease activities in the protein fractions were detected byactivity gel analysis or zymography. The proteins were electropho-retically separated in 10% SDS–polyacrylamide gels containing100 lg/ml salmon sperm DNA. After electrophoresis the gels werewashed five times with 50 mM Tris–HCl (pH 7.0) containing 1 mMDTT at 4 �C, and incubated in the same buffer at 4 �C overnight toallow the renaturation of proteins. To activate nuclease activities,the gels were incubated for 24 h at 37 �C in the same buffer withthe addition of 1 mM MgCl2. The gels were then stained with ethi-dium bromide for 60 min. After illumination of the gels with UVlight, nuclease activities were observed as dark areas on a fluores-cent background.

Comet assay (SCGE-single cell gel electrophoresis)

For detection of DNA fragmentation associated with the pro-cesses of freezing and thawing, an alkaline comet assay was used[45]. Liver samples were minced in ice-cold HBSS buffer (0.14 g/lCaCl2, 0.4 g/l KCl, 0.06 g/l KH2PO4, 0.1 g/l MgCl2�6H2O, 0.1 g/lMgSO4�7H2O, 8.0 g/l NaCl, 0.35 g/l NaHCO3, 0.09 g/l Na2HPO4�7H2O,1.0 g/l D-glucose) containing 20 mM EDTA and 10% DMSO. Thesolution was aspirated from the suspension and fresh solutionwas added. The procedure was repeated three times and the finalcell suspension was collected. Ten microliters of the cell suspen-sion was mixed with 75 ll 1% low melting point agarose (LMPA)in PBS (pH 7.4) buffer (20 mM Na2HPO4, 10 mM KCl, 70 mMKH2PO4, 340 mM NaCl, pH 7.4) and placed onto an agarose coated(1.5% agarose in PBS buffer) microscope slide and covered withcover-glass. After complete solidification (10 min), the cover-glasswas removed and the third protective layer of 75 ll 1% LMPA waspipetted onto the slide. Coverslips were placed and removed aftersolidification. The microscope slides were immersed in cold lysissolution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, 10% DMSO, pH 10.0) and incubated for 2 h at 4 �C. After lysis,the slides were placed in a horizontal electrophoresis unit andincubated for 30 min at 4 �C in electrophoresis buffer (300 mMNaOH, 1 mM EDTA, pH 13.0) before electrophoresis (0.01 kV,139 mA, 30 min). After electrophoresis, the slides were placed inneutralization buffer (0.4 Tris–HCl (pH 7.4)), incubated three timesfor 5 min and subsequently stained with SYBR GREEN I (Sigma–Al-drich). Comets were visualized and captured with a 40� objectivelens mounted on a fluorescence microscope (Leica DMLB with at-tached CCD camera). One hundred comet images per slide wererandomly captured and analyzed by a visual scoring method

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[13]. The images were classified into five categories, defined astypes 0, 1, 2, 3 and 4; 0 indicates no or very low damage, 1, 2and 3 indicate low, medium and long DNA migration, respectively,and 4 indicates the highest level of DNA degradation (presented ascomets with very small heads and long tails). Images were ana-lyzed with TriTekCometScore™ Freeware v1.5 (available at:http://www.AutoComet.com). The tail moment (expressed in arbi-trary units) was selected as a parameter for estimating the extentof DNA degradation as it includes both the tail length and percentof DNA in the comet tail.

LDH (lactate dehydrogenase) release assay

Liver samples (600 mg) were homogenized in cold 0.1 M potas-sium-phosphate buffer, pH 7.4, at 4 �C. The buffer was added toyield a concentration of 30 mg tissue/ml. The homogenates werecentrifuged at 14,000g for 10 min at 4 �C and the supernatantwas examined for LDH activity by a colorimetric assay. Ten micro-liters of supernatant was diluted to 100 ll with 0.1 M potassium-phosphate buffer, pH 7.4, and then mixed with the same volume(100 ll) of staining solution composed of 54 mM calcium L-lactatehydrate, 0.66 mM iodonitrotetrazolium chloride, 0.28 mM phena-zine methosulfate and 1.3 mM NAD in 0.1 M potassium-phosphatebuffer, pH 7.4. The staining solution was freshly prepared and allthe staining procedures were performed in the dark at room tem-perature. The stain was developed during 5 min incubation in thedark and the absorbance was recorded at 492 nm. LDH release innecrotic samples was estimated with respect to the level of the en-zyme released in the control sample, as well as to the total amountof enzyme obtained after homogenization of the control sample inthe presence of 3% Triton X-100.

Genomic DNA isolation

Liver (200 mg) was minced with scissors and homogenized in10 mM Tris–HCl (pH 8.0), 100 mM NaCl and 25 mM EDTA (pH8.0). Proteinase K and SDS were added to the homogenate to finalconcentrations of 100 lg/ml and 0.5%, respectively, and incubatedovernight at 50 �C. DNA was purified by extraction once with phe-nol/chloroform/isoamyl alcohol (25:24:1) and once with chloro-form/isoamyl alcohol (24:1). The extraction was repeated twiceand DNA was precipitated with 1.5 volumes of ethanol overnightat �20 �C. After centrifugation, the pellet containing RNA andDNA was resuspended in TE (10 mM Tris–HCl, 1 mM EDTA (pH8.0)) buffer and incubated with RNase (200 lg/ml) for 2 h at roomtemperature, followed by an incubation with proteinase K (200 lg/ml) for 2 h at 37 �C. Finally, the mixture was re-extracted with phe-nol/chloroform/isoamyl alcohol, precipitated with ethanol, andresuspended in TE buffer. The DNA was separated electrophoreti-cally on a 1.3% agarose gel in 0.045 M Tris–Borate, 0.001 M EDTA(TBE) buffer for 3.5 h. The gel was stained with ethidium bromide.

Zymogram of protease activity

The presence of protease activities in the cytosol protein frac-tion was examined by a protease activity zymogram. Fifty micro-grams of cytosol proteins was subjected to electrophoresis underdenaturing conditions using 12% acrylamide gels containing 0.2%gelatin. The gelatin solution was made up as a 2% stock solutionin distilled water and dissolved by heating. Protein samples wereapplied to the gel in standard SDS-loading buffer containing 0.1%SDS without 2-mercaptoethanol. The samples were not boiled be-fore loading. The gels were run at 200 V for 1 h. Following electro-phoresis, the gel was washed for 1 h at room temperature with2.5% Triton X-100 and then incubated overnight in activation buf-fer (50 mM Tris–HCl, pH 7.6, 0.2 M NaCl, 5 mM CaCl2). Hydrolysis

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of gelatin was detected by staining with Coomassie Brilliant BlueR-250 followed by destaining in methanol/acetic acid/water(5:5:1).

SDS–polyacrylamide gel electrophoresis and Western immunoblotanalysis

Protein concentrations were determined as described [34].Twenty micrograms of proteins was loaded onto a 4% stacking/12% separating slab gel [32]. The gels were stained with CoomassieBrilliant Blue R-250 or electroblotted onto a polyvinylidene difluo-ride (PVDF) membrane. Western immunoblot analysis was per-formed using mouse monoclonal anti-cytochrome c antibody(Santa Cruz Biotechnology, A-8), rabbit polyclonal anti-caspase-3antibody (Santa Cruz Biotechnology, H-277), rabbit polyclonalanti-cleaved caspase-3 antibody (Cell Signaling, #9661), goat poly-clonal anti-lamin B antibody (Santa Cruz Biotechnology, M-20) andrabbit polyclonal anti-PARP-1 antibody (Santa Cruz Biotechnology,H-250). Staining was performed by the chemiluminescent tech-nique according to the manufacturer’s instructions (Santa CruzBiotechnology).

Cathepsin D activity assay

Cathepsin D enzymatic activity in the cytosol was assessed byan activity assay with hemoglobin as a substrate. The reaction mix-ture contained 44 mM citrate buffer (pH 2.8) and 1.1% hemoglobinsolution in deionizied water, equilibrated at 37 �C and adjusted topH 3.0. One milligram of cytosol proteins was then added and thereaction mixture was incubated 10 min at 37 �C. The enzymaticreaction was stopped by adding an equal volume of 5% trichloro-acetic acid. After centrifugation, absorbance of the supernatantcontaining hemoglobin peptides was determined at 280 nm. A unitof cathepsin D activity is defined as the amount of enzyme causingan absorbance change of 1.0 at 280 nm per min under thedescribed experimental conditions. Cathepsin D activity wasexpressed as units of cathepsin D activity per milligram of totalprotein (U/mg).

Fig. 1. Biochemical events that accompany cell destruction after thawing of liver exposfrom control (fresh) liver (lane 1), liver after one (lane 2) and three cycles of freezing a(estimated with respect to the level of the enzyme released in the control sample as wsample in the presence of 3% Triton X-100). (C) Agarose gel electrophoresis of genomic DNof freezing and thawing (lane 3).

Please cite this article in press as: N. Grdovic et al., Proteolytic events in cryon(2010), doi:10.1016/j.cryobiol.2010.01.005

Results

Previously, we described the morphological changes and bio-chemical events that accompany necrotic cell destruction afterthawing whole liver frozen in liquid nitrogen [22]. Essentially thesame molecular events were observed in the course of necrotic celldeath in vivo, induced by repeated freezing and thawing of a part ofthe liver tissue in an anaesthetized rat. Typically, a 23 kDa Mg2+-dependent and sequence non-specific, nuclear matrix-associatedendonuclease (p23) that was characterized earlier [23] became ac-tive. One cycle of freezing and thawing promoted the appearanceof p23 activity. Subjecting the liver to three freezing/thawing cy-cles did not cause further increase in activity of the enzyme, basedon activity gel analysis of nuclear matrix proteins isolated fromfresh liver (Fig. 1A, lane 1) and liver exposed to freezing/thawingcycles (lanes 2 and 3). The appearance of endonucleolytic activitywas accompanied by an increase of LDH activity in liver samplesthat were subjected once or three times to cryogenic temperaturesand subsequently thawed (Fig. 1B). Since LDH is a cytoplasmic en-zyme whose release provides an overall assessment of plasmamembrane integrity, it would seem that the activation of p23 cor-related with cell death.

The observed activation of an enzyme with endonucleolyticactivity implies its involvement in DNA degradation that generallyaccompanies the cell death process. However, when the total DNAfrom livers exposed once or three times to freezing and thawingwas examined by agarose gel electrophoresis, extensive DNA deg-radation was not observed (Fig. 1C), although a slight, high molec-ular weight smear was present in the sample obtained fromexcised liver that was subjected to three cycles of freezing andthawing (Fig. 1C, lane 3). Since the employed agarose gel electro-phoresis detects double-strand DNA breaks, the possibility thatfreezing mostly led to single-strand breaks was examined by thealkaline comet assay (Fig. 2). Examination of cells obtained fromfresh liver and livers exposed to one or three cycles of freezing/thawing revealed that freezing induced DNA damage. Accordingto appearance, five standard classes of comets were observed andscored visually as described [13] (Fig. 2A). In fresh liver cells, thegreat majority of analyzed cells (about 86%) were without DNA

ed to cryogenic temperatures. (A) Activity gel analysis of nuclear matrices isolatednd thawing (lane 3). (B) Assessment of cell membrane destruction by LDH releaseell as to the total amount of enzyme obtained after homogenization of the controlA isolated from control (fresh) liver (lane 1), liver after one (lane 2) and three cycles

ecrotic cell death: Proteolytic activation of endonuclease P23, Cryobiology

Fig. 2. Comet assay of hepatocytes from fresh liver and livers after one or threecycles of freezing and thawing. (A) Comet scoring; (B) representative images ofcomets obtained from fresh liver (a), liver after one (b) or three cycles of freezingand thawing (c). (C) An estimate of the extent of DNA degradation from the tailmoment.

Fig. 3. In vitro activation of p23 and its subsequent association with the nuclearmatrix. Activity gel analyses of pellet fraction after incubation of nuclei isolatedfrom control liver with cytosol fractions isolated from livers that were frozen/thawed once (lane 2), three times (lane 3) and with total lysates of nuclei isolatedfrom livers that were frozen/thawed once (lane 4) and three times (lane 5). Nucleiincubated in incubation buffer (lane 1). Activity gel analyses of pellet fraction afterincubation of nuclei isolated from control liver with the cytosol isolated from liverthat was frozen/thawed once (lane 6); with a cytosol fraction preincubated with acocktail of protease inhibitors (lane 7). Nuclear matrices isolated from nucleiobtained after incubation with cytosol proteins (lane 8) and cytosol proteins pre-treated with protease inhibitors (lane 9).

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tails and were accordingly ascribed a value of 0 (Fig. 2A and Ba).Exposure to cryogenic temperatures and subsequent thawing ofwhole liver resulted in a nearly equal distribution of all five classesof comets, reflecting different degrees of DNA damage (Fig. 2A).The comet from this sample, categorized as class 2, is shown onFig. 2Bb. After three rounds of freezing and thawing almost the en-tire DNA was present in the comet tails. The prevalence (about85%) of class 4 comets showed that the DNA was extensivelydamaged in these samples (Fig. 2A and Bc). Comparison of the tailmoments, the product of the tail length and tail intensity (Fig. 2C),confirmed that DNA damage progressively increased as whole liverwas exposed to additional cycles of freezing and thawing.

Since proteolysis is a common feature of cell death, it is conceiv-able that endonuclease p23 was activated by proteolysis. As p23activity was only observed in different nuclear fractions and sinceit was to a great extent associated with the nuclear matrix [22], itsactivation most probably took place in the nucleus. Two possiblepathways for its activation were considered: (i) that p23 resides

Please cite this article in press as: N. Grdovic et al., Proteolytic events in cryon(2010), doi:10.1016/j.cryobiol.2010.01.005

in an inactive state as a nuclear matrix-bound protein that be-comes activated by an endogenous protease likewise activated byexposure to the physical insult associated with cryogenic temper-atures; (ii) that p23 resides in an inactive state as a soluble proteinand translocates to the nuclear matrix upon activation. Theseassumptions were examined by incubating nuclear matrices andisolated nuclei that were prepared from fresh liver (and therebydevoid of p23 activity), with different soluble protein fractionscontaining proteases that could potentially be responsible for theactivation of a preexisting endonuclease (Fig. 3). The nuclei and nu-clear matrices were, respectively, incubated with either completecytoplasmic or total nuclear lysate proteins prepared from liversthat were exposed to either one or three freezing/thawing steps.The presence of p23 was assessed by activity gel analysis. Endonu-clease p23 activity was not detected on the nuclear matrix (resultsnot shown) but was observed in nuclei that were incubated withcytosol proteins prepared from liver exposed to one freezing/thaw-ing cycle (Fig. 3, lane 2). A slight endonucleolytic activity was alsoobserved after incubation of nuclei with the nuclear lysate pre-pared from liver after a single freezing/thawing (lane 4). We con-cluded that the appearance of p23 was the result of action of anactivation factor present in the cytosol since incubation of isolatednuclei in incubation buffer did not induce p23 activation (lane 1). Itshould be noted that p23 activity was not observed in nuclei incu-bated either with the cytosol or with the total nuclear lysate pre-pared from livers exposed to three freezing/thawing cycles (lanes3 and 5, respectively). Presumably the activation factor had be-come inactivated by repeated exposure to freezing and thawingcycles.

To establish whether the activation of p23 was the result of theactivity of an endogenous protease(s) present in the cytosol frac-tion, the cytosol was treated for 15 min at 30 �C with a proteaseinhibitor mix containing aprotinin, leupeptin, pepstatin and PMSFand then incubated with nuclei prepared from fresh liver as de-scribed above. After the incubation, the nuclei were washed andsubjected to activity gel analysis. Nuclei that were incubated withthe untreated cytosol fraction acquired an associated p23 activity(Fig. 3, lane 6) whereas the nuclei that were incubated with thecytosol fraction that was pre-treated with protease inhibitors were

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devoid of any associated p23 activity (lane 7). This result revealedthat a proteolytic activity that was present in the cytosol fractionwas responsible for the activation of p23. Since the relative abun-dance of p23 in the total nuclear protein population is lower thanin the nuclear matrix, the nuclei from the above experiment, i.e.nuclei that were incubated with cytosol fractions that were un-treated and nuclei that were incubated with the cytosol fractionspre-treated with protease inhibitor, were processed for the nuclearmatrix that was subsequently examined by activity gel analysis.Endonuclease p23 activity was observed in nuclear matrices thatwere prepared from nuclei incubated with the untreated cytosol(Fig. 3, lane 8). In nuclear matrices prepared from nuclei that wereincubated with the protease inhibitor-treated cytosol fraction, p23activity was absent (lane 9). This result clearly showed that an ex-tra-nuclear, cytosolic protease was responsible for the activation ofendonuclease p23 during cryonecrosis and confirmed that after itsactivation p23 translocated to the nuclear matrix. The proteaseinhibitor that prevented the activation of p23 was identified afterincubating the nuclei isolated from fresh liver with cytosolproteins that were separately pre-treated with either aprotinin,leupeptin, pepstatin or PMSF, isolating the respective nuclear

Fig. 4. Characterization of proteolytic events that accompany p23 activation. (A) Activit(lane 1); cytosol proteins prepared from liver after one cycle of freezing and thawing (thawing (lane 3); nuclear matrix proteins isolated from nuclei that were incubated withcytosol fraction preincubated with a cocktail of protease inhibitors (lane 5). Nuclear mapreincubated with aprotinin (lane 6), leupeptin (lane 7), pepstatin (lane 8) and PMSF (la

Please cite this article in press as: N. Grdovic et al., Proteolytic events in cryon(2010), doi:10.1016/j.cryobiol.2010.01.005

matrices and subsequently testing them for the presence of p23activity by activity gel analysis (Fig. 4A). Aprotinin, leupeptin andPMSF did not affect p23 activation (lanes 6, 8 and 9, respectively),while pepstatin completely inhibited the appearance of nuclearmatrix-associated p23 activity (lane 7).

In order to identify the proteolytic process that occurred duringthe in vitro incubation, Western analysis with antibodies to lamin Band PARP-1 proteins was performed (Fig. 4B). Lamin B and PARP-1are nuclear matrix proteins that are widely used as biochemicalmarkers that can distinguish between apoptosis and necrosis astheir proteolysis results in cell death type-specific proteolytic frag-ments. The proteolytic patterns of nuclear matrix proteins ob-tained after in vitro incubation experiments were compared withthe degradation pattern of the nuclear matrix isolated from fro-zen/thawed liver that was previously described as necrotic [22].The nuclear matrix that was isolated from liver after one cycle offreezing and thawing contained the full-length PARP-1 (116 kDa)molecule, as well as the typical 72 and 50 kDa necrotic cleavageproducts (lane 3). The main proteolytic fragment that was ob-served in the nuclear matrix isolated from nuclei that were incu-bated with cytosol proteins from the liver subjected to cryogenic

y gel analyses; (B) Western immunoblot analysis. Nuclei isolated from control liverlane 2); nuclear matrix proteins isolated from liver after one cycle of freezing and

cytosol proteins isolated from liver that was frozen/thawed once (lane 4) and thetrix proteins obtained from nuclei after incubation with cytosol proteins separatelyne 9).

ecrotic cell death: Proteolytic activation of endonuclease P23, Cryobiology

Fig. 5. Proteolytic activities in the cytosol after exposure of the liver to cryogenictemperatures. (A) Zymogram of protease activities of cytosol proteins isolated fromcontrol liver (lane 1) and after one cycle of freezing and thawing (lane 2). (B)Western analysis of cytosol proteins isolated from control liver (lane 1) and afterone cycle of freezing and thawing (lane 2). (C) Cathepsin D activity assay of cytosolproteins isolated from control and liver after one cycle of freezing and thawing.

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temperatures was the apoptotic 89 kDa fragment whereas thenecrotic fragments were present at relatively lower concentrations(lane 4). Virtually the same proteolytic patterns were detected innuclear matrices prepared from nuclei incubated with the cytosolfractions that were separately pre-treated either with aprotinin,leupeptin or PMSF (lanes 6, 8 and 9, respectively). After incubationof nuclei with cytosols that were pre-treated either with a cocktailof protease inhibitors or with pepstatin alone, the isolated nuclearmatrices contained predominantly necrotic fragments of PARP-1while the presence of the apoptotic 89 kDa product was signifi-cantly reduced (lanes 5 and 7). Western analysis with anti-laminB antibody revealed a similar course of events. Namely, in the nu-clear matrix that was isolated from liver after freezing/thawinglamin B was intact, which is typical for the necrotic process (lane3), while the nuclear matrix that was obtained after the in vitroincubation experiment, aside from the full-length protein(66 kDa), also contained the 45 kDa lamin B apoptotic fragment(lane 4). This cleavage product was also present in the nuclearmatrices isolated from nuclei that were incubated with cytosolsseparately pre-treated either with aprotinin, leupeptin or PMSFseparately (lanes 6, 8 and 9, respectively). However, pretreatmentsof cytosol fractions either with the protease inhibitor cocktail orwith pepstatin were sufficient for complete loss of the apoptoticfragment in the nuclear matrices (lanes 5 and 7). To a certain ex-tent these results are inconsistent with the assumption that endo-nuclease p23 is involved in the necrotic process. Although theactivation of p23 was enabled during the in vitro incubation of nu-clei from fresh liver with cytosol proteins from frozen/thawed li-ver, this particular incubation was accompanied by proteolysiswith a hallmark of apoptosis rather than of necrosis. In contrast,freezing and thawing whole liver induced necrotic proteolyticevents and activation of endonuclease p23.

While the apoptotic process has been extensively studied andthe key players involved in its initiation and execution have beenidentified, the biochemical mechanisms underlying the necroticprocess still remain obscure. In order to clarify the proteolytic pro-cesses that had occurred in the in vitro incubation system and inwhole liver during freezing and thawing, proteins from the cytosolfraction were analyzed for the presence of established factors ofcell death pathways. Screening for the presence of protease activityin cytosol fractions obtained from fresh and liver exposed to onecycle of freezing and thawing revealed that the freezing processbrought about qualitative and quantitative changes (Fig. 5A). Per-forming a protease activity zymogram under non-reducing condi-tions that enable the detection of proteases requiring neutral pHfor their activity revealed the presence of only two higher molecu-lar weight proteases in the cytosol prepared from fresh liver(Fig. 5A, lane 1), while the cytosol from frozen/thawed liver exhib-ited additional protease activities beside the increased activity ofpreexisting proteases (lane 2). Under non-reducing conditions re-quired for the protease activity zymogram, the observed proteo-lytic activities could not be linked to precise molecular masses.

The presence of apoptotic proteolytic fragments of PARP-1 andlamin B in the nuclear matrix obtained from nuclei after the in vitroincubation indicates that the apoptotic machinery was induced inliver cells after freezing/thawing. This was confirmed after Wes-tern analysis of cytosol proteins obtained from fresh and frozen/thawed liver with antibody to cytochrome c (Fig. 5B). Cytochromec was detected in the cytosol prepared from liver after freezing/thawing (lane 2) and was absent in the cytosol obtained from freshliver (lane 1). The direct consequence of cytochrome c release fromthe mitochondria is initiation of the cascade of proteolytic eventsand activation of effector caspases. Western analysis with antibodyto caspase-3 (Fig. 5B) revealed a prominent decrease of the level ofprocaspase-3 in the cytosol isolated from liver after freezing/thaw-ing in comparison with the cytosol from fresh liver (lanes 1 and 2).

Please cite this article in press as: N. Grdovic et al., Proteolytic events in cryon(2010), doi:10.1016/j.cryobiol.2010.01.005

The reduction in procaspase-3 was accompanied by an increase ofthe cleaved, activated caspase-3 fragment (Fig. 5B, lane 2).

The necrotic cleavage products are the result of activation of anentirely different proteolytic system presumably involving thecathepsins, lysosomal proteases. Since necrotic PARP-1 cleavageproducts were observed in the nuclear matrix that was isolatedfrom liver exposed to freezing/thawing, as well as in the nuclearmatrix prepared from nuclei after the in vitro incubation with cyto-sol proteins that were pre-treated either with the protease inhibi-tor cocktail or pepstatin alone, our aim was to determine whetherthe cathepsins play an important role in proteolytic processesassociated with freezing. As pepstatin was the only protease inhib-itor that exhibited an effect on proteolysis, we focused our atten-tion on cathepsin D which is completely inhibited in its presence.The activity of cathepsin D in the cytosol from fresh and liver thatwas exposed to freezing/thawing was examined by the cathepsin Denzymatic activity assay (Fig. 5C). That freezing and thawing had acritical effect on lysosomal membrane integrity was revealed bythe finding that the activity of cathepsin D in the cytosol from fro-zen/thawed liver was almost fourfold higher compared to its activ-ity in the cytosol from fresh liver. It can be concluded that freezing/thawing activated both apoptotic and necrotic effectors, making itdifficult to conclude which process was the exclusive executor ofcell death induced by freezing.

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Discussion

During the past 20 years many enzymes with a non-specificendonucleolytic activity have been described and characterizedand for many the involvement in apoptotic DNA degradation hasbeen proposed [33]. Yet, there is no single enzyme that can be con-sidered the endonuclease that cleaves DNA during necrosis. A pos-sible explanation is the generally accepted view that necrotic DNAdegradation is random, the result of lysosomal membrane destruc-tion and activation of non-specific lysosomal nucleases [26]. Anal-ysis of the structure of DNA breaks during the early stages ofnecrosis (including cryonecrosis) suggests that necrotic DNA deg-radation is a non-random event and that a specific nucleolyticactivity is involved [15,27]. We described a nuclear matrix-associ-ated endonuclease p23 [23] in rat liver and based on its activationduring in vivo cryonecrosis and after exposure of the whole liver tocryogenic temperatures, we put forward the assumption that p23was involved in cryonecrotic cell death [22].

The activation of endonuclease p23 after freezing/thawing wasaccompanied by DNA degradation, a fundamental aspect of celldeath. The pattern of DNA degradation was examined by agarosegel electrophoresis (Fig. 1C) which allows for the detection of frag-ments resulting from double-strand breaks, and by the alkaline co-met assay (Fig. 2) which is used for the detection of both single-and double-strand breaks [45]. The presence of clearly delineatedcomets in the treated samples, together with the absence of lowmolecular weight DNA degradation after agarose gel electrophore-sis, points to important conclusions. First, DNA cleavage afterfreezing/thawing resulted in mainly high molecular weight frag-ments. Had the DNA undergone fragmentation to oligonucleoso-mal or other types of low molecular weight fragments, therelatively small pieces of DNA would have readily disappeared dur-ing the comet assay, leaving behind empty ‘‘ghosts” of cells withonly a small percentage of remaining residual DNA fluorescence[13]. Also, low molecular weight fragments would have been ob-served after agarose gel electrophoresis. The presence of highmolecular weight DNA fragments can be correlated with the nucle-ar matrix localization of p23. Higher order chromatin organizationis established through the association of specific DNA regions withthe nuclear matrix [35] and activation of a nuclear matrix-associ-ated endonuclease is expected to generate high molecular weightDNA fragments. The second conclusion drawn from the results ofthe comet assay and agarose gel electrophoresis was that DNA deg-radation was to a great extent the result of single-strand breaks.The non-appearance of DNA degradation in the treated samplesafter agarose gel electrophoresis points to the complete absenceof double-strand breaks in the DNA while the comet assay clearlyshowed extensive DNA degradation which can therefore be con-cluded resulted from single-strand breaks. One of the major differ-ences between apoptosis and necrosis is the mode of DNAcleavage. Apoptosis is accompanied by double-strand DNA breakswhile DNA cleavage during necrosis is characterized by single-strand breaks [21]. The pattern of DNA degradation after freez-ing/thawing is characterized by the presence of single-strandbreaks and can be described as necrotic. These findings supportthe idea that p23, as an endonuclease capable of introducing sin-gle-strand breaks into double-stranded DNA [23], is an active par-ticipant in the cryonecrotic process.

Cryonecrosis has been considered to be a completely disorga-nized process and therefore the molecular mechanisms underlyingit have not been examined in detail. Our aim was to examine, bystudying p23 activation, the main factors or processes implicatedin cryonecrotic cell death. The molecular pathway of p23 activationduring cryonecrosis of rat liver cells was examined by compara-tively simple procedures based on the incubation of the nuclearproteins that were prepared from fresh liver (devoid of p23

Please cite this article in press as: N. Grdovic et al., Proteolytic events in cryon(2010), doi:10.1016/j.cryobiol.2010.01.005

activity), with different soluble protein fractions which potentiallycontained a p23 activation factor. The appearance of p23 in nucleiafter the incubation with the cytosol that was isolated from liverexposed to freezing/thawing suggests that the factor responsiblefor the activation of p23 resided in the cytosol (Fig. 3, lane 2).The potential activator of p23 most probably translocates to thenucleus during cryonecrosis since slight p23 activity was also ob-served after the incubation of nuclei with the nuclear lysate thatwas prepared from the liver after a single freezing/thawing step(Fig. 3, lane 4). As the presence of active p23 in the nucleus wascompletely dependent on the pretreatment of cytosol proteinswith a cocktail of protease inhibitors (Fig. 3, lanes 8 and 9), themain conclusion from these in vitro activation experiments wasthat the activation of endonuclease p23 was the result of a prote-olytic event.

Proteolytic processes execute both apoptotic and necrotic pro-cesses [2]. Activation of the caspase proteolytic machinery duringapoptosis does not lead to an indiscriminate digestion of all cellu-lar proteins. It leads to the proteolysis of particular target proteinsand consequently either to their activation or inactivation [10].Proteolytic cleavage leads to defined biochemical and morpholog-ical changes that are typical for apoptosis. The molecular mecha-nisms that induce the activation of proteases in necrosis and thedownstream cascades and targeted proteins remain to a great ex-tent obscure, although recently the cytosolic calpains and lyso-somal cathepsins were identified as the most important playersin the execution of necrotic cell death [3]. It is unclear whether ne-crotic proteolysis is simply the result of the release of non-specificproteases or of programmed processes that are accompanied bythe specific degradation of key target proteins. The latter is sup-ported by the findings of Casiano et al. [9] that can be summarizedin three main observations: (i) necrosis is accompanied by the spe-cific cleavage of key nuclear substrates, not by a generalized degra-dation of intracellular material; (ii) proteins that are degradedduring apoptosis are also degraded during necrosis; (iii) necroticcleavage patterns differ from apoptotic patterns, suggesting thatdifferent proteases operate in different cell death pathways. Ourfinding that cryonecrotic proteolysis does not lead to a non-specificproteolytic degradation but to the activation of specific proteins –endonuclease p23 – supports the idea that an ordered pathway ofmolecular events underlies cryonecrotic cell death, the only type ofcell death aside from cell death induced by mechanical injury thatis still considered to be unregulated.

At present, several proteins serve as biochemical markers of thenecrotic process, whether based on their unique cleavage products(PARP-1, Topo I, NuMA, SAF-A) or absence of cleavage (lamin B)[8,9,20]. Based on the degradation pattern of PARP-1 and the com-plete absence of lamin B cleavage that are specific to necrosis, weshowed that the proteolytic process that accompanied ex vivoexposure of liver to cryogenic temperatures can be described as ne-crotic (Fig. 4B, lane 3 and [22]). However, using the same criteriawe found that the in vitro incubation of fresh nuclei with the cyto-sol fraction isolated from frozen/thawed liver resulted in theappearance of apoptotic degradation patterns for both PARP-1and lamin B. In order to define the proteolytic activity that wasresponsible for the observed proteolysis, fresh nuclei were incu-bated with the cytosol fraction that was preincubated with differ-ent protease inhibitors. Pepstatin A, a pharmacological inhibitor ofcathepsin D, completely inhibited PARP-1 and lamin B cleavage,implying that cathepsin D played an important role in the appear-ance of apoptotic products after the in vitro incubation experi-ments. Moreover, we found that cathepsin D was alsoresponsible for the activation of endonuclease p23 since its activa-tion was completely repressed in the presence of pepstatin A. Thecentral role of cathepsin D is supported by the observation thatcathepsin D activity was increased almost fourfold in the cytosol

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isolated from liver exposed to a single freezing/thawing cycle com-pared to the cytosol obtained from fresh liver. The rise in cathepsinD activity probably resulted from the loss of lysosomal membraneintegrity, which is expected during the cryonecrotic process[15,39]. Increased cathepsin D activity is in agreement with pro-posed role of lysosomal proteases, in particular of cathepsins B, Dand L in necrotic proteolysis [20,41].

Analysis of cytosol proteins from fresh liver and liver exposed tofreezing and thawing revealed that, aside from the increase incathepsin D activity, the cytosol isolated from frozen/thawed livercontained cytochrome c and active caspase-3 (Fig. 5B), which couldexplain the appearance of the apoptotic degradation patterns ofPARP-1 and lamin B that were obtained in the in vitro incubationexperiment. Cytochrome c is released into the cytosol from mito-chondria in both apoptotic and necrotic processes [7]. However,the presence of the apoptotic effector protease caspase-3 pointsto the involvement of apoptotic events during freezing/thawing.It has been shown that cryoinjury induces apoptosis by disruptingmitochondrial integrity [25,54]. The authors found that sublethalcryoinjury of colorectal cancer cells in vitro was accompanied bythe translocation of Bax to the mitochondrial membrane, the re-lease of cytochrome c and activation of caspase-3. The likely triggerof mitochondrial damage during cryoinjury could have beencathepsin D. Evidence has been provided that lysosomal cathepsinsinfluence the activities of different apoptotic factors and are thusinvolved in apoptotic regulation [12,18,24,43,47]. Cathepsin Dwas shown to activate Bax in T cells [6] and to be involved in therelease of cytochrome c from mitochondria in fibroblasts [29,44].Pepstatin A, a cathepsin D inhibitor, has been shown to block therelease of mitochondrial cytochrome c and caspase activation incardiomyocytes and fibroblasts [29,30]. In addition, cathepsin D di-rectly activates caspase-8 which in turn results in caspase-3 activa-tion in neutrophils [14]. We propose that increased activity ofcathepsin D induced cytochrome c release which led to caspase-3activation and determined the apoptotic proteolytic pattern ob-served in the in vitro incubation experiments (Fig. 4B, lane 4).

Obviously, there is a marked discrepancy between proteolysisthat occurred during freezing/thawing and proteolysis that was in-duced in our in vitro system. A possible explanation could be thefact that many cathepsins become irreversibly inactivated in vitroat the pH of the cytoplasm [51], whereas under these conditionscaspases are maximally active [46]. Therefore, our in vitro incuba-tion experiment would favor the caspase proteolytic pattern whilethe activity of the cathepsins was reduced. Nonetheless, the pres-ence of active cathepsin D, as well as active caspase-3 in the cyto-sol obtained from liver exposed to freezing/thawing revealed thepotential of the cells for executing both types of cell death. Whichspecific protease is the executor probably depends on the injury le-vel, i.e. on the freezing temperature. Apoptosis is a type of celldeath that occurs only when the temperature that induces cryoin-jury is above �36 �C while at lower temperatures cells die bynecrosis [25]. Thus, the central region of a cryogenic lesion is ex-pected to be completely necrotic, while the periphery of the lesionwhere the temperature is higher contains a mixture of lethally-and sub-lethally-damaged cells [5,25]. In this region, cells woulddie by apoptosis. The temperature-sensitive factor in cells couldbe the cellular energy status, i.e. the cellular availability of ATPwhich appears to be the key determinant of the apoptosis-necrosisswitch [11]. Freezing induces a significant decrease in the ATP con-tent [36,50]. Thus, it is possible that a cryoinjured cell possessesthe potential to die either by apoptosis or necrosis and that the fi-nal decision is made by the ATP content of the cell.

In this paper, we suggest that cells exposed to cryogenic tem-peratures have the potential to die either via the apoptotic or thenecrotic pathway and that the lysosomal protease cathepsin Dplays a central role in both processes. Cathepsin D is also involved

Please cite this article in press as: N. Grdovic et al., Proteolytic events in cryon(2010), doi:10.1016/j.cryobiol.2010.01.005

in the proteolytic activation of endonuclease p23 that accompaniesliver cell damage after exposure to cryogenic temperature.

Despite the widespread view of necrosis as an uncontrolledform of cell death, certain types of cell death with necrotic mor-phology are mediated by a stereotyped, evolutionary-preservedsequence of biochemical events [17,56] described following ische-mia, hypoxia, in Alzheimer’s disease, Parkinson’s disease, embryo-nal development and in pathogen-induced cell death [3,57]. In theabsence of an adequate, precise terminology and nomenclaturethat would distinguish between different forms of non-apoptoticand non-autophagic cell deaths, ‘‘programmed necrosis” has beenused to describe cell death that proceeds along a fixed pathwayof molecular events [17,56]. Thus, acute cell breakdown due to di-rect action or damaging stimuli such as extreme cold or heat andmechanical injuries remains the only type of cell death that is con-sidered to be unregulated. Cryonecrosis which is induced by aphysical stimulus rather than a molecular signal is a good exampleof this type of cell death. Ultrastructural examinations of rat liverafter freezing revealed that cytoplasmic changes consisting ofalterations of mitochondrial and endoplasmic reticulum structuresprecede changes in the plasma membrane [50,58,59]. The directconsequence of these alterations is decreased ATP content and in-creased Ca2+ concentration, two of the main mediators of ‘‘pro-grammed necrosis”. Hence it appears that the cell possesses alimited number of options in response to damaging stimuli. To-gether with our finding that cryonecrosis is accompanied by theactivation of endonucleases p23 by proteolysis, this suggests thatan ordered pathway of molecular events underlies the executionof cryonecrotic cell death. It could be concluded that terms suchas uncontrolled or unregulated should be used with caution sinceit appears that each process that occurs in a cell is a part of a pre-defined pattern.

The likelihood that an ordered pathway of molecular eventsunderlies cell death induced by cryogenic temperatures allowsfor a better control of cell death inside the cryogenic lesion. Under-standing the mechanisms that are responsible for the induction oflow temperature cell death is the first step in the potential use ofcell death inducers in addition to cryogenic temperatures thatcould enhance the efficacy of cryosurgery.

Acknowledgment

This work was supported by the Ministry of Science of theRepublic of Serbia, Grant No. 143002B.

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