Journal of Surgical Research 139, 143–156 (2007)

RESEARCH REVIEW

Screening and Detection of Apoptosis

Sergio Huerta, M.D.,1 Emily J. Goulet, B.S., Sara Huerta-Yepez, Ph.D.,and Edward H. Livingston, M.D., F.A.C.S.

UT Southwestern Medical Center/VA North Texas Health Care System, Department of Gastrointestinal and Endocrine Surgery,Dallas, Texas

Submitted for publication April 27, 2006

doi:10.1016/j.jss.2006.07.034

Since programmed cell death was first described bythe electron microscopic cellular changes demonstrat-ing an organized form of cell death over 30 years ago,it has undergone a great deal of scrutiny as a potentialtarget for several diseases including cancer. The tech-niques for the study of apoptosis have evolved accord-ingly. Methodologies for the study of apoptosis wereexamined by a MEDLINE search of the English-language literature and are summarized in this re-view. This review discusses the various ways to studyapoptosis with specific assays, reagents, and mole-cules. The particular advantages and disadvantages ofeach method are reviewed. © 2007 Elsevier Inc. All rights

reserved.

Key Words: caspases; Bcl-2; Apaf-1; death receptorpathway; AIF; proteosome.

INTRODUCTION

Apoptosis refers to an active energy dependent pro-cess initiated by a variety of stimuli, either intracellu-lar or extracellular, in which living cells participate intheir own death in an organized and efficient manner.Apoptosis, in contrast with the unorganized cell deathof necrosis, is a carefully orchestrated process. Apopto-sis was originally described by Kerr and Wyllie in 1972[1], who observed the unique changes in cells undergo-ing organized cell death by electron microscopy [2] asshown in Fig. 1 [3]. Because of its importance in devel-opment and many disease processes, including cancer,apoptosis has undergone extensive examination in thepast 30 years and the methods to screen and study

1 To whom correspondence and reprint requests should be addressedat 4500 Lancaster Road, Dallas, TX 75216. E-mail: Sergio.Huerta@

UTSouthwestern.edu.

143

programmed cell death have evolved accordingly. Thepurpose of this report is to provide a brief and compres-sive review of the process of apoptosis with direct ap-plicability to an investigator interested in this impor-tant biological process of cell suicide.

REVIEW OF THE PROCESS OF APOPTOSIS

Apoptosis involves a dynamic interplay of severalmolecules with up-regulatory and down-regulatoryproperties. Stimulation of pro-apoptotic molecules orinhibition anti-apoptotic factors are dependent on thecell type and the form of insult. It is unlikely that theactivation or inactivation of a single component willalter the ultimate fate of the cell and lead to apoptosis.A classical review of the process of apoptosis is depictedin Fig. 2 and reviewed below.

In the classical form of apoptosis, activation of execu-tioner proteolytic enzymes called cysteinyl aspartate-specific proteases (caspases) destines the cell to undergocell death. Caspases are grouped into initiator caspases(8 and 10), and execution caspases (3, 6, and 7). Acti-vation of executioner caspases occurs via type 1 and/ortype 2 apoptosis (Fig. 2).

Type 1 Apoptosis

The extrinsic pathway or the death receptor pathwayof apoptosis is initiated by binding of: 1) the TNFligand to the TNF (tumor necrosis factor) receptor, theTRAIL ligand (TNF-related apoptosis inducing ligand)to the DR4 and DR5 receptors, or the FasL ligand tothe Fas receptors. This association leads to recruit-ment of adaptor molecules such as FADD or TRADDresulting in activation of initiator caspases 8 and 10,

which in turn cleaves and activates executioner

0022-4804/07 $32.00© 2007 Elsevier Inc. All rights reserved.

144 JOURNAL OF SURGICAL RESEARCH: VOL. 139, NO. 1, MAY 1, 2007

caspases 3, 6, and 7, culminating in apoptosis (seeTable 1 for a glossary of terms) [4, 5].

Type 2 Apoptosis

The intrinsic or mitochondrial pathway is activatedby release of cytochrome c from the mitochondrial in-termembrane into the cytosol. Once released, cyto-chrome c interacts with Apaf-1, ATP, and pro-caspase 9to form the apoptosome. The apoptosome cleaves andactivates caspase 9, which leads to caspase 3, 6, and 7activity stimulating apoptosis [6]. Activation of caspase9 without involvement of the apoptosome has also beendescribed [7, 8].

The permeability of the mitochondrial membrane tocytochrome c is determined by the relative ratio ofpro-apoptotic and anti-apoptotic mediators. When pro-apoptotic molecules BAX and/or Bak are translocatedfrom the mitochondrial intermembrane, there is a netincrease of cytochrome c available to form the apopto-some. Pro-apoptotic molecules, such as BAX or Bak,

FIG. 1. Changes in cell morphology as detected by transmissionelectron microscopy. (A) Normal cell. (B) Late phase apoptosis asindicated by nuclear fragmentation. Images from Zeigler U et al.used with permission [3].

effects are mediated by altering mitochondrial mem-

brane permeability. Because these proteins have theability to insert themselves into membranes, it hasbeen hypothesized that these molecules function byinsertion into the membranes and creating channels orpores allowing the mitochondrial matrix to leakthrough [9]. Another proposal for the mechanism ofaction of BAX or Bak is that they cause a significantconformational change in the voltage dependent anionchannel (VDAC) thus allowing large proteins to passthrough also [9]. Coupling of pro-apoptotic moleculeswith anti-apoptotic factors (i.e., Bcl-2, Bcl-xL, andMcl-1) neutralizes their anti-apoptotic actions. Thus,the relative ratio of pro-apoptotic and anti-apoptoticmediators determines the relative amount of cyto-chrome c available to form the apoptosome.

The mitochondrial pathway also results in stimula-tion of apoptosis by release of SMAC/DIABLO andOmi/HTRA-2. The role of these factors is to neutralizethe actions of inhibitors of apoptosis (IAP) such ascIAP1, cIAP2, and XIAP [10].

Synthesis and/or activation of IAPs are under controlof the transcription factor nuclear factor kappa B(NF�B). NF�B is present in the cytosol in an inactiveform bound to a second molecule I�B. Coupling ofNF�B and I�B prevents the NF�B subunits (p50and/or p65) from translocating into the nucleus.Phosphorylation leads to ubiquitination and subse-quent degradation of IkB, which frees either one orboth units of NF�B. NF�B (p50 and/or p65) thentranslocates into the nucleus where it initiates tran-scription of IAPs [11].

Communication between type 1 and type 2 apoptosiscan occur at various stages. Activation of caspase 8results in stimulation of Bid, which leads to release ofcytochrome c and apoptosome activation (in type 2cells) [12]. Similarly, downstream stimulation ofcaspase 6 may feed back and activate caspase 8 [13].

Other mechanisms of apoptosis independent of thecaspase cascade have been described. These mecha-nisms may involve proteases, apoptosis inducing fac-tors (AIF), endonuclease G, calpains, and cathepsins[14]. AIF is released into the cytosol from the mitochon-drial intermembrane where it interacts with cyclophi-lin A and becomes a DNAase. Once translocation to thenucleus occurs, DNA fragmentation is initiated [41].

Several studies are available to determine whethercells are undergoing programmed cell death versus ne-crosis. The study of apoptosis can be compartmental-ized in various ways

1. Time progression: Early versus Late (Fig. 3)2. Extrinsic versus Intrinsic pathways (Fig. 2)3. Compartmentalization: Nucleus, Cytosol, Mito-

chondria, Cell membrane (Fig. 4)

The specific type of test depends on the particularstudy to be performed and the type of specimen to be

used (i.e., tissue versus cells). Apoptosis occurs in a

145HUERTA ET AL.: SCREENING AND DETECTION OF APOPTOSIS

sequential manner with the first morphologicalchanges appearing at the cell membrane. Cell to celladhesion decreases and either the cytosolic or mito-chondrial proteins are altered resulting in nuclearchanges. This process takes 1 to 3 h [15]. The ultimatedeterminant of apoptosis is an orderly form of intranu-cleosomal DNA fragmentation (180–200 bp). Becausethe biochemical hallmark of programmed cell death isinitiation of a caspase cascade that ultimately leads toDNA fragmentation, both caspase activation and DNAfragmentation needs to be demonstrated to establish ifa cell has undergone the process of apoptosis. For in-stance, activation of caspase-3 and other morphologicalchanges in apoptosis may be observed without DNAfragmentation; this would not be apoptosis [16]. Sev-eral kits for the detection of apoptosis are commercially

FIG. 2. Mechanisms of apoptosis. The death receptor (extrinsstimulatory effects. (Dashed) Inhibitory effects.

available [17, 18]. Each method to detect and verify

apoptosis has advantages and disadvantages and it isbest to confirm apoptosis with multiple complementarytechniques. For example, microscopic observation ofmorphological changes combined with DNA fragmen-tation assays are two reliable independent techniquesthat define cell death occurring by apoptosis. The mostreliable technique, however, remains the observationof morphological changes as cells undergo programmedcell death.

SCREENING FOR APOPTOSIS

Morphological Changes

Electron Microscopy

The differences in the specific morphological changes

and the mitochondrial (intrinsic) pathway of apoptosis. (Arrow)

ic)

that cells underwent in apoptosis in comparison to

146 JOURNAL OF SURGICAL RESEARCH: VOL. 139, NO. 1, MAY 1, 2007

necrosis was first observed by electron microscopy(EM). As such it is the gold-standard test to distinguishbetween the two. The specific morphological changes inearly and late apoptosis that can be detected with EMare depicted in Fig. 5. Electron microscopy is a highlyspecific and sensitive test for the detection of apoptosisand can be augmented by staining cell DNA fragmen-tation by TUNEL (EM-TUNEL). Cells are: fixed,stained, dehydrated, embedded, and cut into thin sec-tions with an ultramicrotome, all of which can be per-formed in a general laboratory. Cell sections are pickedup on copper grids, post-stained, then viewed underand electron microscope. EM requires expensive equip-ment and specialized training [19]. Thus, EM is notwidely used as a common technique for apoptosisscreening because it is time consuming and screeningof large samples is difficult.

Light Microscopy and Fluorescence Microscopy

Light microscopy (LM) detects nuclear condensationand apoptotic bodies in cells stained with hematoxylin

TABLE 1

Glossary of Apotosis Terms

AIF Apoptosis-inducing factorApaf-1 Apoptosis activating factor-1APO-1 � Fas Apoptosis inducing receptor-1Bad Bcl-2 antagonist of cell deathBak Bcl-2 antagonist/KillerBAX Bcl-2 associated X proteinBcl-2 B-cell lymphoma -2BH3 Bcl-2 Homology 3 domain only proteinsBid Bcl interacting domainBim Bcl-2 interacting mediator of cell deathBir Baculoviral inhibitor of apoptosis repeatBRUCE Bir-repeat containing ubiquitin conjugate enzymeCARD Caspase recruitment domainCARP Caspase associated RING-proteinCaspase Cysteinyl aspartate-specific proteasec-FLIP Cellular FLICE-inhibitory proteinDED Death effector domainDIABLO Direct IAP binding protein with low plDISC Death inducing signaling complexFADD Fas Associated Death DomainIAP Inhibitor of apoptosisICE Interleukin 1-� converting enzymeNF�B Nuclear Factor Kappa BNOXA Name for damageSMAC/D SMAC/DIABLO � second mitochondrial-derived

activatorPARP Poly(ADP-ribose) polymerasePUMA p53 up-regulated modulator of apoptosisRING Really Interesting New GeneTdT Terminal deoxynucelotidyl transferaseTRADD Tumor Associated Death DomainTRAIL Tumor necrosis factor-related apoptosis-inducing

ligandTUNEL TdT-mediated dUTP-biotin nick end-labelingXIAP X-linked IAP

and eosin (H&E). LM has a low ability for detecting

apoptotic cells. However, it can be augmented by nu-clear staining of cells with propidium iodide (PI), whichdifferentiates necrotic cells. The acuity of light micros-copy can also be enhanced by the use of fluorescence.Nuclear fluorescent dyes such as Hoechst 33258 can beused to visualize nuclear changes. These microscopictechniques can only be used to detect the late events ofapoptosis and their use is also limited by the inabilityto screen a large number of samples. All microscopictechniques, including LM, fluorescence microscopy(FM), and EM, can detect apoptosis in vitro but arelimited by the fact that they can only detect apoptosisat a single point in time. Thus, it is possible to miss thecharacteristic apoptotic bodies by microscopy to estab-lish apoptosis.

DNA FRAGMENTATION

TUNEL

The terminal deoxynucleotidyl transferase-dUTPnick end labeling (TUNEL) assays were introduced byGavrieli et al. in 1992 [15]. TUNEL has rapidly becomethe most widely used in situ test for the study of apo-ptosis. TUNEL is based on the specific binding of ter-minal deoxynuclotidyl transferase (TdT) to 3=-OH endsof fragmented DNA. Following proteolytic treatment ofhistological sections, TdT incorporates X-dUTP (X �biotin, DIG, or fluorescein) at sites of DNA breaks.Termini modified nucleotides Avidin-peroxidase ampli-fies the signal and allows for examination of labeledcells under light or fluorescent microscopy, flow cytom-etry, or immunohistochemistry (Fig. 6) [15]. There area few limitations of this method. First, fixation andhandling of tissue can significantly alter the results ofthe TUNEL assay [20–22]. Secondly, when formalin-fixed, paraffin-embedded sections have been shown toprovide inaccurate rates of apoptosis if fixation is pro-

FIG. 3. The typical methods to study early versus late apoptosis.

opt

147HUERTA ET AL.: SCREENING AND DETECTION OF APOPTOSIS

longed [23]. In addition, measurement of apoptosis at asingle time point in tissue sections may be an inaccu-rate representation of the true rate of programmed celldeath for that tissue. Finally, TUNEL is not specific forapoptosis; it indicates DNA cleavage from any form ofcell death and necrotic cells may also be labeled by thistechnique [24]. Despite these limitations, there areseveral key benefits of the TUNEL assay. One is thatquantification of apoptosis is possible by determiningthe ratio of positive nuclei over the number of cellscounted resulting in the apoptotic index (AI). Further-more, TUNEL assays can be used for cells in grown inculture, adherent cells grown in slides, frozen tissuesections and formalin-fixed, paraffin-embedded tissuesections. All in all, TUNEL is an accepted assay toestablish apoptosis in vitro and in situ and when con-firmed with other methods it is a reliable test for apo-ptosis.

In Situ End-Labeling Technique

The in situ end-labeling technique (ISEL) is a mod-ification of the TUNEL assay introduced in 1993 byWijsman et al. [25] Instead of TdT, ISEL uses DNApolymerase I to label the 3=-OH DNA nicks. Quantifi-cation is also possible with ISEL. As with TUNEL,dUTP can be linked to biotin, DIG, or fluorescein en-abling the use of automated image cytometry such thatthe study of a large number of samples can be per-formed (Fig. 6). ISEL is less sensitive and takes longerto perform than TUNEL.

FIG. 4. Study of aspects of ap

FIG. 5. Morphological changes occurr

Field Inversion Gel Electrophoresis

Field inversion gel electrophoresis (FIGE) is basedon the observation that DNA fragmentation occurs as aresult of chromatin loop domains forming 300 and/or50 bp DNA segments. This occurs before internucleo-somal fragmentation (180–200 bp segments) [26]. Theearlier event can occur without the later.

Flow Cytometry and ELISA

DNA fragmentation can be identified with flow cy-tometry based assays using the monoclonal antibodyB-F6, which detects internucleosomal breaks [27]. Bythe same principle, histone associated DNA fragmentscan be detected using the enzyme-linked immunosor-bent assay (ELISA) kits available. This assay is par-ticularly useful for non-replicating cells that do notincorporate labeled nucleotides. ELISA tests can beused with: adherent cells, cells in suspension culture,cell culture supernatant, serum, plasma, and lysates ofcells obtained ex vivo [28].

DNA Laddering

The internucleosomal fragmentation produced byendonucleases at expected intervals of 180 base pairsto 200 base pairs is part of the biochemical hallmark ofapoptosis. Standard DNA extraction techniques areused to obtain the nucleic acids from either cells orhomogenized tissue. DNA is then electrophoresed in anagarose gel that demonstrates the characteristic DNA

osis by various compartments.

ing during early and late apoptosis.

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laddering pattern (Fig. 7). DNA fragmentation kits arecommercially available [17]. The major limitations ofthis technique is the need for a large quantity of DNAto produce the laddering pattern. Thus, a large number ofcells undergoing apoptosis are required (low sensitivity).DNA laddering demonstrates apoptosis in a cell popula-tion and does not assess the degree of apoptosis ofindividual cells. This technique, therefore, is not quanti-tative. DNA laddering is labor-intensive and time-consuming.

DNA Fragmentation Measurement

Apoptotic cells undergoing DNA fragmentation con-tain high percentages of low molecular weight (LMW)DNA compared to non-apoptotic cells with high per-centages of high molecular weight (HMW) DNA. LMWDNA can be separated from HMW DNA by filtrationand centrifugation. In this technique, replicating cells arepre-labeled with a nucleotide analogue such as 5-bromo-2=-deoxyuridine (BrdU) before induction of apoptosis.The relative amounts of labeled LMW DNA and HMWDNA are quantitated. Because this technique is basedon the incorporation of a nucleotide analogue, apopto-sis can only measured in replicating cells in vitro.

APO ssDNA

A new technique takes advantage of the fact thatlarge stretches of single stranded DNA (ssDNA) occurin heat denatured apoptotic cells. The APO ssDNAassay uses antibodies specific to the damaged DNA. Itis quick and easy to use, gives qualitative or quantita-tive results and can be analyzed by flow cytometry, a96 well plate reader or fluorescence microscopy. Kitsare commercially available.

DNA STAINING ASSAYS

Dye Exclusion

The cell membrane of dead cells becomes permeable

FIG. 6. TUNEL and ISEL.

to dyes and can be stained. In contrast, viable cells can

be excluded by dyes that will not penetrate the intactplasma membrane. Trypan blue is a typical exclusiondye that can be used to differentiate viable and deadcells by light microscopy such that blue-stained cellscan be distinguished by the viable non-stained cells.

DNA Binding Dyes

The fluorescent dye, propidium iodide (PI), becomeshighly fluorescent upon binding of DNA. PI only stainsnon-viable cells. Stained and unstained cells can becounted by flow cytometry. During apoptosis and DNAfragmentation, the amount of stained DNA contentdecreases in cells. The percentage of cells in the G1

phase naturally decreases as apoptosis progresses. Themajor disadvantage of PI staining by flow cytometry isthat this technique does not distinguish between theG1 and G2 DNA content. Decreased G2 DNA contentmay falsely represent cells undergoing apoptosis.Thus, this is not a very specific test for apoptosis. Onceapoptosis has been established by another method,PI-flow cytometry is a powerful technique to studyvarious drugs and drug concentration effects on apo-ptosis.

Hoechst 33342, 4=,6-diamidino-2-phenylindole(DAPI), and YOPRO-1 incorporate into DNA in via-ble cells and become highly fluorescent. These DNAmarkers make the chromatin condensation readily vis-ible by FM and simplify the detection of morphologicalchanges during apoptosis.

Annexin V

Annexin V assays are based on the observation thatduring induction of apoptosis, phosphatidylserine (PS)

FIG. 7. DNA Laddering.

149HUERTA ET AL.: SCREENING AND DETECTION OF APOPTOSIS

is translocated from the inner leaflet to the outer leaf-let of the plasma membrane. This early event in apo-ptosis allows for recognition of phagocytic cells suchthat apoptotic cells can be eliminated without cell pro-teolytic enzyme leakage [29]. Annexin V, a 35-kDaCa2�-binding protein fastens with high affinity to phos-phatidylserine [30]. Annexin V conjugated with fluoro-chromes allows for the use of this assay with fluores-cent microscopy. Dyes for light and electron microscopyare also available for the use of annexin V. Thus, thisassay can be used both in vivo and in vitro. Annexin Vassays take advantage of the ability to usebiotinylated-annexin, which can label cells in all stagesof apoptosis as measured by flow cytometry. Annexin Vbound to fluorescent dyes can be used in real timeimaging. The notable disadvantage of annexin V is thatit can label necrotic rather than apoptotic cells therebymaking it less specific, but this can be compensated inflow cytometry by using annexin V in conjunction witha DNA binding dye to differentiate between the two.

ANTIBODIES AGAINST TARGETS OF EXECUTIONERCASPASES

PARP

Activation of executioner caspases 3 and 7 results incleavage and inactivation of proteins involved in DNArepair mechanisms. These proteins include: DNA-dependent protein kinase and poly(ADP-ribose) poly-merase (PARP). PARP is a 113-kDa protein that bindsat specific DNA strand breaks. Enzymatic cleavage ofPARP results in 89 kDa and 24 kDa fragments. Amonoclonal antibody is commercially available thatrecognizes the 89 kDa cleaved form of PARP. Themonoclonal antibody can be used in western or immu-nohistochemical analyses as well as flow cytometry.

MP30

MP30 is a commercially available [17, 18] monoclo-nal antibody that specifically binds to cytokeratin-18,another target molecule of executioner caspases. Thisantibody is useful in detecting apoptosis in single cellsand tissue sections by immunohistochemistry. Addi-tionally, when combined with fluorescein, MP30 can beused in conjunction with FM and flow cytometry.

Flow Cytometry

Flow cytometry is currently being used extensivelyto detect the early changes of apoptosis. Cell cycleanalysis including apoptosis can be measure by PIstaining by measuring DNA content through the cellcycle. Several antibodies can be labeled with FITC andused in conjunction with flow cytometry. These include

active caspases, annexin V, TRAIL, and so forth.

SPECIFIC MEDIATORS OF APOPTOSIS

Plasma Membrane

Fas, FasL, TRAIL-L, TRAIL-R (DR4, DR5), TNF-�

Fas is a 45 to 52 kDa type 1 transmembrane receptorwidely expressed in cells, which can also exist in thesoluble form by differently splicing the transmembranedomain. Fas mediated apoptosis is initiated by bindingof the receptor to its ligand FasL, which demonstratesa more restricted expression. There are two forms ofFasL: membrane bound FasL and soluble FasL. Thesoluble form is generated by action of a metallopro-tease [5]. TRAIL is another member of the of the TNFrelated proteins that mediates apoptosis upon bindingof its ligand; TRAIL-L.

Fas/TRAIL mediated apoptosis can be induced byuse of monoclonal agonistic antibodies CH-11 and sol-uble recombinant TRAIL/Ap2L (rTRAIL) respectively.Similarly, the recombinant form of TNF-� (rTNF-�)can be used to induce TNF-mediated apoptosis. Mono-clonal antibodies against the membrane bound recep-tors Fas, TRAIL, and TNF are commercially availableand can be used for immunohistochemistry, or conju-gated with FITC, it can be used in conjunction withflow cytometry. Immunohistochemistry can be used bytypical laboratory techniques to detect presence ofthese antibodies along the membrane of cells.

Phosphatidyl Serine

Phosphatidyl serine (PS) is externalized early duringapoptosis from the inner leaflet of the plasma mem-brane to its outer leaflet such that apoptotic cells canbe recognized by phagocytic cells. Annexin V binds PSwith high affinity and can be used in conjunction withfluorochromes for the detection of apoptotic cells withflow cytometry or fluorescence microscopy [30].

Ceramide

Ceramide is the byproduct of the catalytic hydrolysisof sphingomyelin. Ceramide is an intracellular secondmessenger which activates serine and threonine pro-tein kinases and phosphoprotein phosphatase [31, 32]leading to DNA fragmentation and cell death [33]. Thisresponse is mediated, at least in part, by coupling ofTNF-� to sphingomyelinase in the plasma membrane[33]. Ceramide also increases the activity of NuclearFactor Kappa B (NF�B). Methods for the detection ofceramide and phospholipids have been previously de-scribed [34]. Sphingomyelinase-free phospholipase (Ba-cillus cerus) and 1,2-Diacylglycerol are both commer-

cially available.

150 JOURNAL OF SURGICAL RESEARCH: VOL. 139, NO. 1, MAY 1, 2007

Mitochondria

Mitochondrial Potential

Mitochondrial dysfunction occurs early in apoptosisand it is accompanied by a decreased membrane poten-tial. Changes in mitochondrial potential can be exam-ined by fluorescence of the cationic lipophilic dyeCMTMRos and other fluorochromes, which arepotential-sensitive dyes and can be used in conjunctionwith flow cytometry [35].

Cytochrome C

Cytochrome c is a water-soluble protein which func-tions in the oxidative respiration chain and triggerscaspase 3 cleavage by activation of caspase 9 duringthe formation of the apoptosome (Fig. 2). Cytochrome cis transcribed in the cell nucleus, translated in thecytosol and then transported to the intermitochondrialspace by chaperone proteins. The protein product is a13-kDa size protein and monoclonal antibodies for itsdetection are commercially available.

SMAC/DIABLO

SMAC/DIABLO is a mitochondrial protein that pro-motes apoptosis by neutralizing the activity of IAPs.Upon induction of apoptosis, SMAC/DIABLO is se-creted from the intermitochondrial membrane, whereit binds the BIR domain of IAPs, thereby blocking theability of IAPs to inhibit active caspase 3, 7, and 9

FIG. 8. Mechanism by which the inhibitors of apoptosis (IAPs)block caspase 3 and 7 from activating their substrates. SMAC/DIABLO neutralized the action of IAPs, thereby stimulating ap-optosis.

FIG. 9. The Bcl-2 fa

(Fig. 8) [36]. Like cytochrome c, SMAC/DIABLO istranslated in the cytosol and imported into the mito-chondrial intermembrane. SMAC/DIABLO release,like cytochrome c, is also inhibited by Bcl-2. SMAC/DIABLO is able to interact with all mammalian IAPs[36, 37]. Monoclonal antibodies for the 21-kDa proteinare commercially available for immunohistochemistry.Western blot analysis for SMAC/DIABLO may requireisolation and mitochondrial preparation as previouslydescribed [37].

Ceramide

Ceramide also plays an important role in apoptosisat the level of the mitochondria. Mitochondrial perme-ability is increased by formation of ceramide channelsthereby allowing the release of cytochrome c and otherproapoptotic proteins from the inner mitochondrialmembrane resulting in apoptosis. The mitochondriacontains enzymes responsible for the synthesis andhydrolysis of ceramide; there exists a mechanism forregulating the level of ceramide in mitochondria. Inaddition, mitochondrial ceramide levels have beenshown to be elevated before the induction phase ofapoptosis [38]. Measurement of mitochondrial perme-ability accompanied by cellular levels of ceramide dur-ing the induction of apoptosis permits examination ofthe relative role of mitochondrial ceramide during ap-optosis.

Bcl-2 and Its Family

The B-cell lymphoma family of proteins consists ofover a dozen members typically categorized into threefunctional groups depending on the number of Bcl-2homology domains (BH) [39, 40]. The most completeform of the members contains four BH domains (BH1-BH4) in addition to a transmembrane domain thatanchors these proteins to either the outer mitochon-drial membrane or the endoplasmic reticulum. Theproteins have anti-apoptotic properties and consist ofBcl-2, Bcl-xL, and Bcl-M. The most potent anti-apoptotic member in this family is Bcl-xL (Fig. 9). Thesecond group of Bcl-2 proteins lacks the BH4 domain.The proteins have proapoptotic properties and consistof BAX and Bak. The BH3 only domain proteins con-

mily of proteins.

spa

151HUERTA ET AL.: SCREENING AND DETECTION OF APOPTOSIS

sisting of Bid and Bik also have pro-apoptotic proper-ties. The relative stimulation of pro- and anti-apoptoticmitochondrial Bcl-2 members determines the relativepermeability of the mitochondrial membrane to cyto-chrome c resulting in stimulation of apoptosis by for-mation of the apoptosome. All of the members of theBcl-2 family can be studied by standard laboratorytechniques and monoclonal antibodies are commer-cially available for Western blot analysis and/or immu-nohistochemistry.

Apoptosis Inducing Factor

Substantial evidence continues to accumulate todemonstrate that apoptosis occurs via non-caspase me-diated mechanisms. Several mediators have been de-scribed and these include: proteases, apoptosis induc-ing factor (AIF), endonuclease G, calpains, andcathepsins, which are able to induce DNA fragmenta-tion [14]. AIF is released into the cytosol from themitochondrial intermembrane where it interacts withcyclophilin A and becomes a DNAase. Once transloca-tion to the nucleus occurs, DNA fragmentation is ini-tiated [41]. Monocolonal antibodies against AIF arecommercially available and can be used for immuno-histochemistry and Western blot analysis.

Cytosol

Caspases

The typical executioners of apoptosis are the proteo-lytic enzymes called cysteinyl aspartate specific pro-teases (caspases; Table 2). Caspases are grouped intoinitiator caspases (8 and 10), characterized by a longN-terminal, and execution caspases (3, 6, and 7), charac-terized by a short N-terminal. Because caspases are

TAB

Summary of Caspase

Function

Caspase-1 Interleukin converting enzyme 1 (ICE)Caspase-4Caspase-5Caspase-11Caspase-12 Released by ER stress and activated by calciumCaspase-13Caspase-14Caspase-2 Mediates mitochondrial cytochrome c releaseCaspase-8 Extrinsic pathwayCaspase-9 Intrinsic pathwayCaspase-10 Extrinsic pathwayCaspase-3 Once activated the cell is destined to undergo apCaspase-6Caspase-7

Activator caspases are short-N terminal caspases. Executioner ca

essential effectors molecules of apoptosis, assaying for

cleaved caspases allows one to detect early apoptosis.Monoclonal antibodies are available against all of thecaspases for immunocytochemistry, immunohisto-chemistry, ELISA, and even some for flow cytometry(i.e., active caspase 3).

The most elucidatory assay for the activation ofcaspases involves detection of proteolytic cleavage oftheir target molecules such as PARP by Western blotanalysis or by immunohistochemistry as well as flowcytometry. Anti-PARP monoclonal antibodies are com-mercially available.

Another target molecule of executioner caspases iscytokeratin 18 (CK-18). A commercially available an-tibody M30 recognizes a specific caspase cleavagesite within CK-18. This antibody is useful in detect-ing apoptosis in single cells and tissue sections byimmunohistochemistry. Additionally, when com-bined with fluorescein, M30 can be used in conjunc-tion with FM and flow cytometry.

The Apoptosome

The interaction of Apoptosis protease activatingfactor-1 (Apaf-1) with cytochrome c (Apaf-2), caspase 9(Apaf-3), and ATP leads to the formation of the apop-tosome. This activates caspase 9, resulting in cleavageof caspase 3 and induction of apoptosis. Apaf-1 is a130-kDa protein and a monoclonal antibody is commer-cially available.

Inhibitors of Apoptosis

The inhibitors of apoptosis (IAP) family of proteinsare under regulation of NF�B (Fig. 10). The mecha-nism of action of IAPs is by direct inhibition of activecaspase 3, 7, and 9. Seven mammalian IAPs (Fig. 11)

2

volved in Apoptosis

Regulatoryunit

Cytokine processors CARDCARD

ulates caspase 3

Activator caspases CARDDEDCARDDED

osis Executioners caspases

ses are long-N terminal caspases.

LE

s In

stim

opt

have been described and these are differentiated from

s.

152 JOURNAL OF SURGICAL RESEARCH: VOL. 139, NO. 1, MAY 1, 2007

each other by their BIR and RING-Zn domains (seeglossary of terms). The IAP surviving is emerging as aninhibitor of apoptosis important in several malignan-cies. Survivin is present in almost all transformed cellsand cancers tested today [36, 42]. Monoclonal antibod-ies against all of the members of the IAP family arecommercially available for immunohistochemistry andor Western blot analysis.

Heath Shock Proteins

The death receptor and mitochondrial pathway areinfluenced by heat shock proteins (Hsp), which mayhave pro-apoptotic and anti-apoptotic properties [43].Anti-apoptotic heat shock proteins include Hsp27,which inhibits release of cytochrome c, and Hsp70 andHsp90, which bind to Apaf-1 thereby inhibiting theapoptosome. Pro-apoptotic Hsp60 and HSP90 directlystimulate caspase 3 [44]. Specific antibodies againstthe Hsps are commercially available.

NF�B

Nuclear factor kappa B (NF�B) is a transcriptionfactor typically found in the cytoplasm in an inactive

FIG. 10. NF�B is typically bound to I�B, phosphorylation (p) ltranslocates to the nucleus where they initiate transcription of IAP

FIG. 11. Family of IAPs proteins. Protein s

form bound to I�B. Phosphorylation of I�B leads to itsenzymatic degradation. Uncoupling of I�B and NF�Brenders activation of NF�B. NF�B is composed of twosubunits, p50 and p65, which can function indepen-dently or as a duo by translocating into the nucleuswhere it promotes transcription of IAPs leading to ex-ecutor caspase inhibition (Fig. 10). NF�B participatesin transcriptional regulation of Bcl-xL [45]. Inhibitionof NF�B sensitizes cells to TRAIL mediated apoptosis.The potent inhibition of apoptosis mediated by NF�Boccurs, therefore, via both the mitochondrial and thedeath receptor pathway. Specific antibodies for the de-tection of NF�B are commercially available. Localiza-tion of NF�B in the cell is important. If NF�B proteinis detected localized within the nucleus, NF�B hasbeen activated. Therefore, immunohistochemistryanalysis may be superior to Western blot analysis indetermining the specific role of NF�B in apoptosis.Alternatively, the function of NK�B can be determinedby EMSA, which stands for Electrophoretic MobilityShift Assay (EMSA). EMSA is a useful tool for identi-fying proteins that interact with DNA and the methodsfor EMSA have previously been described [46–49].

s to ubiquitin (UBQ) labeling and degradation. p50 and p65 then

ead

ize and known mRNA sizes are depicted.

153HUERTA ET AL.: SCREENING AND DETECTION OF APOPTOSIS

Several inhibitors of NF�B are commercially available:Bay 11-1075, Bay 11-7085, and Bay 11-7082. SN50inhibits translocation of the NF-�B active complex intothe nucleus.

The Proteosome

The proteosome is a 26S protease that possessesinhibitory and stimulatory properties in the processesof apoptosis. By proteolysis of ubiquitin-labeled de-struction, the proteosome keeps the levels of SMAC/DIABLO and Omi/HtrA2 low in the cytosol. Stimula-tion of apoptosis leads to destruction of the active unitof the proteosome (19S unit), which in turn leads toaccumulation of SMAC/DIABLO and Omi/HtrA2 re-sulting is a further stimulus of apoptosis [50–52]. Theproteosome can effectively be inhibited by Velcade(bortezomib) to assess its role in the process of apopto-sis in a given system. Isolation and purification of theproteasome has been previously described [53]. Severalmonoclonal antibodies that detect the various frag-ments of the proteasome after caspase activity arecommercially available [51].

Microarray Analysis

With the advent of large-scale analysis of gene ex-pression, many apoptotic genes can be studied simul-taneously. Microarray analysis is a powerful techniqueto study thousands of genes concurrently in a singleexperiment. On the GeneChip oligonucleotide array agiven gene is represented by 15 to 20 different 25-meroligonucleotides that serve as unique sequence-sequence detectors. An additional control element onthese arrays is used as a mismatch sequence (MM).These are probes that are designed to be complemen-tary to the reference sequence except for a homomericbase mismatch at the central position. The presence ofthe mismatched oligonucleotide allows cross-hybridization and local background to be estimatedand subtracted from the perfect match (p.m.) signal. Inthe GeneChip expression assay, eukaryotic mRNA isconverted to biotinylated cRNA from oligo-dT-primedcDNA [54]. Each sample is hybridized to a separatearray. Transcript levels are calculated by reference tocRNA spikes of known concentration added to the hy-bridization mixture. Differences in mRNA levels be-tween samples are determined by comparison of anytwo hybridization patterns produced on separate ar-rays of the same array type. This requires RNA extrac-tion, which can be obtained from tissue or from cells bystandard laboratory techniques.

The first measure is the model-based expressionvalue that summarizes the approximate expressionlevel over the 20 individual probes. The second mea-sure is the present/absent call. The microarray tech-nique relies strongly on statistical analysis. There are

several statistical programs available [55, 56]. This

typical analysis utilizes a number of details of theprobe pairs to determine if there was or was not anymRNAs corresponding to that particular gene in thesample. Genes were considered to have significant dif-ferential expression if they met the following criteria

1. A significant main effect in the ANOVA model2. A fold change greater than 2 in one of the

comparisons

P values for the ANOVA coefficients are based ontwo-sided tests and have not been adjusted for multiplecomparisons. A major disadvantage of this technique isthat statistical significance does not correlate with bi-ological significance. Simply stated, it has been notedin the apoptosis pathway that microarray analysis onseveral genes do not meet criteria for inclusion in ex-perimental models [57] but further studies by RT-PCRand Western blot analysis has uncovered biologicallyactive genes in the process of apoptosis. However, mi-croarray analysis has been used to identify apoptoticgenes in specific malignanancies [58–60].

RNA Interference Studies

The study of the relative role of specific genes inprocess of apoptosis has been made possible by RNAinterference studies. RNA interference is a techniquewhereby protein synthesis is prevented by silencing ofthe mRNA through chromatin remodeling. RNAase IIIprocesses dsRNA once they exit the nucleus by slicingit into short nucleotide duplexes of 21-28 base pairs inlength, which are referred to as short interfering RNAs(siRNA). Gene silencing occurs when siRNA moleculesinteract with a multi-protein referred to as the RNA-inducing silencing complex containing the endonucleo-lytic silencer Argonaute-2. The target mRNA is thencleaved resulting in silencing of the gene. This processis highly specific and requires only a small (seven con-tiguous complementary base pairs) homologous se-quence of the target mRNA to effectively result insilencing of protein expression [61, 62]. This techniquehas been used for the study of several apoptotic pro-teins including FLIP, [63] Blc-2, [64–67] Bcl-xL, [67–69] Survivin, [70–75] and XIAP [65, 76]. siRNA kitsare commercially available. The specific kit to be usedis dependent of the type of RNA to be silenced. Microar-ray in combination with siRNA has also been used toelucidate important genes in colon carcinogenesis [60].

Induction of Apoptosis

1. Starvation of cells by growing them in serum freemedia can effectively induce apoptosis in rapidly grow-ing cells in vitro.

2. TGF-�1 can also be used to induce apoptosis. De-pending on the cell line, the dose will vary between 10

and 100 ng/mL.

154 JOURNAL OF SURGICAL RESEARCH: VOL. 139, NO. 1, MAY 1, 2007

3. Chemotherapeutic agents affect the mitochon-drial pathway. Dosage depends on the cell line, buttypically varies between 5 to 50 �g/mL.

i. Cisplatinumii. Adriamyciniii. Etoposide

Death Receptor Pathway

1. TNF� (40 ng/ml) induces apoptosis via the deathreceptor pathway.

ii. FasL CH-11, a monoclonal antibody specific forthe Fas receptor, can be used to mimic the effects ofFasL and induces apoptosis via the death receptorpathway.

iii. rTRAIL dosage depends on the cell line, but typ-ically ranges between 10 to 50 ng/mL.

Radiation Therapy

Irradiation, IR (500 Cgy) delivered with a 137Csdual source cell irradiator effectively induces apoptosisin cells grown in culture [77].

We conclude that the methods of investigating apo-ptosis should be tailored to the specific biochemicalprocess under investigation. The detection of apoptosisshould consist of at least two methods in a given sys-tem. Once apoptosis has been established in a partic-ular system, a less specific and more sensitive tech-nique can be used to study several samplessimultaneously. Current techniques focusing on theproteasome, AIFs, and siRNA studies hold a great dealof potential in elucidating new perspectives in the fas-cinating process of regulated cell destruction.

ACKNOWLEDGMENTS

This work was supported by a grant from the Dallas VeteransAdministration Research Fund and the Hudson-Penn Surgery Fund.

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