Apoptosis: A Review of Programmed Cell...

Toxicologic Pathology, 35:495–516, 2007Copyright C© by the Society of Toxicologic PathologyISSN: 0192-6233 print / 1533-1601 onlineDOI: 10.1080/01926230701320337

Invited Review

Apoptosis: A Review of Programmed Cell Death

SUSAN ELMORE

NIEHS, Laboratory of Experimental Pathology, Research Triangle Park, North Carolina 27709, USA

ABSTRACT

The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependentbiochemical mechanisms. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development andfunctioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Inappropriate apoptosis(either too little or too much) is a factor in many human conditions including neurodegenerative diseases, ischemic damage, autoimmune disordersand many types of cancer. The ability to modulate the life or death of a cell is recognized for its immense therapeutic potential. Therefore, researchcontinues to focus on the elucidation and analysis of the cell cycle machinery and signaling pathways that control cell cycle arrest and apoptosis. Tothat end, the field of apoptosis research has been moving forward at an alarmingly rapid rate. Although many of the key apoptotic proteins have beenidentified, the molecular mechanisms of action or inaction of these proteins remain to be elucidated. The goal of this review is to provide a generaloverview of current knowledge on the process of apoptosis including morphology, biochemistry, the role of apoptosis in health and disease, detectionmethods, as well as a discussion of potential alternative forms of apoptosis.

Keywords. Apoptosis; programmed cell death; intrinsic/extrinsic pathway; granzyme A/B; perforin; autophagy.

INTRODUCTION

The term apoptosis (a-po-toe-sis) was first used in a now-classic paper by Kerr, Wyllie, and Currie in 1972 to describea morphologically distinct form of cell death, although cer-tain components of the apoptosis concept had been explicitlydescribed many years previously (Kerr et al., 1972; Paweletz,2001; Kerr, 2002). Our understanding of the mechanisms in-volved in the process of apoptosis in mammalian cells tran-spired from the investigation of programmed cell death thatoccurs during the development of the nematode Caenorhab-ditis elegans (Horvitz, 1999). In this organism 1090 somaticcells are generated in the formation of the adult worm, ofwhich 131 of these cells undergo apoptosis or “programmedcell death.” These 131 cells die at particular points during thedevelopment process, which is essentially invariant betweenworms, demonstrating the remarkable accuracy and controlin this system. Apoptosis has since been recognized and ac-cepted as a distinctive and important mode of “programmed”cell death, which involves the genetically determined elim-ination of cells. However, it is important to note that otherforms of programmed cell death have been described andother forms of programmed cell death may yet be discovered(Formigli et al., 2000; Sperandio et al., 2000; Debnath et al.,2005).

Apoptosis occurs normally during development and agingand as a homeostatic mechanism to maintain cell populationsin tissues. Apoptosis also occurs as a defense mechanism suchas in immune reactions or when cells are damaged by disease

Address correspondence to: Susan Elmore, NIEHS, P.O. Box 12233,Research Triangle Park, NC 27709, USA; e-mail: [email protected]

or noxious agents (Norbury and Hickson, 2001). Althoughthere are a wide variety of stimuli and conditions, both phys-iological and pathological, that can trigger apoptosis, not allcells will necessarily die in response to the same stimulus.Irradiation or drugs used for cancer chemotherapy results inDNA damage in some cells, which can lead to apoptotic deaththrough a p53-dependent pathway. Some hormones, such ascorticosteroids, may lead to apoptotic death in some cells(e.g., thymocytes) although other cells are unaffected or evenstimulated.

Some cells express Fas or TNF receptors that can lead toapoptosis via ligand binding and protein cross-linking. Othercells have a default death pathway that must be blocked bya survival factor such as a hormone or growth factor. Thereis also the issue of distinguishing apoptosis from necrosis,two processes that can occur independently, sequentially, aswell as simultaneously (Hirsch, 1997; Zeiss, 2003). In somecases it’s the type of stimuli and/or the degree of stimuli thatdetermines if cells die by apoptosis or necrosis. At low doses,a variety of injurious stimuli such as heat, radiation, hypoxiaand cytotoxic anticancer drugs can induce apoptosis but thesesame stimuli can result in necrosis at higher doses. Finally,apoptosis is a coordinated and often energy-dependent pro-cess that involves the activation of a group of cysteine pro-teases called “caspases” and a complex cascade of events thatlink the initiating stimuli to the final demise of the cell.

Morphology of ApoptosisLight and electron microscopy have identified the various

morphological changes that occur during apoptosis (Hacker,2000). During the early process of apoptosis, cell shrinkage

495

at U DE G (CUCSH) on September 14, 2015tpx.sagepub.comDownloaded from

http://tpx.sagepub.com/

496 ELMORE TOXICOLOGIC PATHOLOGY

and pyknosis are visible by light microscopy (Kerr et al.,1972). With cell shrinkage, the cells are smaller in size, the cy-toplasm is dense and the organelles are more tightly packed.Pyknosis is the result of chromatin condensation and this isthe most characteristic feature of apoptosis. On histologicexamination with hematoxylin and eosin stain, apoptosis in-volves single cells or small clusters of cells. The apoptoticcell appears as a round or oval mass with dark eosinophiliccytoplasm and dense purple nuclear chromatin fragments(Figure 1). Electron microscopy can better define the sub-cellular changes. Early during the chromatin condensationphase, the electron-dense nuclear material characteristicallyaggregates peripherally under the nuclear membrane al-though there can also be uniformly dense nuclei (Figures2A, 2B).

Extensive plasma membrane blebbing occurs followed bykaryorrhexis and separation of cell fragments into apoptoticbodies during a process called “budding.” Apoptotic bodiesconsist of cytoplasm with tightly packed organelles with orwithout a nuclear fragment (Figure 2C). The organelle in-tegrity is still maintained and all of this is enclosed withinan intact plasma membrane. These bodies are subsequentlyphagocytosed by macrophages, parenchymal cells, or neo-plastic cells and degraded within phagolysosomes (Figure2D). Macrophages that engulf and digest apoptotic cells arecalled “tingible body macrophages” and are frequently foundwithin the reactive germinal centers of lymphoid follicles oroccasionally within the thymic cortex. The tingible bodiesare the bits of nuclear debris from the apoptotic cells. Thereis essentially no inflammatory reaction associated with theprocess of apoptosis nor with the removal of apoptotic cellsbecause: (1) apoptotic cells do not release their cellular con-stituents into the surrounding interstitial tissue; (2) they arequickly phagocytosed by surrounding cells thus likely pre-venting secondary necrosis; and, (3) the engulfing cells donot produce anti-inflammatory cytokines (Savill and Fadok,2000; Kurosaka et al., 2003).

Distinguishing Apoptosis from NecrosisThe alternative to apoptotic cell death is necrosis, which

is considered to be a toxic process where the cell is a passivevictim and follows an energy-independent mode of death. Butsince necrosis refers to the degradative processes that occurafter cell death, it is considered by some to be an inappro-priate term to describe a mechanism of cell death. Oncosisis therefore used to describe a process that leads to necrosiswith karyolysis and cell swelling whereas apoptosis leads tocell death with cell shrinkage, pyknosis, and karyorrhexis.Therefore the terms “oncotic cell death” and “oncotic necro-sis” have been proposed as alternatives to describe cell deaththat is accompanied by cell swelling, but these terms are notwidely used at this time (Majno and Joris, 1995; Levin et al.,1999).

Although the mechanisms and morphologies of apopto-sis and necrosis differ, there is overlap between these twoprocesses. Evidence indicates that necrosis and apoptosisrepresent morphologic expressions of a shared biochemi-cal network described as the “apoptosis-necrosis continuum”(Zeiss, 2003). For example, two factors that will convert anongoing apoptotic process into a necrotic process include adecrease in the availability of caspases and intracellular ATP

TABLE 1.—Comparison of morphological features of apoptosis and necrosis.

Apoptosis Necrosis

Single cells or small clusters of cells Often contiguous cellsCell shrinkage and convolution Cell swellingPyknosis and karyorrhexis Karyolysis, pyknosis, and

karyorrhexisIntact cell membrane Disrupted cell membraneCytoplasm retained in apoptotic bodies Cytoplasm releasedNo inflammation Inflammation usually present

(Leist et al., 1997; Denecker et al., 2001). Whether a cell diesby necrosis or apoptosis depends in part on the nature of thecell death signal, the tissue type, the developmental stage ofthe tissue and the physiologic milieu (Fiers et al., 1999; Zeiss,2003).

Using conventional histology, it is not always easy to dis-tinguish apoptosis from necrosis, and they can occur simul-taneously depending on factors such as the intensity and du-ration of the stimulus, the extent of ATP depletion and theavailability of caspases (Zeiss, 2003). Necrosis is an uncon-trolled and passive process that usually affects large fields ofcells whereas apoptosis is controlled and energy-dependentand can affect individual or clusters of cells. Necrotic cellinjury is mediated by two main mechanisms; interferencewith the energy supply of the cell and direct damage to cellmembranes.

Some of the major morphological changes that occur withnecrosis include cell swelling; formation of cytoplasmic vac-uoles; distended endoplasmic reticulum; formation of cyto-plasmic blebs; condensed, swollen or ruptured mitochondria;disaggregation and detachment of ribosomes; disrupted or-ganelle membranes; swollen and ruptured lysosomes; andeventually disruption of the cell membrane (Kerr et al., 1972;Majno and Joris, 1995; Trump et al., 1997). This loss of cellmembrane integrity results in the release of the cytoplasmiccontents into the surrounding tissue, sending chemotatic sig-nals with eventual recruitment of inflammatory cells. Becauseapoptotic cells do not release their cellular constituents intothe surrounding interstitial tissue and are quickly phagocy-tosed by macrophages or adjacent normal cells, there is es-sentially no inflammatory reaction (Savill and Fadok, 2000;Kurosaka et al., 2003). It is also important to note that pykno-sis and karyorrhexis are not exclusive to apoptosis and canbe a part of the spectrum of cytomorphological changes thatoccurs with necrosis (Cotran et al., 1999). Table 1 comparessome of the major morphological features of apoptosis andnecrosis.

Is Apoptosis an Irreversible Process?Many of the genes that control the killing and engulfment

processes of programmed cell death have been identified, andthe molecular mechanisms underlying these processes haveproven to be evolutionarily conserved (Metzstein et al., 1998).Until recently, apoptosis has traditionally been consideredan irreversible process with caspase activation committing acell to death and the engulfment genes serving the purposeof dead cell removal. However, the uptake and clearance ofapoptotic cells by macrophages may involve more than justthe removal of cell debris. Hoeppner et al. have shown thatblocking engulfment genes in C. elegans embryos enhancescell survival when cells are subjected to weak pro-apoptoticsignals (Hoeppner et al., 2001).



Vol. 35, No. 4, 2007 REVIEW OF APOPTOSIS 497

FIGURE 1.—Figure 1A is a photomicrograph of a section of exocrine pancreas from a B6C3F1 mouse. The arrows indicate apoptotic cells that are shrunken withcondensed cytoplasm. The nuclei are pyknotic and fragmented. Note the lack of inflammation. Figure 1B is an image of myocardium from a 14 week-old rat treatedwith ephedrine (25 mg/kg) and caffeine (30 mg/kg). Within the interstitial space there are apoptotic cells with condensed cytoplasm, condensed and hyperchromaticchromatin and fragmented nuclei (long arrows). Admixed with the apoptotic bodies are macrophages, some with engulfed apoptotic bodies (arrowheads) (Howdenet al., 2004, by permission of the American Journal of Physiology). Figure 1C is a photomicrograph of normal thymus tissue from a control Sprague–Dawley ratfor comparison. Figure ID illustrates sheets of apoptotic cells in the thymus from a rat that was treated with dexamethasone to induce lymphocyte apoptosis. Underphysiological conditions, apoptosis typically affects single cells or small clusters of cells. However, the degree of apoptosis in this treated rat is more severe due to theamount of dexamethasone administered (1 mg/kg bodyweight) and the time posttreatment (12 hours). The majority of lymphocytes are apoptotic although there area few interspersed cells that are morphologically normal and most likely represent lymphoblasts or macrophages. The apoptotic lymphocytes are small and deeplybasophilic with pyknotic and often-fragmented nuclei. Macrophages are present with engulfed cytoplasmic apoptotic bodies (arrows). H&E.




FIGURE 1.—(Continued)

Reddien et al. demonstrated that, in C. elegans, mutationsthat cause partial loss of function of killer genes allow the sur-vival of some cells that are programmed to die via apoptosis,and mutations in engulfment genes enhance the frequency ofthis cell survival. Moreover, mutations in engulfment genes

alone allowed the survival and differentiation of some cellsthat were otherwise destined to die via apoptosis (Reddienet al., 2001). These findings suggest that genes that medi-ate corpse removal can also function to actively kill cells. Inother words, the engulfing cells may act to ensure that cells




FIGURE 2.—Figure 2A is a transmission electron micrograph (TEM) of the normal thymus tissue depicted in Figure 1C. The lymphocytes are closely packed, havelarge nuclei and scant cytoplasm. Figure 2B is a TEM of apoptotic thymic lymphocytes in an early phase of apoptosis with condensed and peripheralized chromatin.The cytoplasm is beginning to condense and the cell outlines are irregular. The arrow indicates a fragmented section of nucleus and the arrowhead most likely indicatesan apoptotic body that seems to contain predominantly cytoplasm without organelles or nuclear material. Figure 2C illustrates an apoptotic lymphocyte in the processof “budding” or extrusion of membrane-bound cytoplasm containing organelles (arrow). Once the budding has occurred, this extruded fragment will be an “apoptoticbody.” These apoptotic bodies are membrane-bound and thus do not release cytoplasmic contents into the interstitium. Macrophages or other adjacent healthy cellssubsequently engulf the apoptotic bodies. For these reasons, apoptosis does not incite an inflammatory reaction. Figure 2D is a TEM of a section of thymus withlymphocytes in various stages of apoptosis. The large cell in the center of the photomicrograph is a macrophage with engulfed intracytoplasmic apoptotic bodies.This macrophage is also called a “tingible body macrophage.” The arrowhead indicates a lymphocyte in an advanced stage of apoptosis with nuclear fragmentation.




FIGURE 2.—(Continued)

triggered to undergo apoptosis will die rather than recoverafter the initial stages of death.

In vertebrates, there is some evidence of a potential rolefor macrophages in promoting the death of cells in some tis-sues. Elimination of macrophages in the anterior chamberof the rat eye resulted in the survival of vascular endothe-lial cells that normally undergo apoptosis (Diez-Roux and

Lang, 1997). Other studies have demonstrated that inhibitionof macrophages can disrupt the remodeling of tissues in themouse eye or in the tadpole tail during regression; two pro-cesses that involve apoptosis (Lang and Bishop, 1993; Littleand Flores, 1993). Geske and coworkers (2001) demonstratedthat early p53-induced apoptotic cells can be rescued fromthe apoptotic program if the apoptotic stimulus is removed.




Their research suggests that DNA repair is activated early inthe p53-induced apoptotic process and that this DNA repairmay be involved in reversing the cell death pathway in somecircumstances.

Mechanisms of ApoptosisThe mechanisms of apoptosis are highly complex and so-

phisticated, involving an energy-dependent cascade of molec-ular events (Figure 3). To date, research indicates that thereare two main apoptotic pathways: the extrinsic or deathreceptor pathway and the intrinsic or mitochondrial path-way. However, there is now evidence that the two path-ways are linked and that molecules in one pathway caninfluence the other (Igney and Krammer, 2002). There isan additional pathway that involves T-cell mediated cyto-toxicity and perforin-granzyme-dependent killing of the cell.The perforin/granzyme pathway can induce apoptosis via ei-ther granzyme B or granzyme A. The extrinsic, intrinsic, andgranzyme B pathways converge on the same terminal, or ex-ecution pathway. This pathway is initiated by the cleavageof caspase-3 and results in DNA fragmentation, degradationof cytoskeletal and nuclear proteins, cross-linking of pro-teins, formation of apoptotic bodies, expression of ligandsfor phagocytic cell receptors and finally uptake by phago-cytic cells. The granzyme A pathway activates a parallel,caspase-independent cell death pathway via single strandedDNA damage (Martinvalet et al., 2005).

Biochemical Features: Apoptotic cells exhibit severalbiochemical modifications such as protein cleavage, proteincross-linking, DNA breakdown, and phagocytic recognitionthat together result in the distinctive structural pathology de-scribed previously (Hengartner, 2000). Caspases are widelyexpressed in an inactive proenzyme form in most cells and

FIGURE 3.—Schematic representation of apoptotic events. The two main pathways of apoptosis are extrinsic and intrinsic as well as a perforin/granzyme pathway.Each requires specific triggering signals to begin an energy-dependent cascade of molecular events. Each pathway activates its own initiator caspase (8, 9, 10)which in turn will activate the executioner caspase-3. However, granzyme A works in a caspase-independent fashion. The execution pathway results in characteristiccytomorphological features including cell shrinkage, chromatin condensation, formation of cytoplasmic blebs and apoptotic bodies and finally phagocytosis of theapoptotic bodies by adjacent parenchymal cells, neoplastic cells or macrophages.

once activated can often activate other pro-caspases, allow-ing initiation of a protease cascade. Some pro-caspases canalso aggregate and autoactivate. This proteolytic cascade, inwhich one caspase can activate other caspases, amplifies theapoptotic signaling pathway and thus leads to rapid cell death.

Caspases have proteolytic activity and are able to cleaveproteins at aspartic acid residues, although different caspaseshave different specificities involving recognition of neigh-boring amino acids. Once caspases are initially activated,there seems to be an irreversible commitment towards celldeath. To date, ten major caspases have been identified andbroadly categorized into initiators (caspase-2,-8,-9,-10), ef-fectors or executioners (caspase-3,-6,-7) and inflammatorycaspases (caspase-1,-4,-5) (Cohen, 1997; Rai et al., 2005).The other caspases that have been identified include caspase-11, which is reported to regulate apoptosis and cytokinematuration during septic shock, caspase-12, which mediatesendoplasmic-specific apoptosis and cytotoxicity by amyloid-β, caspase-13, which is suggested to be a bovine gene, andcaspase-14, which is highly expressed in embryonic tissuesbut not in adult tissues (Hu et al., 1998; Nakagawa et al.,2000, Koenig et al., 2001; Kang et al., 2002).

Extensive protein cross-linking is another characteristic ofapoptotic cells and is achieved through the expression andactivation of tissue transglutaminase (Nemes et al., 1996).DNA breakdown by Ca2+-and Mg2+-dependent endonucle-ases also occurs, resulting in DNA fragments of 180 to 200base pairs (Bortner et al., 1995). A characteristic “DNA lad-der” can be visualized by agarose gel electrophoresis with anethidium bromide stain and ultraviolet illumination.

Another biochemical feature is the expression of cell sur-face markers that result in the early phagocytic recogni-tion of apoptotic cells by adjacent cells, permitting quick




TABLE 2.—Extrinsic pathway proteins, abbreviations, and alternate nomenclature.

Abbreviation Protein Name Select Alternate Nomenclature

TNF-α Tumor necrosis factor alpha TNF ligand, TNFA, cachectinTNFR1 Tumor necrosis factor receptor 1 TNF receptor, TNFRSF1A, p55 TNFR, CD120aFasL Fatty acid synthetase ligand Fas ligand, TNFSF6, Apo1, apoptosis antigen ligand 1, CD95L, CD178, APT1LG1FasR Fatty acid synthetase receptor Fas receptor, TNFRSF6, APT1, CD95Apo3L Apo3 ligand TNFSF12, Apo3 ligand, TWEAK, DR3LGDR3 Death receptor 3 TNFRSF12, Apo3, WSL-1, TRAMP, LARD, DDR3Apo2L Apo2 ligand TNFSF10, TRAIL, TNF-related apoptosis inducing ligandDR4 Death receptor 4 TNFRSF10A, TRAILR1, APO2DR5 Death receptor 5 TNFRS10B, TRAIL-R2, TRICK2, KILLER, ZTNFR9FADD Fas-associated death domain MORT1TRADD TNF receptor-associated death domain TNFRSF1A associated via death domainRIP Receptor-interacting protein RIPK1DED Death effector domain Apoptosis antagonizing transcription factor, CHE1caspase-8 Cysteinyl aspartic acid-protease 8 FLICE, FADD-like Ice, Mach-1, Mch5c-FLIP FLICE-inhibitory protein Casper, I-FLICE, FLAME-1, CASH, CLARP, MRIT

References:1. Human Protein Reference Database 〈http://www.hprd.org/〉.2. ExPASy Proteomics Server 〈http://ca.expasy.org/〉.

phagocytosis with minimal compromise to the surround-ing tissue. This is achieved by the movement of the nor-mal inward-facing phosphatidylserine of the cell’s lipidbilayer to expression on the outer layers of the plasma mem-brane (Bratton et al., 1997). Although externalization ofphosphatidylserine is a well-known recognition ligand forphagocytes on the surface of the apoptotic cell, recent stud-ies have shown that other proteins are also be exposed on thecell surface during apoptotic cell clearance. These includeAnnexin I and calreticulin.

Annexin V is a recombinant phosphatidylserine-bindingprotein that interacts strongly and specifically with phos-phatidylserine residues and can be used for the detection ofapoptosis (Van Engeland et al., 1998; Arur et al., 2003). Cal-reticulin is a protein that binds to an LDL-receptor relatedprotein on the engulfing cell and is suggested to cooperatewith phosphatidylserine as a recognition signal (Gardai et al.,2005). The adhesive glycoprotein, thrombospondin-1, can beexpressed on the outer surface of activated microvascular en-dothelial cells and, in conjunction with CD36, caspase-3-likeproteases and other proteins, induce receptor-mediated apop-tosis (Jimenez et al., 2000).

Extrinsic Pathway: The extrinsic signaling pathways thatinitiate apoptosis involve transmembrane receptor-mediatedinteractions. These involve death receptors that are membersof the tumor necrosis factor (TNF) receptor gene superfamily(Locksley et al., 2001). Members of the TNF receptor fam-ily share similar cyteine-rich extracellular domains and havea cytoplasmic domain of about 80 amino acids called the“death domain” (Ashkenazi and Dixit, 1998). This death do-main plays a critical role in transmitting the death signal fromthe cell surface to the intracellular signaling pathways. Todate, the best-characterized ligands and corresponding deathreceptors include FasL/FasR, TNF-α/TNFR1, Apo3L/DR3,Apo2L/DR4 and Apo2L/DR5 (Chicheportiche et al., 1997;Ashkenazi et al., 1998; Peter and Kramer, 1998; Sulimanet al., 2001; Rubio-Moscardo et al., 2005).

The sequence of events that define the extrinsic phaseof apoptosis are best characterized with the FasL/FasR andTNF-α/TNFR1 models. In these models, there is clusteringof receptors and binding with the homologous trimeric lig-and. Upon ligand binding, cytplasmic adapter proteins are re-

cruited which exhibit corresponding death domains that bindwith the receptors. The binding of Fas ligand to Fas receptorresults in the binding of the adapter protein FADD and thebinding of TNF ligand to TNF receptor results in the bind-ing of the adapter protein TRADD with recruitment of FADDand RIP (Hsu et al., 1995; Grimm et al., 1996; Wajant, 2002).FADD then associates with procaspase-8 via dimerization ofthe death effector domain. At this point, a death-inducingsignaling complex (DISC) is formed, resulting in the auto-catalytic activation of procaspase-8 (Kischkel et al., 1995).

Once caspase-8 is activated, the execution phase of apop-tosis is triggered. Death receptor-mediated apoptosis can beinhibited by a protein called c-FLIP which will bind to FADDand caspase-8, rendering them ineffective (Kataoka et al.,1998; Scaffidi, 1999). Another point of potential apoptosisregulation involves a protein called Toso, which has beenshown to block Fas-induced apoptosis in T cells via inhibi-tion of caspase-8 processing (Hitoshi et al., 1998). Table 2lists the major extrinsic pathway proteins with common ab-breviations and some of the alternate nomenclature used foreach protein.

Perforin/granzyme Pathway: T-cell mediated cytotoxic-ity is a variant of type IV hypersensitivity where sensitizedCD8+ cells kill antigen-bearing cells. These cytotoxic T lym-phocytes (CTLs) are able to kill target cells via the extrinsicpathway and the FasL/FasR interaction is the predominantmethod of CTL-induced apoptosis (Brunner et al., 2003).However, they are also able to exert their cytotoxic effectson tumor cells and virus-infected cells via a novel pathwaythat involves secretion of the transmembrane pore-formingmolecule perforin with a subsequent exophytic release of cy-toplasmic granules through the pore and into the target cell(Trapani and Smyth, 2002). The serine proteases granzymeA and granzyme B are the most important component withinthe granules (Pardo et al., 2004).

Granzyme B will cleave proteins at aspartate residues andwill therefore activate pro-caspase-10 and can cleave factorslike ICAD (Inhibitor of Caspase Activated DNAse) (Sakahiraet al., 1998). Reports have also shown that granzyme B canutilize the mitochondrial pathway for amplification of thedeath signal by specific cleavage of Bid and induction ofcytochrome c release (Barry and Bleackley, 2002; Russell and




Ley, 2002). However, granzyme B can also directly activatecaspase-3. In this way, the upstream signaling pathways arebypassed and there is direct induction of the execution phaseof apoptosis.

It is suggested that both the mitochondrial pathway anddirect activation of caspase-3 are critical for granzyme B-induced killing (Goping et al., 2003). Recent findings indi-cate that this method of granzyme B cytotoxicity is criticalas a control mechanism for T cell expansion of type 2 helperT (Th2) cells (Devadas et al., 2006). Moreover, findings in-dicate that neither death receptors nor caspases are involvedwith the T cell receptor-induced apoptosis of activated Th2cells because blocking their ligands has no effect on apop-tosis. On the other hand, Fas-Fas ligand interaction, adapterproteins with death domains and caspases are all involved inthe apoptosis and regulation of cytotoxic Type 1 helper cellswhereas granzyme B has no effect.

Granzyme A is also important in cytotoxic T cell inducedapoptosis and activates caspase independent pathways. Oncein the cell, granzyme A activates DNA nicking via DNAseNM23-H1, a tumor suppressor gene product (Fan et al.,2003). This DNAse has an important role in immune surveil-lance to prevent cancer through the induction of tumor cellapoptosis. The nucleosome assembly protein SET normallyinhibits the NM23-H1 gene. Granzyme A protease cleavesthe SET complex thus releasing inhibition of NM23-H1, re-sulting in apoptotic DNA degradation. In addition to inhibit-ing NM23-H1, the SET complex has important functionsin chromatin structure and DNA repair. The proteins thatmake up this complex (SET, Ape1, pp32, and HMG2) seemto work together to protect chromatin and DNA structure(Lieberman and Fan, 2003). Therefore, inactivation of thiscomplex by granzyme A most likely also contributes to apop-tosis by blocking the maintenance of DNA and chromatinstructure integrity.

Intrinsic Pathway: The intrinsic signaling pathways thatinitiate apoptosis involve a diverse array of non-receptor-mediated stimuli that produce intracellular signals that actdirectly on targets within the cell and are mitochondrial-initiated events. The stimuli that initiate the intrinsic pathwayproduce intracellular signals that may act in either a positiveor negative fashion. Negative signals involve the absence ofcertain growth factors, hormones and cytokines that can leadto failure of suppression of death programs, thereby trig-gering apoptosis. In other words, there is the withdrawal offactors, loss of apoptotic suppression, and subsequent activa-tion of apoptosis. Other stimuli that act in a positive fashioninclude, but are not limited to, radiation, toxins, hypoxia,hyperthermia, viral infections, and free radicals.

All of these stimuli cause changes in the inner mito-chondrial membrane that results in an opening of the mi-tochondrial permeability transition (MPT) pore, loss ofthe mitochondrial transmembrane potential and release oftwo main groups of normally sequestered pro-apoptoticproteins from the intermembrane space into the cytosol(Saelens et al., 2004). The first group consists of cytochromec, Smac/DIABLO, and the serine protease HtrA2/Omi (Caiet al., 1998; Du et al., 2000; Loo et al., 2002; Garridoet al., 2005). These proteins activate the caspase-dependentmitochondrial pathway. Cytochrome c binds and activates

Apaf-1 as well as procaspase-9, forming an “apoptosome”(Chinnaiyan, 1999; Hill et al., 2004).

The clustering of procaspase-9 in this manner leads tocaspase-9 activation. Smac/DIABLO and HtrA2/Omi are re-ported to promote apoptosis by inhibiting IAP (inhibitors ofapoptosis proteins) activity (van Loo et al., 2002a; Schimmer,2004). Additional mitochondrial proteins have also beenidentified that interact with and suppress the action of IAPhowever gene knockout experiments suggest that binding toIAP alone may not be enough evidence to label a mitochon-drial protein as “pro-apoptotic” (Ekert and Vaux, 2005).

The second group of pro-apoptotic proteins, AIF, endonu-clease G and CAD, are released from the mitochondria dur-ing apoptosis, but this is a late event that occurs after thecell has committed to die. AIF translocates to the nucleusand causes DNA fragmentation into ∼50–300 kb pieces andcondensation of peripheral nuclear chromatin (Joza et al.,2001). This early form of nuclear condensation is referredto as “stage I” condensation (Susin et al., 2000). Endonu-clease G also translocates to the nucleus where it cleavesnuclear chromatin to produce oligonucleosomal DNA frag-ments (Li et al., 2001). AIF and endonuclease G both functionin a caspase-independent manner. CAD is subsequently re-leased from the mitochondria and translocates to the nucleuswhere, after cleavage by caspase-3, it leads to oligonucleo-somal DNA fragmentation and a more pronounced and ad-vanced chromatin condensation (Enari et al., 1998). This laterand more pronounced chromatin condensation is referred toas “stage II” condensation (Susin et al., 2000).

The control and regulation of these apoptotic mitochon-drial events occurs through members of the Bcl-2 family ofproteins (Cory and Adams, 2002). The tumor suppressor pro-tein p53 has a critical role in regulation of the Bcl-2 familyof proteins, however the exact mechanisms have not yet beencompletely elucidated (Schuler and Green, 2001). The Bcl-2family of proteins governs mitochondrial membrane perme-ability and can be either pro-apoptotic or anti-apoptotic. Todate, a total of 25 genes have been identified in the Bcl-2 fam-ily. Some of the anti-apoptotic proteins include Bcl-2, Bcl-x,Bcl-XL, Bcl-XS, Bcl-w, BAG, and some of the pro-apoptoticproteins include Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, andBlk. These proteins have special significance since they candetermine if the cell commits to apoptosis or aborts the pro-cess. It is thought that the main mechanism of action of theBcl-2 family of proteins is the regulation of cytochrome c re-lease from the mitochondria via alteration of mitochondrialmembrane permeability.

A few possible mechanisms have been studied but nonehave been proven definitively. Mitochondrial damage in theFas pathway of apoptosis is mediated by the caspase-8 cleav-age of Bid (Li et al., 1998; Esposti, 2002). This is one exam-ple of the “cross-talk” between the death-receptor (extrinsic)pathway and the mitochondrial (intrinsic) pathway (Igneyand Krammer, 2002). Serine phosphorylation of Bad is asso-ciated with 14-3-3, a member of a family of multifunctionalphosphoserine binding molecules. When Bad is phosphory-lated, it is trapped by 14-3-3 and sequestered in the cytosolbut once Bad is unphosphorylated, it will translocate to themitochondria to release cytochrome C (Zha, et al., 1996).

Bad can also heterodimerize with Bcl-Xl or Bcl-2, neutral-izing their protective effect and promoting cell death (Yang




TABLE 3.—Intrinsic pathway proteins, abbreviations, and alternate nomenclature.

Abbreviation Protein Name Select Alternate Nomenclature

Smac/DIABLO Second mitochondrial activator of caspases/direct IAPbinding protein with low PI

None

HtrA2/Omi High-temperature requirement Omi stress regulated endoprotease, serine protease Omiprotein A2

IAP Inhibitor of Apoptosis Proteins XIAP, API3, ILP, HILP, HIAP2, cIAP1, API1, MIHB,NFR2-TRAF signaling complex protein

Apaf-1 Apoptotic protease activating factor APAF1Caspase-9 Cysteinyl aspartic acid-protease-9 ICE-LAP6, Mch6, Apaf-3AIF Apoptosis Inducing Factor Programmed cell death protein 8, mitochondrialCAD Caspase-Activated DNAse CAD/CPAN/DFF40Bcl-2 B-cell lymphoma protein 2 Apoptosis regulator Bcl-2Bcl-x BCL2 like 1 BCL2 related proteinBcl-XL BCL2 related protein, long isoform BCL2L protein, long form of Bcl-xBcl-XS BCL2 related protein, short isoformBcl-w BCL2 like 2 protein Apoptosis regulator BclWBAG BCL2 associated athanogene BAG family molecular chaperone regulatorBcl-10 B-cell lymphoma protein 10 mE10, CARMEN, CLAP, CIPERBAX BCL2 associated X protein Apoptosis regulator BAXBAK BCL2 antagonist killer 1 BCL2L7, cell death inhibitor 1BID BH3 interacting domain death agonist p22 BIDBAD BCL2 antagonist of cell death BCL2 binding protein, BCL2L8, BCL2 binding

component 6, BBC6, Bcl-XL/Bcl-2 associated deathpromoter

BIM BCL2 interacting protein BIM BCL2 like 11BIK BCL2 interacting killer NBK, BP4, BIP1, apoptosis inducing NBKBlk Bik-like killer protein B lymphoid tyrosine kinase, p55-BLK, MGC10442Puma BCL2 binding component 3 JFY1, PUMA/JFY1, p53 up-regulated modulator of

apoptosisNoxa Phorbol-12-myristate-13-acetate-induced protein 1 PMA induced protein 1, APR14-3-3 Tyrosine 3-monooxygenase/tryptophan

5-monooxygenase activation protein14-3-3 eta, theta, zeta, beta, epsilon, sigma, gamma

Aven Cell death regulator Aven NoneMyc Oncogene Myc c-myc, Myc proto-oncogene protein

References1. Human Protein Reference Database 〈http://www.hprd.org/〉.2. ExPASy Proteomics Server 〈http://ca.expasy.org/〉.

et al., 1995). When not sequestered by Bad, both Bcl-2 andBcl-Xl inhibit the release of cytochrome C from the mito-chondria although the mechanism is not well understood. Re-ports indicate that Bcl-2 and Bcl-XL inhibit apoptotic deathprimarily by controlling the activation of caspase proteases(Newmeyer et al., 2000). An additional protein designated“Aven” appears to bind both Bcl-Xl and Apaf-1, thereby pre-venting activation of procaspase-9 (Chau et al., 2000). Thereis evidence that overexpression of either Bcl-2 or Bcl-Xl willdown-regulate the other, indicating a reciprocal regulationbetween these two proteins.

Puma and Noxa are two members of the Bcl2 family thatare also involved in pro-apoptosis. Puma plays an impor-tant role in p53-mediated apoptosis. It was shown that, invitro, overexpression of Puma is accompanied by increasedBAX expression, BAX conformational change, transloca-tion to the mitochondria, cytochrome c release and reduc-tion in the mitochondrial membrane potential (Liu et al.,2003). Noxa is also a candidate mediator of p53-inducedapoptosis. Studies show that this protein can localize to themitochondria and interact with anti-apoptotic Bcl-2 fam-ily members, resulting in the activation of caspase-9 (Odaet al., 2000). Since both Puma and Noxa are induced by p53,they might mediate the apoptosis that is elicited by geno-toxic damage or oncogene activation. The Myc oncoproteinhas also been reported to potentiate apoptosis through bothp53-dependent and -independent mechanisms (Meyer et al.,2006).

Further elucidation of these pathways should have impor-tant implications for tumorigenesis and therapy. Table 3 lists

the major intrinsic pathway proteins with common abbrevi-ations and some of the alternate nomenclature used for eachprotein.

Execution PathwayThe extrinsic and intrinsic pathways both end at the point

of the execution phase, considered the final pathway of apop-tosis. It is the activation of the execution caspases that beginsthis phase of apoptosis. Execution caspases activate cyto-plasmic endonuclease, which degrades nuclear material, andproteases that degrade the nuclear and cytoskeletal proteins.Caspase-3, caspase-6, and caspase-7 function as effector or“executioner” caspases, cleaving various substrates includingcytokeratins, PARP, the plasma membrane cytoskeletal pro-tein alpha fodrin, the nuclear protein NuMA and others, thatultimately cause the morphological and biochemical changesseen in apoptotic cells (Slee et al., 2001).

Caspase-3 is considered to be the most important of theexecutioner caspases and is activated by any of the initiatorcaspases (caspase-8, caspase-9, or caspase-10). Caspase-3specifically activates the endonuclease CAD. In proliferatingcells CAD is complexed with its inhibitor, ICAD. In apop-totic cells, activated caspase-3 cleaves ICAD to release CAD(Sakahira et al., 1998). CAD then degrades chromosomalDNA within the nuclei and causes chromatin condensation.Caspase-3 also induces cytoskeletal reorganization and disin-tegration of the cell into apoptotic bodies. Gelsolin, an actinbinding protein, has been identified as one of the key sub-strates of activated caspase-3.




Gelsolin will typically act as a nucleus for actin polymer-ization and will also bind phosphatidylinositol biphosphate,linking actin organization and signal transduction. Caspase-3will cleave gelsolin and the cleaved fragments of gelsolin, inturn, cleave actin filaments in a calcium independent manner.This results in disruption of the cytoskeleton, intracellulartransport, cell division, and signal transduction (Kothakotaet al., 1997).

Phagocytic uptake of apoptotic cells is the last componentof apoptosis. Phospholipid asymmetry and externalizationof phosphatidylserine on the surface of apoptotic cells andtheir fragments is the hallmark of this phase. Although themechanism of phosphatidylserine translocation to the outerleaflet of the cell during apoptosis is not well understood, ithas been associated with loss of aminophospholipid translo-case activity and nonspecific flip-flop of phospholipids ofvarious classes (Bratton et al., 1997). Research indicates thatFas, caspase-8, and caspase-3 are involved in the regulationof phosphatidylserine externalization on oxidatively stressederythrocytes however caspase-independent phosphatidylser-ine exposure occurs during apoptosis of primary T lympho-cytes (Ferraro-Peyret et al., 2002; Mandal et al., 2005).

The appearance of phosphotidylserine on the outer leafletof apoptotic cells then facilitates noninflammatory phago-cytic recognition, allowing for their early uptake and disposal(Fadok et al., 2001). This process of early and efficient uptakewith no release of cellular constituents, results in essentiallyno inflammatory response. Table 4 lists the major proteins inthe execution pathway with common abbreviations and someof the alternate nomenclature used for each protein.

PHYSIOLOGIC APOPTOSIS

The role of apoptosis in normal physiology is as significantas that of its counterpart, mitosis. It demonstrates a comple-mentary but opposite role to mitosis and cell proliferation inthe regulation of various cell populations. It is estimated thatto maintain homeostasis in the adult human body, around 10billion cells are made each day just to balance those dyingby apoptosis (Renehan et al., 2001). And that number can in-crease significantly when there is increased apoptosis duringnormal development and aging or during disease.

Apoptosis is critically important during various devel-opmental processes. As examples, both the nervous sys-tem and the immune system arise through overproductionof cells. This initial overproduction is then followed by thedeath of those cells that fail to establish functional synaptic

TABLE 4.—Execution pathway proteins, abbreviations, and alternate nomenclature.

Abbreviation Protein name Select alternate nomenclature

Caspase-3 Cysteinyl aspartic acid-protease-3 CPP32, Yama, Apopain, SCA-1, LICECaspase-6 Cysteinyl aspartic acid-protease-6 Mch-2Caspase-7 Cysteinyl aspartic acid-protease-7 Mch-3, ICE-LAP-3, CMH-1Caspase-10 Cysteinyl aspartic acid-protease-10 Mch4, FLICE-2PARP Poly (ADP-ribose) polymerase ADP ribosyl transferase, ADPRT1, PPOLAlpha fodrin Spectrin alpha chain Alpha-II spectrin, fodrin alpha chainNuMA Nuclear mitotic apparatus protein SP-H antigenCAD Caspase-activated DNAse DNA fragmentation factor subunit beta, DFF-40, caspase-activated nuclease, CPANICAD Inhibitor of CAD DNA fragmentation factor subunit alpha, DFF-45

References1. Human Protein Reference Database 〈http://www.hprd.org/〉2. ExPASy Proteomics Server 〈http://ca.expasy.org/〉

connections or productive antigen specificities, respectively(Nijhawan et al., 2000; Opferman and Korsmeyer, 2003).

Apoptosis is also necessary to rid the body of pathogen-invaded cells and is a vital component of wound healing inthat it is involved in the removal of inflammatory cells and theevolution of granulation tissue into scar tissue (Greenhalgh,1998). Dysregulation of apoptosis during wound healing canlead to pathologic forms of healing such as excessive scarringand fibrosis. Apoptosis is also needed to eliminate activatedor auto-aggressive immune cells either during maturation inthe central lymphoid organs (bone marrow and thymus) or inperipheral tissues (Osborne, 1996).

Additionally, apoptosis is central to remodeling in theadult, such as the follicular atresia of the postovulatory fol-licle and post-weaning mammary gland involution, to namea couple of examples (Tilly, 1991; Lund et al., 1996). Fur-thermore, as organisms grow older, some cells begin to de-teriorate at a faster rate and are eliminated via apoptosis.One theory is that oxidative stress plays a primary role in thepathophysiology of age-induced apoptosis via accumulatedfree-radical damage to mitochondrial DNA (Harman, 1992;Ozawa, 1995). It is clear that apoptosis has to be tightly regu-lated since too little or too much cell death may lead to pathol-ogy, including developmental defects, autoimmune diseases,neurodegeneration, or cancer.

PATHOLOGIC APOPTOSIS

Abnormalities in cell death regulation can be a significantcomponent of diseases such as cancer, autoimmune lym-phoproliferative syndrome, AIDS, ischemia, and neurode-generative diseases such as Parkinson’s disease, Alzheimer’sdisease, Huntington’s disease, and Amyotrophic Lateral Scle-rosis. Some conditions feature insufficient apoptosis whereasothers feature excessive apoptosis.

Cancer is an example where the normal mechanisms of cellcycle regulation are dysfunctional, with either an overprolif-eration of cells and/or decreased removal of cells (King andCidlowski, 1998). In fact, suppression of apoptosis duringcarcinogenesis is thought to play a central role in the devel-opment and progression of some cancers (Kerr et al., 1994).There are a variety of molecular mechanisms that tumor cellsuse to suppress apoptosis.

Tumor cells can acquire resistance to apoptosis by theexpression of anti-apoptotic proteins such as Bcl-2 or by thedown-regulation or mutation of pro-apoptotic proteins suchas Bax. The expression of both Bcl-2 and Bax is regulated by




the p53 tumor suppressor gene (Miyashita, 1994). Certainforms of human B cell lymphoma have overexpression ofBcl-2, and this is one of the first and strongest lines ofevidence that failure of cell death contributes to cancer (Vauxet al., 1988). Another method of apoptosis suppression incancer involves evasion of immune surveillance (Smyth et al.,2001).

Certain immune cells (T cells and natural killer cells) nor-mally destroy tumor cells via the perforin/granzyme B path-way or the death-receptor pathway. In order to evade immunedestruction, some tumor cells will diminish the response ofthe death receptor pathway to FasL produced by T cells. Thishas been shown to occur in a variety of ways including down-regulation of the Fas receptor on tumor cells. Other mech-anisms include expression of nonfunctioning Fas receptor,secretion of high levels of a soluble form of the Fas receptorthat will sequester the Fas ligand or expression of Fas lig-and on the surface of tumor cells (Cheng et al., 1994; Cefaiet al., 2001; Elnemr et al., 2001). In fact, some tumor cellsare capable of a Fas ligand-mediated “counterattack” thatresults in apoptotic depletion of activated tumor infiltratinglymphocytes (Koyama et al., 2001).

Alterations of various cell signaling pathways can resultin dysregulation of apoptosis and lead to cancer. The p53tumor suppressor gene is a transcription factor that regulatesthe cell cycle and is the most widely mutated gene in humantumorigenesis (Wang and Harris, 1997). The critical role ofp53 is evident by the fact that it is mutated in over 50% of allhuman cancers. p53 can activate DNA repair proteins whenDNA has sustained damage, can hold the cell cycle at theG1/S regulation point on DNA damage recognition, and caninitiate apoptosis if the DNA damage proves to be irrepara-ble (Pientenpol and Stewart, 2002). Tumorigenesis can occurif this system goes awry. If the p53 gene is damaged, thentumor suppression is severely reduced. The p53 gene can bedamaged by radiation, various chemicals, and viruses such asthe Human papillomavirus (HPV). People who inherit onlyone functional copy of this gene will most likely developLi–Fraumeni syndrome, which is characterized by the devel-opment of tumors in early adulthood (Varley et al., 1997; Guet al., 2001).

The ataxia telangiectasia-mutated gene (ATM) has alsobeen shown to be involved in tumorigenesis via the ATM/p53signaling pathway (Kitagawa and Kastan, 2005). The ATMgene encodes a protein kinase that acts as a tumor suppres-sor. ATM activation, via ionizing radiation damage to DNA,stimulates DNA repair and blocks cell cycle progression.One mechanism through which this occurs is ATM depen-dent phosphorylation of p53 (Kurz and Lees-Miller, 2004).

As mentioned previously, p53 then signals growth arrestof the cell at a checkpoint to allow for DNA damage repairor can cause the cell to undergo apoptosis if the damage can-not be repaired. This system can also be inactivated by anumber of mechanisms including somatic genetic/epigeneticalterations and expression of oncogenic viral proteins such asthe HPV, leading to tumorigenesis (Bolt et al., 2005). Othercell signaling pathways can also be involved in tumor de-velopment. For example, upregulation of the phosphatidyli-nositol 3-kinase/AKT pathway in tumor cells renders themindependent of survival signals. In addition to regulation ofapoptosis, this pathway regulates other cellular processes,

such as proliferation, growth, and cytoskeletal rearrangement(Vivanco and Sawyers, 2002).

In addition to cancer, too little apoptosis can also resultin diseases such as autoimmune lymphoproliferative syn-drome (ALPS) (Worth et al., 2006). This occurs when thereis insufficient apoptosis of auto-aggressive T cells, resultingin multiple autoimmune diseases. An overproliferation of Bcells occurs as well, resulting in excess immunoglobulin pro-duction, leading to autoimmunity. Some of the common dis-eases of ALPS include hemolytic anemia, immune-mediatedthrombocytopenia, and autoimmune neutropenia. The differ-ent types of this condition are caused by different mutations.Type 1A results from a mutation in the death domain of theFas receptor, Type 1B results from a mutation in Fas ligandand Type 2 results from a mutation in caspase-10, reducingits activity.

Excessive apoptosis may also be a feature of some condi-tions such as autoimmune diseases, neurodegenerative dis-eases, and ischemia-associated injury. Autoimmune defi-ciency syndrome (AIDS) is an example of an autoimmunedisease that results from infection with the human immun-odeficiency virus (HIV) (Li et al., 1995). This virus infectsCD4+ T cells by binding to the CD4 receptor. The virus issubsequently internalized into the T cell where the HIV Tatprotein is thought to increase the expression of the Fas recep-tor, resulting in excessive apoptosis of T cells.

Alzheimer’s disease is a neurodegenerative condition thatis thought to be caused by mutations in certain proteins suchas APP (amyloid precursor protein) and presenilins. Prese-nilins are thought to be involved in the processing of APP toamyloid β. This condition is associated with the depositionof amyloid β in extracellular deposits known as plaques andamyloid β is thought to be neurotoxic when found in aggre-gated plaque form. Amyloid β is thought to induce apoptosisby causing oxidative stress or by triggering increased Fasligand expressions in neurons and glia. It may also activatemicroglia, which would result in TNFα secretion and activa-tion of the TNF-R1, leading to apoptosis (Ethell and Buhler,2003).

Excessive apoptosis is also thought to play an importantrole in various ischemia-associated injuries. One example ismyocardial ischemia caused by an insufficient blood supply,leading to a decrease in oxygen delivery to, and subsequentdeath of, the cardiomyocytes. Although necrosis does occur,overexpression of BAX has been detected in ischemic my-ocardial tissue and therapy aimed at reducing apoptosis hasshown some success in reducing the degree of tissue damage(Hochhauser et al., 2003). One hypothesis is that the damageproduced by ischemia is capable of initiating apoptosis butif ischemia is prolonged, necrosis occurs. If energy produc-tion is restored, as with reperfusion, the apoptotic cascadethat was initiated by ischemia may proceed (Freude et al.,2000). Although the extent to which apoptosis is involvedin myocardial ischemia remains to be clarified, there is clearevidence that supports a role for this mode of cell death.

INHIBITION OF APOPTOSIS

There are many pathological conditions that feature ex-cessive apoptosis (neurodegenerative diseases, AIDS, is-chemia, etc.) and thus may benefit from artificially inhibit-ing apoptosis. As our understanding of the field evolves, the




identification and exploitation of new targets remains a con-siderable focus of attention (Nicholson, 2000). A short list ofpotential methods of anti-apoptotic therapy includes stimu-lation of the IAP (inhibitors of apoptosis proteins) family ofproteins, caspase inhibition, PARP (poly [ADP-ribose] poly-merase) inhibition, stimulation of the PKB/Akt (protein ki-nase B) pathway, and inhibition of Bcl-2 proteins.

The IAP family of proteins is perhaps the most impor-tant regulators of apoptosis due to the fact that they regulateboth the intrinsic and extrinsic pathways (Deveraux and Reed,1999). Eight human IAP proteins have now been identifiedalthough XIAP (X-linked mammalian inhibitor of apopto-sis protein) and survivin remain the better-known members(Silke et al., 2002; Colnaghi et al., 2006). So far, membersof the IAP family have been investigated as therapeutic tar-gets for the treatment of stroke, spinal cord injuries, multiplesclerosis as well as cancer. The synthetic nonspecific cas-pase inhibitor z-VAD-fmk was shown to reduce the severityof myocardial reperfusion injury in rat and mouse modelsof myocardial infarction (Mocanu et al., 2000). Specific in-hibitors of caspase activity may also prove beneficial. ICE(Interleukin-1 beta-converting enzyme), also called caspaseI, is a cysteine protease that appears to mediate intracellu-lar protein degradation during apoptosis (Livingston, 1997).ICE inhibitors have been developed to treat rheumatoid arthri-tis and other inflammatory conditions via reduction of inter-leukin 1β (Le and Abbenante, 2005).

Due to the dual role of PARP-1 in both DNA repair andapoptosis, the pharmacological use of PARP-1 inhibitors maybe able to attenuate ischemic and inflammatory cell and or-gan injury or may be able to enhance the cytotoxicity of an-titumor agents (Graziani and Szabo, 2005). Recent researchwith PARP-1 knockout mice indicates that the use of PARP-1inhibitors may be an effective therapy for the injury associ-ated with myocardial ischemia and reperfusion injury (Zhouet al., 2006). Infusion of insulin-like growth factor-1 (IGF-1), which stimulates PKB/Akt signaling and promotes cellsurvival, was shown to be beneficial in animal models ofmyocardial ischemia (Fujio et al., 2000).

Other studies with transgenic models of cardiac ischemiaand global brain ischemia indicate that inhibiting the expres-sion and/or function of Bax can prevent cytochrome c releasefrom mitochondria, inhibit the decrease in the mitochon-drial membrane potential, and protect cells against apoptosis(Hochhauser et al., 2003; Hetz et al., 2005). The potentialtherapeutic modalities mentioned here represent just a few ofthe past and current research efforts in this field. As the molec-ular and biochemical complexities of apoptosis continue to beelucidated, new therapeutic strategies will continue to evolve.

ASSAYS FOR APOPTOSIS

Since apoptosis occurs via a complex signaling cascadethat is tightly regulated at multiple points, there are many op-portunities to evaluate the activity of the proteins involved.As the activators, effectors and regulators of this cascade con-tinue to be elucidated, a large number of apoptosis assays aredevised to detect and count apoptotic cells. However, manyfeatures of apoptosis and necrosis can overlap, and it is there-fore crucial to employ two or more distinct assays to confirmthat cell death is occurring via apoptosis. One assay may de-tect early (initiation) apoptotic events and a different assay

may target a later (execution) event. The second assay, usedto confirm apoptosis, is generally based on a different prin-ciple. Multiplexing, which is the ability to gather more thanone set of data from the same sample, is another method-ology for apoptosis detection that is becoming increasinglypopular. There are a large variety of assays available, but eachassay has advantages and disadvantages which may make itacceptable to use for one application but inappropriate for an-other application (Watanabe et al., 2002; Otsuki et al., 2003).Therefore, when choosing methods of apoptosis detection incells, tissues or organs, understanding the pros and cons ofeach assay is crucial.

Understanding the kinetics of cell death in each modelsystem is also critical. Some proteins, such as caspases, areexpressed only transiently. Cultured cells undergoing apop-tosis in vitro will eventually undergo secondary necrosis.Apoptotic cells in any system can die and disappear relativelyquickly. The time from initiation of apoptosis to completioncan occur as quickly as 2–3 hours. Therefore a false negativecan occur if the assay is done too early or too late. More-over, apoptosis can occur at low frequency or in specific siteswithin organs, tissues and cultures. In such cases, the abilityto rapidly survey large areas could be useful. In general, if de-tailed information on the mechanism of cell death is desired,the duration of toxin exposure, the concentration of the testcompound and the choice of assay endpoint become critical.

A detailed description of all methodologies and assaysfor detecting apoptosis is beyond the scope of this article.However, some of the most commonly employed assays arementioned and briefly described. Apoptosis assays, based onmethodology, can be classified into six major groups and asubset of the available assays in each group is indicated andbriefly discussed:

1. Cytomorphological alterations2. DNA fragmentation3. Detection of caspases, cleaved substrates, regulators and

inhibitors4. Membrane alterations5. Detection of apoptosis in whole mounts6. Mitochondrial assays.

Cytomorphological AlterationsThe evaluation of hematoxylin and eosin-stained tissue

sections with light microscopy does allow the visualizationof apoptotic cells. Although a single apoptotic cell can bedetected with this method, confirmation with other methodsmay be necessary. Because the morphological events of apop-tosis are rapid and the fragments are quickly phagocytized,considerable apoptosis may occur in some tissues before itis histologically apparent. Additionally, this method detectsthe later events of apoptosis, so cells in the early phase ofapoptosis will not be detected.

Semi-ultrathin sections from an epoxy-resin-embeddedblock can be stained with toluidine blue or methylene blueto reveal intensely stained apoptotic cells when evaluatedby standard light microscopy. This methodology depends onthe nuclear and cytoplasmic condensation that occurs dur-ing apoptosis. The tissue and cellular details are preservedwith this technique and surveys of large tissue regions aredistinct advantages. However, smaller apoptotic bodies will




not be detected and healthy cells with large dense intracel-lular granules can be mistaken for apoptotic cells or debris.Additionally, there is loss of antigenicity during processingso that immunohistological or enzyme assays cannot be per-formed on the same tissue. However, this tissue may be usedfor transmission electron microscopy (TEM).

TEM is considered the gold standard to confirm apop-tosis. This is because categorization of an apoptotic cell isirrefutable if the cell contains certain ultrastructural morpho-logical characteristics (White and Cinti, 2004). These char-acteristics are: (1) electron-dense nucleus (marginalizationin the early phase); (2) nuclear fragmentation; (3) intact cellmembrane even late in the cell disintegration phase; (4) disor-ganized cytoplasmic organelles; (5) large clear vacuoles; and(6) blebs at the cell surface. As apoptosis progresses, thesecells will lose the cell-to-cell adhesions and will separatefrom neighboring cells. During the later phase of apoptosis,the cell will fragment into apoptotic bodies with intact cellmembranes and will contain cytoplasmic organelles with orwithout nuclear fragments. Phagocytosis of apoptotic bodiescan also be appreciated with TEM. The main disadvantagesof TEM are the cost, time expenditure, and the ability to onlyassay a small region at a time. Other disadvantages includethe difficulty in detecting apoptotic cells due to their transientnature and the inability to detect apoptotic cells at the earlieststages.

DNA FragmentationThe DNA laddering technique is used to visualize the en-

donuclease cleavage products of apoptosis (Wyllie, 1980).This assay involves extraction of DNA from a lysed cellhomogenate followed by agarose gel electrophoresis. Thisresults in a characteristic “DNA ladder” with each band inthe ladder separated in size by approximately 180 base pairs.This methodology is easy to perform, has a sensitivity of 1 ×106 cells (i.e., level of detection is as few as 1,000,000 cells),and is useful for tissues and cell cultures with high numbersof apoptotic cells per tissue mass or volume, respectively. Onthe other hand, it is not recommended in cases with low num-bers of apoptotic cells. There are other disadvantages to thisassay. Since DNA fragmentation occurs in the later phaseof apoptosis, the absence of a DNA ladder does not elimi-nate the potential that cells are undergoing early apoptosis.Additionally, DNA fragmentation can occur during prepara-tion making it difficult to produce a nucleosome ladder andnecrotic cells can also generate DNA fragments.

The TUNEL (Terminal dUTP Nick End-Labeling) methodis used to assay the endonuclease cleavage products by en-zymatically end-labeling the DNA strand breaks (Kresseland Groscurth, 1994; Ito and Otsuki, 1998). Terminal trans-ferase is used to add labeled UTP to the 3′-end of the DNAfragments. The dUTP can then be labeled with a varietyof probes to allow detection by light microscopy, fluores-cence microscopy or flow cytometry (Figure 4). The assaysare available as kits and can be acquired from a variety ofcompanies. This assay is also very sensitive, allowing detec-tion of a single cell via fluorescence microscopy or as few as∼100 cells via flow cytometry. It is also a fast technique andcan be completed within 3 hours. The disadvantages are costand the unknown parameter of how many DNA strand breaksare necessary for detection by this method. This method is

also subject to false positives from necrotic cells and cells inthe process of DNA repair and gene transcription. For thesereasons, it should be paired with another assay.

Detection of Caspases, Cleaved Substrates, Regulators,and Inhibitors

There are more than 13 known caspases (procaspases oractive cysteine caspases) that can be detected using varioustypes of caspase activity assays (Gurtu et al., 1997). Thereare also immunohistochemistry assays that can detect cleavedsubstrates such as PARP and known cell modifications suchas phosphorylated histones (Figure 5A) (Love et al., 1999;Talasz et al., 2002). Fluorescently conjugated caspase in-hibitors can also be used to label active caspases within cells(Grabarek et al., 2002). Caspase activation can be detected ina variety of ways including western blot, immunoprecipita-tion and immunohistochemistry (Figure 5B). Both polyclonaland monoclonal antibodies are available to both pro-caspasesand active caspases.

One method of caspase detection requires cell lysis in or-der to release the enzymes into the solution, coating of mi-crowells with anti-caspases; followed by detection with afluorescent labeled substrate. Detection of caspase activityby this method usually requires 1 × 105 cells. This tech-nique allows selection for individual initiator or executioncaspases. It also allows for rapid and consistent quantifica-tion of apoptotic cells. The major disadvantage is that theintegrity of the sample is destroyed thereby eliminating thepossibility of localizing the apoptotic event within the tissueor determining the type of cell that is undergoing apoptosis.Another disadvantage is that caspase activation does not nec-essarily indicate that apoptosis will occur. Moreover, there istremendous overlap in the substrate preferences of the mem-bers of the caspase family, affecting the specificity of theassay.

Apoptosis PCR microarray is a relatively new methodol-ogy that uses real-time PCR to profile the expression of atleast 112 genes involved in apoptosis (Hofmann et al., 2001;Vallat et al., 2003). These PCR microarrays are designedto determine the expression profile of genes that encode keyligands, receptors, intracellular modulators, and transcriptionfactors involved in the regulation of programmed cell death.Genes involved in anti-apoptosis can also be assessed withthis methodology. Comparison of gene expression in cells ortissues can be performed between test samples and controls.This type of assay allows for the evaluation of the expressionof a focused panel of genes related to apoptosis and severalcompanies offer apoptosis pathway-specific gene panels.

Hierarchical cluster analysis of genes can reveal distincttemporal expression patterns of transcriptional activationand/or repression. However, interpretation of the results canbe confounded by the large number of analyzed genes andby the methodological complexity. This methodology usesa 96-well plate and as little as 5 nanograms of total RNA.Every mRNA, or transcript, is labeled with a marker, such asa fluorescent dye. A real-time PCR instrument is used for ex-pression profiling. The location and intensity of the resultingsignals give an estimate of the quantity of each transcript inthe sample. The microarray test should be combined with adifferent methodology to confirm apoptosis.




FIGURE 4.—There are many ways of detecting apoptosis at different stages on histological sections. One commonly used method is called TUNEL (Terminaldeoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling). One of the major characteristics of apoptosis is the degradation of DNA after the activation ofCa/Mg dependent endonucleases. This DNA cleavage leads to strand breaks within the DNA. However, necrosis can also result in similar DNA cleavage. Thereforean additional method should be used to confirm apoptosis. The TUNEL method identifies cells with DNA strand breaks in situ by using terminal deoxynucleotidyltransferase (TdT) to transfer biotin-dUTP to the cleaved ends of the DNA. The biotin-labeled cleavage sites are then detected by reaction with HRP (horse radishperoxidase) conjugated streptavidin and visualized by DAB (diaminobenzidine), which gives a brown color. The tissue is typically counterstained with Toluidineblue to allow evaluation of tissue architecture. Figure 4A a photomicrograph of a section of testes from a B6C3F1 mouse that was in a National Toxicology Programbioassay. The TUNEL assay was used on this section of tissue to detect apoptotic cells. The seminiferous tubule is cut in cross section and has sperm, spermatogoniaand spermatocytes in various stages of development. The arrows point to labeled (dark brown) apoptotic spermatogonia in the basal layer of the seminiferousepithelium. Figure 4B is an image of myocardium from 14 week-old rat treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg). There are multiple apoptoticcells that have stained brown with the TUNEL method (arrows).




FIGURE 5.—When histological changes suggestive of apoptosis are detected in H&E stained tissues (i.e., Figure 1B), confirmation of apoptosis can be obtainedwith a variety of additional stains. The images in Figure 5 are of myocardium from 14-week-old rats treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg).Figure 5A is the interventricular septum stained by anti-phospho-H2A.X, a histone variant that becomes phosphorylated during apoptosis; note the strongly stainedapoptotic bodies (arrows). Figure 5B demonstrates the negative control of the same section shown in (A); nonimmune rabbit IgG was used at equivalent conditions inplace of the primary antibody. Note the unstained apoptotic bodies (arrows). Figure 5C illustrates cleaved caspase-3 staining for apoptosis revealing the presence ofintracytoplasmic positive myofibers (small arrows) located in the interventricular septum; large arrow indicates an apoptotic body. Figure 5D is a negative control ofthe same heart demonstrated in 5C. The small arrows demonstrate degenerating myofibers; large arrows indicate apoptotic bodies. Nyska et al., 2005, by permission2 of Oxford University Press.

Membrane AlterationsExternalization of phosphatidylserine residues on the outer

plasma membrane of apoptotic cells allows detection via An-nexin V in tissues, embryos or cultured cells (Bossy-Wetzeland Green, 2000). Once the apoptotic cells are bound withFITC-labeled Annexin V, they can be visualized with fluo-rescent microscopy. The advantages are sensitivity (can de-tect a single apoptotic cell) and the ability to confirm theactivity of initiator caspases. The disadvantage is that themembranes of necrotic cells are labeled as well. Therefore acritical control is to demonstrate the membrane integrity ofthe phosphatidylserine-positive cells. Since loss of membraneintegrity is a pathognomonic feature of necrotic cell death,necrotic cells will stain with specific membrane-impermeantnucleic acid dyes such as propidium iodide and trypan blue.Likewise, the membrane integrity of apoptotic cells can bedemonstrated by the exclusion of these dyes. The transfer ofphosphatidylserine to the outside of the cell membrane willalso permit the transport of certain dyes into the cell in a uni-

directional manner. As the cell accumulates dye and shrinksin volume, the cell dye content becomes more concentratedand can be visualized with light microscopy. This dye-uptakebioassay works on cell cultures, does not label necrotic cells,and has a high level of sensitivity (can detect a single apop-totic cell).

Detection of Apoptosis in Whole MountsApoptosis can also be visualized in whole mounts of em-

bryos or tissues using dyes such as acridine orange (AO),Nile blue sulfate (NBS), and neutral red (NR) (Zucker et al.,2000). Since these dyes are acidophilic, they are concen-trated in areas of high lysosomal and phagocytotic activity.The results would need to be validated with other apoptosisassays because these dyes cannot distinguish between lyso-somes degrading apoptotic debris from degradation of otherdebris such as microorganisms. Although all of these dyes arefast and inexpensive, they have certain disadvantages. AO istoxic and mutagenic and quenches rapidly under standard




conditions whereas NBS and NR do not penetrate thick tis-sues and can be lost during preparation for sectioning. Lyso-Tracker Red is another dye that acts in a similar way; howeverthis dye can be used with laser confocal microscopy to pro-vide 3-dimensional imaging of apoptotic cells. This dye isstable during processing, penetrates thick tissues and is re-sistant to quenching. This dye can be used for cell culture aswell as whole mounts of embryos, tissues, or organs.

Mitochondrial AssaysMitochondrial assays and cytochrome c release allow the

detection of changes in the early phase of the intrinsic path-way. Laser scanning confocal microscopy (LSCM) createssubmicron thin optical slices through living cells that can beused to monitor several mitochondrial events in intact singlecells over time (Bedner et al., 1999; Darzynkiewicz et al.,1999). Mitochondrial permeability transition (MPT), depo-larization of the inner mitochondrial membrane, Ca2+ fluxes,mitochondrial redox status, and reactive oxygen species canall be monitored with this methodology. The main disadvan-tage is that the mitochondrial parameters that this method-ology monitors can also occur during necrosis. The elec-trochemical gradient across the mitochondrial outer mem-brane (MOM) collapses during apoptosis, allowing detectionwith a fluorescent cationic dye (Poot and Pierce, 1999). Inhealthy cells this lipophilic dye accumulates in the mitochon-dria, forming aggregates that emit a specific fluorescence. Inapoptotic cells the MOM does not maintain the electrochem-ical gradient and the cationic dye diffuses into the cytoplasmwhere it emits a fluorescence that is different from the aggre-gated form.

Other mitochondrial dyes can be used that measure theredox potential or metabolic activity of the mitochondria incells. However, these dyes do not address the mechanismof cell death and should be used in conjunction with otherapoptosis detection methods such as a caspase assay.

Cytochrome c release from the mitochondria can also beassayed using fluorescence and electron microscopy in livingor fixed cells (Scorrano et al., 2002). However, cytochromec becomes unstable once it is released into the cytoplasm(Goldstein et al., 2000). Therefore a non-apoptotic controlshould be used to ensure that the staining conditions used areable to detect any available cytochrome c.

Apoptotic or anti-apoptotic regulator proteins such as Bax,Bid, and Bcl-2 can also be detected using fluorescence andconfocal microscopy (Tsien, 1998; Zhang et al., 2002). How-ever, the fluorescent protein tag may alter the interaction ofthe native protein with other proteins. Therefore, other apop-tosis assays should be used to confirm the results.

OTHER FORMS OF PROGRAMMED CELL DEATH

There is evidence of other forms of non-apoptotic pro-grammed cell death that should also be considered since theymay lead to new insights into cell death programs and revealtheir potentially unique roles in development, homeostasis,neoplasia and degeneration. It has become increasingly ap-parent that cell death mechanisms include a highly diversearray of phenotypes and molecular mechanisms. And there isevidence that modulation of one form of cell death may leadto another. Because other types of cell death may require geneactivation and function in an energy dependent manner, they

are also considered to be forms of “programmed cell death.”Therefore, there is some resistance to the exclusive use ofthe term “programmed cell death” to specifically describeapoptosis.

There are necrotic-like phenotypes that require gene ac-tivation and protein synthesis so they are, strictly speaking,forms of programmed cell death (Proskuryakov et al., 2003).These forms of cell death that have certain morphological fea-tures of both necrosis and apoptosis have been given the term“aponecrosis” (Formigli et al., 2000). By affecting the mito-chondrial respiratory chain with antimycin A, Formigli andcoworkers induced a type of cell death that shared dynamic,molecular, and morphological features with both apoptosisand necrosis.

Caspase-independent mechanisms of neuronal cell deathhave also been identified. This specific type of programmedcell death may involve specific mitochondrial factors. Inexperimental models, apoptosis-inducing factor (AIF) andendonuclease G promote this type of cell death; however,Smac/DIABLO and HtrA2/Omi may also contribute (Rav-agnan et al., 2002; van Loo et al., 2002b). Oppenheim andcoworkers (2001) have shown that programmed cell deathoccurs in developing mammalian neurons, even after the ge-netic deletion of caspases. Other research has shown thatinhibition of the caspase execution machinery may only tem-porarily rescue damaged neurons and that classical apoptoticfeatures can still appear in caspase-inhibited neurons (Vol-bracht et al., 2001). It appears that caspase-dependent andcaspase-independent mechanisms of neuronal cell death maydepend on brain region, cell type, and age.

Sperandio and coworkers (2000) have described a form ofprogrammed cell death that is morphologically and biochem-ically distinct from apoptosis, dubbed “paraptosis.” Althoughthis form of cell death does not respond to caspase inhibitorsor BCL-XL, it is driven by an alternative form of caspase-9activity that is Apaf-1 independent. This alternative form ofprogrammed cell death is reported to occur during develop-ment and in transgenic models of Huntington’s disease andhuman amyotrophic lateral sclerosis (Dal Canto and Gurney,1994; Turmaine et al., 2000).

There is speculation that “autophagy” represents anothermechanism for programmed cell death and, similar to apop-tosis, has important roles in developmental processes, hu-man diseases and cellular responses to nutrient deprivation(Schwartz et al., 1993; Gozuacik and Kimchi, 2004; Debnathet al., 2005). Other terms used synonymously are “macroau-tophagy” and “autophagic type II cell death” (Klionsky andEmr, 2000). Autophagic cell death is characterized by thesequestration of cytoplasm and organelles in double or mul-timembrane vesicles and delivery to the cells own lysosomesfor subsequent degradation (Noda et al., 2002). In a sense,the cell “cannibalizes” itself. The mechanisms and morphol-ogy of autophagy are evolutionarily conserved with strongsimilarities between organisms as diverse as animals, plantsand yeast. The process of autophagy depends on both contin-uous protein synthesis and the continuous presence of ATP.The molecular mechanisms have been extensively studied inyeast and mammalian orthologues continue to be elucidated(Ohsumi, 2001; Huang and Klionsky, 2002).

This distinction of a autophagic programmed cell deathwas made because it was determined that some cells would




undergo caspase-independent gene-activated cell death butwould display few of the ultrastructural features character-istic of apoptosis (Table 1) and would not exhibit DNAladdering (Cohen, 1991). However these cells do requirede novo gene expression with an increase in expression ofthe polyubiquitin gene, similar to apoptosis (Ohsumi, 2001;Gozuacik and Kimchi, 2004). Specifically, autophagy occursin all eukaryotic cells and involves the dynamic rearrange-ment of subcellular membranes to sequester cytoplasm andorganelles for delivery to the lysosome or vacuole wheredegradation occurs. This is considered to be the major in-ducible pathway for the general turnover of cytoplasmiccomponents.

A unique ubiquitin-like protein conjugation system anda protein complex that directs membrane docking and fu-sion at the lysosome or vacuole are important componentsof autophagy. In general, the process of autophagy can bedivided into 4 steps: (1) induction, (2) formation of the au-tophagosome, (3) fusion with the lysosome or vacuole, and(4) autophagic body breakdown and recycling. Although themolecular details are still being elucidated, the regulationof this process occurs through various kinases, phosphatasesand guanosine triphosphatases (GTPases).

There are some settings where autophagy and apopto-sis seem to be interconnected and the idea of “molecularswitches” between the two processes has been introduced(Piacentini et al., 2003). This is similar to the concept ofthe necrosis-apoptosis “continuum” or “paradox” which sug-gests that both apoptosis and necrosis represent morpho-logic expressions of a shared biochemical network in whichthe route of cell death depends on a variety of factors suchas the physiologic milieu, developmental stage, tissue type,and the nature of the cell death signal (Hirsch et al., 1997;Zeiss, 2003). However, the core apoptotic pathway can bediverted to induce a necrotic phenotype by alteration ofthe availability of intracellular ATP and the availability ofcaspases.

A similar relationship may occur between apoptosis andautophagy. It has been suggested that mitochondria may becentral organelles that integrate both apoptosis and autophagy(Elmore et al., 2001). Moreover, some of the same signals thatare involved in apoptosis may also be involved in autophagy.For example, in both apoptosis and autophagy, there is thecoordinated regulation of Akt (protein kinase B) and p70S6kinase. Other proteins that may be part of the network con-necting the two types of cell death include DAPk, Beclin 1,BNIP3, HSpin1, or protymosin-α (Klionsky and Emr, 2000).Malignant transformation is another link between autophagyand apoptosis (Gozuacik and Kimchi, 2004).

The role of genetic alterations in the pathways thatcontrol cellular proliferation/apoptosis in cancer develop-ment has been well established. Similarly, a correlationbetween reduced autophagy and cancer has also been doc-umented. Studies have indicated that during malignant trans-formation several proteins and pathways that are related toautophagy signaling are deregulated resulting in reduced au-tophagocytic activity. This suggests that, in some circum-stances, autophagy may function as a safeguard mechanismthat restricts uncontrolled cell growth. Autophagy has alsobeen considered a protective mechanism against apopto-sis. Lemasters and coworkers (1998) observed that depo-

larized mitochondria, a feature of apoptosis, are rapidlyeliminated by autophagy in primary hepatocytes. Eliminat-ing damaged mitochondria prevents the release of proapop-totic substances from the mitochondria, thus preventingapoptosis.

Autophagy is considered the major cellular mechanism fordisposing of long-lived proteins and cytoplasmic organelles;however, the concept of autophagic cell death has been a mat-ter of debate within the scientific community. Since there isa distinct advantage of increased autophagy in various phys-iological and stress conditions, it has been suggested thatautophagy represents an important adaptive mechanism thatattempts to rescue cells from death. In other words, the pres-ence of autophagic vesicles in dying cells may reflect anadaptive response to maintain cell survival under stress con-ditions rather than a reflection of “autophagic cell death.”Alternatively, are apoptosis and autophagic cell death mu-tually exclusive or can both apply in a situation similar tothe apoptosis-necrosis continuum? It may be that the type ofcell death depends on the severity of the response, the influ-ences of cellular constituents and/or the influences of othersignaling pathways.

There are reports that apoptosis and autophagic pro-grammed cell death are not mutually exclusive and the diversemorphologies are attributed, in part, to distinct biochemicaland molecular events (Gozuacik and Kimchi, 2004). In cer-tain cells and under certain conditions the morphological fea-tures of autophagy may occur prior to apoptotic cell death,representing an early phase of apoptosis. The controversystill remains, however interest in the field of autophagic celldeath is constantly increasing with the emergence of newassays and markers for elucidating the molecular basis ofautophagy and its possible implications in programmed celldeath and malignant cell transformation.

CONCLUSIONS

Apoptosis is regarded as a carefully regulated energy-dependent process, characterized by specific morphologicaland biochemical features in which caspase activation playsa central role. Although many of the key apoptotic proteinsthat are activated or inactivated in the apoptotic pathwayshave been identified, the molecular mechanisms of actionor activation of these proteins are not fully understood andare the focus of continued research. The importance of un-derstanding the mechanistic machinery of apoptosis is vi-tal because programmed cell death is a component of bothhealth and disease, being initiated by various physiologic andpathologic stimuli. Moreover, the widespread involvement ofapoptosis in the pathophysiology of disease lends itself totherapeutic intervention at many different checkpoints. Un-derstanding the mechanisms of apoptosis, and other variantsof programmed cell death, at the molecular level providesdeeper insight into various disease processes and may thusinfluence therapeutic strategy.

ACKNOWLEDGEMENTS

The authors wish to acknowledge Elizabeth Ney of the Na-tional Institute of Environmental Health Sciences for graphicdesign of Figure 3.




REFERENCES

Arur, S., Uche, U. E., Rezaul, K., Fong, M., Scranton, V., Cowan, A. E., Mohler,W., and Han, D. K. (2003). Annexin I is an endogenous ligand that mediatesapoptotic cell engulfment. Dev Cell 4, 587–98.

Ashkenazi, A., and Dixit, V. M. (1998). Death receptors: signaling and modu-lation. Science 281, 1305–8.

Barry, M., and Bleackley, R. C. (2002). Cytotoxic T lymphocytes: all roads leadto death. Nat Rev Immunol 2, 401–9.

Bedner, E., Li, X., Gorczyca, W., Melamed, M. R., and Darzynkiewicz, Z.(1999). Analysis of apoptosis by laser scanning cytometry. Cytometry 35,181–95.

Bortner, C. D., Oldenburg, N. B., and Cidlowski, J. A. (1995). The role of DNAfragmentation in apoptosis. Trends Cell Biol 5, 21–6.

Bossy-Wetzel, E., and Green, D. R. (2000). Detection of apoptosis by AnnexinV labeling. Methods Enzymol 322, 15–18.

Bratton, D. L., Fadok, V. A., Richter, D. A., Kailey, J. M., Guthrie, L. A.,and Henson, P. M. (1997). Appearance of phosphatidylserine on apop-totic cells requires calcium-mediated nonspecific flip-flop and is enhancedby loss of the aminophospholipid translocase. J Biol Chem 272, 26159–65.

Brunner, T., Wasem, C., Torgler, R., Cima, I., Jakob, S., and Corazza, N. (2003).Fas (CD95/Apo-1) ligand regulation in T cell homeostasis, cell-mediatedcytotoxicity and immune pathology. Semin Immunol 15, 167–76.

Chau, B. N., Cheng, E. H., Kerr, D. A., and Hardwick, J. M. (2000). Aven, anovel inhibitor of caspase activation, binds Bcl-xL and Apaf-1. Mol Cell6, 31–40.

Cheng, J., Zhou, T., Liu, C., Shapiro, J. P., Brauer, M. J., Kiefer, M. C., Barr,P. J., and Mountz, J. D. (1994). Protection from Fas-mediated apoptosisby a soluble form of the Fas molecule. Science 263, 1759–62.

Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu, Y. M., Scott, H., Hession, C.,Garcia, I., and Browning, J. L. (1997). TWEAK, a new secreted ligandin the tumor necrosis factor family that weakly induces apoptosis. J BiolChem 272, 32401–10.

Chinnaiyan, A. M. (1999). The apoptosome: heart and soul of the cell deathmachine. Neoplasia 1, 5–15.

Cohen, G. M. (1997). Caspases: the executioners of apoptosis. Biochem J 326(Pt 1), 1–16.

Cohen, J. J. (1991). Programmed cell death in the immune system. Adv Immunol50, 55–85.

Cory, S., and Adams, J. M. (2002). The Bcl2 family: regulators of the cellularlife-or-death switch. Nat Rev Cancer 2, 647–56.

Cotran, R. S., Kumar, V., Collins, T. (1999) Cellular pathology I: cell injuryand cell death. In: Robbins Pathologic Basis of Disease (R. S. Cortan, V.Kumar and T. Collins, eds.), Sixth edition, pp. 1–29. W.B. Saunders Co.,Philadelphia, PA.

Dal Canto, M. C., and Gurney, M. E. (1994). Development of central nervoussystem pathology in a murine transgenic model of human amyotrophiclateral sclerosis. Am J Pathol 145, 1271–9.

Darzynkiewicz, Z., Bedner, E., Li, X., Gorczyca, W., and Melamed, M. R.(1999). Laser-scanning cytometry: A new instrumentation with many ap-plications. Exp Cell Res 249, 1–12.

Debnath, J., Baehrecke, E. H., and Kroemer, G. (2005). Does autophagy con-tribute to cell death? Autophagy 1, 66–74.

Denecker, G., Vercammen, D., Declercq, W., and Vandenabeele, P. (2001).Apoptotic and necrotic cell death induced by death domain receptors. CellMol Life Sci 58, 356–70.

Devadas, S., Das, J., Liu, C., Zhang, L., Roberts, A. I., Pan, Z., Moore, P. A., Das,G., and Shi, Y. (2006). Granzyme B is critical for T cell receptor-inducedcell death of type 2 helper T cells. Immunity 25, 237–47.

Deveraux, Q. L., and Reed, J. C. (1999). IAP family proteins—suppressors ofapoptosis. Genes Dev 13, 239–52.

Diez-Roux, G., and Lang, R. A. (1997). Macrophages induce apoptosis in normalcells in vivo. Development 124, 3633–8.

Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000). Smac, a mitochondrialprotein that promotes cytochrome c-dependent caspase activation by elim-inating IAP inhibition. Cell 102, 33–42.

Ekert, P. G., and Vaux, D. L. (2005). The mitochondrial death squad: hard-ened killers or innocent bystanders? Curr Opin Cell Biol 17, 626–30.

Elmore, S. P., Qian, T., Grissom, S. F., and Lemasters, J. J. (2001). The mi-tochondrial permeability transition initiates autophagy in rat hepatocytes.FASEB J 15, 2286–7.

Elnemr, A., Ohta, T., Yachie, A., Kayahara, M., Kitagawa, H., Ninomiya, I.,Fushida, S., Fujimura, T., Nishimura, G., Shimizu, K., and Miwa, K.(2001). Human pancreatic cancer cells express non-functional Fas recep-tors and counterattack lymphocytes by expressing Fas ligand; a potentialmechanism for immune escape. Int J Oncol 18, 33–9.

Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S.(1998). A caspase-activated DNase that degrades DNA during apoptosis,and its inhibitor ICAD. Nature 391, 43–50.

Esposti, M. D. (2002). The roles of Bid. Apoptosis 7, 433–40.Ethell, D. W., and Buhler, L. A. (2003). Fas ligand-mediated apoptosis in de-

generative disorders of the brain. J Clin Immunol 23, 439–46.Fadok, V. A., de Cathelineau, A., Daleke, D. L., Henson, P. M., and Bratton,

D. L. (2001). Loss of phospholipid asymmetry and surface exposureof phosphatidylserine is required for phagocytosis of apoptotic cells bymacrophages and fibroblasts. J Biol Chem 276, 1071–7.

Fan, Z., Beresford, P. J., Oh, D. Y., Zhang, D., and Lieberman, J. (2003). Tu-mor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its in-hibitor. Cell 112, 659–72.

Ferraro-Peyret, C., Quemeneur, L., Flacher, M., Revillard, J. P., and Genestier, L.(2002). Caspase-independent phosphatidylserine exposure during apopto-sis of primary T lymphocytes. J Immunol 169, 4805–10.

Fiers, W., Beyaert, R., Declercq, W., and Vandenabeele, P. (1999). More thanone way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene18, 7719–30.

Formigli, L., Papucci, L., Tani, A., Schiavone, N., Tempestini, A., Orlandini, G.E., Capaccioli, S., and Orlandini, S. Z. (2000). Aponecrosis: morphologicaland biochemical exploration of a syncretic process of cell death sharingapoptosis and necrosis. J Cell Physiol 182, 41–9.

Freude, B., Masters, T. N., Robicsek, F., Fokin, A., Kostin, S., Zimmermann,R., Ullmann, C., Lorenz-Meyer, S., and Schaper, J. (2000). Apoptosis isinitiated by myocardial ischemia and executed during reperfusion. J MolCell Cardiol 32, 197–208.

Fujio, Y., Nguyen, T., Wencker, D., Kitsis, R. N., and Walsh, K. (2000). Akt pro-motes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation 101, 660–7.

Gardai, S. J., McPhillips, K. A., Frasch, S. C., Janssen, W. J., Starefeldt, A.,Murphy-Ullrich, J. E., Bratton, D. L., Oldenborg, P. A., Michalak, M., andHenson, P. M. (2005). Cell-surface calreticulin initiates clearance of viableor apoptotic cells through trans-activation of LRP on the phagocyte. Cell123, 321–34.

Garrido, C., Galluzzi, L., Brunet, M., Puig, P. E., Didelot, C., and Kroemer,G. (2006). Mechanisms of cytochrome c release from mitochondria. CellDeath Differ 13, 1423–33.

Goldstein, J. C., Waterhouse, N. J., Juin, P., Evan, G. I., and Green, D. R. (2000).The coordinate release of cytochrome c during apoptosis is rapid, completeand kinetically invariant. Nat Cell Biol 2, 156–62.

Goping, I. S., Barry, M., Liston, P., Sawchuk, T., Constantinescu, G., Michalak,K. M., Shostak, I., Roberts, D. L., Hunter, A. M., Korneluk, R., and Bleack-ley, R. C. (2003). Granzyme B-induced apoptosis requires both directcaspase activation and relief of caspase inhibition. Immunity 18, 355–65.

Gozuacik, D., and Kimchi, A. (2004). Autophagy as a cell death and tumorsuppressor mechanism. Oncogene 23, 2891–906.

Grabarek, J., Amstad, P., and Darzynkiewicz, Z. (2002). Use of fluorescentlylabeled caspase inhibitors as affinity labels to detect activated caspases.Hum Cell 15, 1–12.

Graziani, G., and Szabo, C. (2005). Clinical perspectives of PARP inhibitors.Pharmacol Res 52, 109–18.

Greenhalgh, D. G. (1998). The role of apoptosis in wound healing. Int J BiochemCell Biol 30, 1019–30.




Gu, J., Kawai, H., Wiederschain, D., and Yuan, Z. M. (2001). Mechanism offunctional inactivation of a Li-Fraumeni syndrome p53 that has a mutationoutside of the DNA-binding domain. Cancer Res 61, 1741–6.

Gurtu, V., Kain, S. R., and Zhang, G. (1997). Fluorometric and colorimetricdetection of caspase activity associated with apoptosis. Anal Biochem 251,98–102.

Hacker, G. (2000). The morphology of apoptosis. Cell Tissue Res 301, 5–17.Harman, D. (1992). Role of free radicals in aging and disease. Ann N Y Acad

Sci 673, 126–41.Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770–6.Hetz, C., Vitte, P. A., Bombrun, A., Rostovtseva, T. K., Montessuit, S., Hiver,

A., Schwarz, M. K., Church, D. J., Korsmeyer, S. J., Martinou, J. C.,and Antonsson, B. (2005). Bax channel inhibitors prevent mitochondrion-mediated apoptosis and protect neurons in a model of global brain is-chemia. J Biol Chem 280, 42960–70.

Hill, M. M., Adrain, C., Duriez, P. J., Creagh, E. M., and Martin, S. J. (2004).Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes. Embo J 23, 2134–45.

Hirsch, T., Marchetti, P., Susin, S. A., Dallaporta, B., Zamzami, N., Marzo, I.,Geuskens, M., and Kroemer, G. (1997). The apoptosis-necrosis paradox.Apoptogenic proteases activated after mitochondrial permeability transi-tion determine the mode of cell death. Oncogene 15, 1573–81.

Hitoshi, Y., Lorens, J., Kitada, S. I., Fisher, J., LaBarge, M., Ring, H. Z., Francke,U., Reed, J. C., Kinoshita, S., and Nolan, G. P. (1998). Toso, a cell surface,specific regulator of Fas-induced apoptosis in T cells. Immunity 8, 461–71.

Hochhauser, E., Kivity, S., Offen, D., Maulik, N., Otani, H., Barhum, Y., Pannet,H., Shneyvays, V., Shainberg, A., Goldshtaub, V., Tobar, A., and Vidne, B.A. (2003). Bax ablation protects against myocardial ischemia-reperfusioninjury in transgenic mice. Am J Physiol Heart Circ Physiol 284, H2351–9.

Hoeppner, D. J., Hengartner, M. O., and Schnabel, R. (2001). Engulfment genescooperate with ced-3 to promote cell death in Caenorhabditis elegans.Nature 412, 202–6.

Hofmann, W. K., de Vos, S., Tsukasaki, K., Wachsman, W., Pinkus, G. S., Said,J. W., and Koeffler, H. P. (2001). Altered apoptosis pathways in mantle celllymphoma detected by oligonucleotide microarray. Blood 98, 787–94.

Horvitz, H. R. (1999). Genetic control of programmed cell death in the nematodeCaenorhabditis elegans. Cancer Res 59, 1701s–1706s.

Howden, R., Hanlon, P. R., Petranka, J. G., Kleeberger, S., Bucher, J., Dunnick,J., Nyska, A., and Murphy, E. (2005). Ephedrine plus caffeine causesage-dependent cardiovascular responses in Fischer 344 rats. Am J PhysiolHeart Circ Physiol 288, 2219–24.

Hsu, H., Xiong, J., and Goeddel, D. V. (1995). The TNF receptor 1-associatedprotein TRADD signals cell death and NF-kappa B activation. Cell 81,495–504.

Hu, S., Snipas, S. J., Vincenz, C., Salvesen, G., and Dixit, V. M. (1998). Caspase-14 is a novel developmentally regulated protease. J Biol Chem 273, 29648–53.

Huang, W. P., and Klionsky, D. J. (2002). Autophagy in yeast: a review of themolecular machinery. Cell Struct Funct 27, 409–20.

Igney, F. H., and Krammer, P. H. (2002). Death and anti-death: tumour resistanceto apoptosis. Nat Rev Cancer 2, 277–88.

Jimenez, B., Volpert, O. V., Crawford, S. E., Febbraio, M., Silverstein, R. L.,and Bouck, N. (2000). Signals leading to apoptosis-dependent inhibitionof neovascularization by thrombospondin-1. Nat Med 6, 41–8.

Joza, N., Susin, S. A., Daugas, E., Stanford, W. L., Cho, S. K., Li, C. Y., Sasaki,T., Elia, A. J., Cheng, H. Y., Ravagnan, L., Ferri, K. F., Zamzami, N.,Wakeham, A., Hakem, R., Yoshida, H., Kong, Y. Y., Mak, T. W., Zuniga-Pflucker, J. C., Kroemer, G., and Penninger, J. M. (2001). Essential roleof the mitochondrial apoptosis-inducing factor in programmed cell death.Nature 410, 549–54.

Kang, S. J., Wang, S., Kuida, K., and Yuan, J. (2002). Distinct downstreampathways of caspase-11 in regulating apoptosis and cytokine maturationduring septic shock response. Cell Death Differ 9, 1115–25.

Kataoka, T., Schroter, M., Hahne, M., Schneider, P., Irmler, M., Thome, M.,Froelich, C. J., and Tschopp, J. (1998). FLIP prevents apoptosis induced bydeath receptors but not by perforin/granzyme B, chemotherapeutic drugs,and gamma irradiation. J Immunol 161, 3936–42.

Kelliher, M. A., Grimm, S., Ishida, Y., Kuo, F., Stanger, B. Z., and Leder, P.(1998). The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity 8, 297–303.

Kerr, J. F. (2002). History of the events leading to the formulation of the apoptosisconcept. Toxicology 181–182, 471–4.

Kerr, J. F., Winterford, C. M., and Harmon, B. V. (1994). Apoptosis. Its signifi-cance in cancer and cancer therapy. Cancer 73, 2013–26.

Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972). Apoptosis: a basic biolog-ical phenomenon with wide-ranging implications in tissue kinetics. Br JCancer 26, 239–57.

King, K. L., and Cidlowski, J. A. (1998). Cell cycle regulation and apoptosis.Annu Rev Physiol 60, 601–17.

Kischkel, F. C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, M., Krammer,P. H., and Peter, M. E. (1995). Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) withthe receptor. Embo J 14, 5579–88.

Klionsky, D. J., and Emr, S. D. (2000). Autophagy as a regulated pathway ofcellular degradation. Science 290, 1717–21.

Koenig, U., Eckhart, L., and Tschachler, E. (2001). Evidence that caspase-13is not a human but a bovine gene. Biochem Biophys Res Commun 285,1150–4.

Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry,T. J., Kirschner, M. W., Koths, K., Kwiatkowski, D. J., and Williams, L. T.(1997). Caspase-3-generated fragment of gelsolin: effector of morpholog-ical change in apoptosis. Science 278, 294–8.

Koyama, S., Koike, N., and Adachi, S. (2001). Fas receptor counterattack againsttumor-infiltrating lymphocytes in vivo as a mechanism of immune escapein gastric carcinoma. J Cancer Res Clin Oncol 127, 20–6.

Kressel, M., and Groscurth, P. (1994). Distinction of apoptotic and necrotic celldeath by in situ labelling of fragmented DNA. Cell Tissue Res 278, 549–56.

Kurosaka, K., Takahashi, M., Watanabe, N., and Kobayashi, Y. (2003). Silentcleanup of very early apoptotic cells by macrophages. J Immunol 171,4672–9.

Kurz, E. U., and Lees-Miller, S. P. (2004). DNA damage-induced activationof ATM and ATM-dependent signaling pathways. DNA Repair (Amst) 3,889–900.

Lang, R. A., and Bishop, J. M. (1993). Macrophages are required for celldeath and tissue remodeling in the developing mouse eye. Cell 74, 453–62.

Le, G. T., and Abbenante, G. (2005). Inhibitors of TACE and Caspase-1 asanti-inflammatory drugs. Curr Med Chem 12, 2963–77.

Leist, M., Single, B., Castoldi, A. F., Kuhnle, S., and Nicotera, P. (1997). In-tracellular adenosine triphosphate (ATP) concentration: a switch in thedecision between apoptosis and necrosis. J Exp Med 185, 1481–6.

Lemasters, J. J., Nieminen, A. L., Qian, T., Trost, L. C., Elmore, S. P., Nishimura,Y., Crowe, R. A., Cascio, W. E., Bradham, C. A., Brenner, D. A., and Her-man, B. (1998). The mitochondrial permeability transition in cell death: acommon mechanism in necrosis, apoptosis and autophagy. Biochim Bio-phys Acta 1366, 177–96.

Levin, S., Bucci, T. J., Cohen, S. M., Fix, A. S., Hardisty, J. F., LeGrand, E. K.,Maronpot, R. R., and Trump, B. F. (1999). The nomenclature of cell death:recommendations of an ad hoc Committee of the Society of ToxicologicPathologists. Toxicol Pathol 27, 484–90.

Li, C. J., Friedman, D. J., Wang, C., Metelev, V., and Pardee, A. B. (1995).Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein.Science 268, 429–31.

Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998). Cleavage of BID by caspase-8mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell94, 491–501.

Li, L. Y., Luo, X., and Wang, X. (2001). Endonuclease G is an apoptotic DNasewhen released from mitochondria. Nature 412, 95–9.

Lieberman, J., and Fan, Z. (2003). Nuclear war: the granzyme A-bomb. CurrOpin Immunol 15, 553–9.

Little, G. H., and Flores, A. (1993). Inhibition of programmed cell death bycatalase and phenylalanine methyl ester. Comp Biochem Physiol CompPhysiol 105, 79–83.




Liu, F. T., Newland, A. C., and Jia, L. (2003). Bax conformational change is acrucial step for PUMA-mediated apoptosis in human leukemia. BiochemBiophys Res Commun 310, 956–62.

Livingston, D. J. (1997). In vitro and in vivo studies of ICE inhibitors. J CellBiochem 64, 19–26.

Locksley, R. M., Killeen, N., and Lenardo, M. J. (2001). The TNF and TNFreceptor superfamilies: integrating mammalian biology. Cell 104, 487–501.

Love, S., Barber, R., and Wilcock, G. K. (1999). Increased poly(ADP-ribosyl)ation of nuclear proteins in Alzheimer’s disease. Brain 122 (Pt2), 247–53.

Lund, L. R., Romer, J., Thomasset, N., Solberg, H., Pyke, C., Bissell, M. J.,Dano, K., and Werb, Z. (1996). Two distinct phases of apoptosis in mam-mary gland involution: proteinase-independent and -dependent pathways.Development 122, 181–93.

Majno, G., and Joris, I. (1995). Apoptosis, oncosis, and necrosis. An overviewof cell death. Am J Pathol 146, 3–15.

Mandal, D., Mazumder, A., Das, P., Kundu, M., and Basu, J. (2005). Fas-,caspase-8-, and caspase-3-dependent signaling regulates the activity of theaminophospholipid translocase and phosphatidylserine externalization inhuman erythrocytes. J Biol Chem 280, 39460–7.

Martinvalet, D., Zhu, P., and Lieberman, J. (2005). Granzyme A induces caspase-independent mitochondrial damage, a required first step for apoptosis.Immunity 22, 355–70.

Metzstein, M. M., Stanfield, G. M., and Horvitz, H. R. (1998). Genetics ofprogrammed cell death in C. elegans: past, present and future. TrendsGenet 14, 410–6.

Meyer, N., Kim, S. S., and Penn, L. Z. (2006). The Oscar-worthy role of Mycin apoptosis. Semin Cancer Biol 16, 275–87.

Miyashita, T., Krajewski, S., Krajewska, M., Wang, H. G., Lin, H. K., Lieber-mann, D. A., Hoffman, B., and Reed, J. C. (1994). Tumor suppressor p53 isa regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene9, 1799–805.

Mocanu, M. M., Baxter, G. F., and Yellon, D. M. (2000). Caspase inhibition andlimitation of myocardial infarct size: protection against lethal reperfusioninjury. Br J Pharmacol 130, 197–200.

Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan,J. (2000). Caspase-12 mediates endoplasmic-reticulum-specific apoptosisand cytotoxicity by amyloid-beta. Nature 403, 98–103.

Nemes, Z., Jr., Friis, R. R., Aeschlimann, D., Saurer, S., Paulsson, M., andFesus, L. (1996). Expression and activation of tissue transglutaminase inapoptotic cells of involuting rodent mammary tissue. Eur J Cell Biol 70,125–33.

Newmeyer, D. D., Bossy-Wetzel, E., Kluck, R. M., Wolf, B. B., Beere, H. M.,and Green, D. R. (2000). Bcl-xL does not inhibit the function of Apaf-1.Cell Death Differ 7, 402–7.

Nicholson, D. W. (2000). From bench to clinic with apoptosis-based therapeuticagents. Nature 407, 810–6.

Nijhawan, D., Honarpour, N., and Wang, X. (2000). Apoptosis in neural devel-opment and disease. Annu Rev Neurosci 23, 73–87.

Noda, T., Suzuki, K., and Ohsumi, Y. (2002). Yeast autophagosomes: denovo formation of a membrane structure. Trends Cell Biol 12, 231–5.

Norbury, C. J., and Hickson, I. D. (2001). Cellular responses to DNA damage.Annu Rev Pharmacol Toxicol 41, 367–401.

Nyska, A., Murphy, E., Foley, J. F., Collins, B. J., Petranka, J., Howden, R.,Hanlon, P., and Dunnick, J. K. (2005). Acute hemorrhagic myocardialnecrosis and sudden death of rats exposed to a combination of ephedrineand caffeine. Toxicol Sci 83, 388–96.

Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., Tokino,T., Taniguchi, T., and Tanaka, N. (2000). Noxa, a BH3-only member ofthe Bcl-2 family and candidate mediator of p53-induced apoptosis. Science288, 1053–8.

Ohsumi, Y. (2001). Molecular dissection of autophagy: two ubiquitin-like sys-tems. Nat Rev Mol Cell Biol 2, 211–6.

Opferman, J. T., and Korsmeyer, S. J. (2003). Apoptosis in the development andmaintenance of the immune system. Nat Immunol 4, 410–5.

Oppenheim, R. W., Flavell, R. A., Vinsant, S., Prevette, D., Kuan, C. Y., and Ra-kic, P. (2001). Programmed cell death of developing mammalian neuronsafter genetic deletion of caspases. J Neurosci 21, 4752–60.

Osborne, B. A. (1996). Apoptosis and the maintenance of homoeostasis in theimmune system. Curr Opin Immunol 8, 245–54.

Otsuki, Y., Li, Z., and Shibata, M. A. (2003). Apoptotic detection methods–frommorphology to gene. Prog Histochem Cytochem 38, 275–339.

Ozawa, T. (1995). Mechanism of somatic mitochondrial DNA mutations asso-ciated with age and diseases. Biochim Biophys Acta 1271, 177–89.

Paweletz, N. (2001). Walther Flemming: pioneer of mitosis research. Nat RevMol Cell Biol 2, 72–5.

Peter, M. E., and Krammer, P. H. (1998). Mechanisms of CD95 (APO-1/Fas)-mediated apoptosis. Curr Opin Immunol 10, 545–51.

Piacentini, M., Evangelisti, C., Mastroberardino, P. G., Nardacci, R., and Kroe-mer, G. (2003). Does prothymosin-alpha act as molecular switch betweenapoptosis and autophagy? Cell Death Differ 10, 937–9.

Pietenpol, J. A., and Stewart, Z. A. (2002). Cell cycle checkpoint signaling: cellcycle arrest versus apoptosis. Toxicology 181–182, 475–81.

Poot, M., and Pierce, R. H. (1999). Detection of changes in mitochondrial func-tion during apoptosis by simultaneous staining with multiple fluorescentdyes and correlated multiparameter flow cytometry. Cytometry 35, 311–7.

Proskuryakov, S. Y., Konoplyannikov, A. G., and Gabai, V. L. (2003). Necrosis:a specific form of programmed cell death? Exp Cell Res 283, 1–16.

Rai, N. K., Tripathi, K., Sharma, D., and Shukla, V. K. (2005). Apoptosis: abasic physiologic process in wound healing. Int J Low Extrem Wounds 4,138–44.

Ravagnan, L., Roumier, T., and Kroemer, G. (2002). Mitochondria, the killerorganelles and their weapons. J Cell Physiol 192, 131–7.

Reddien, P. W., Cameron, S., and Horvitz, H. R. (2001). Phagocytosis promotesprogrammed cell death in C. elegans. Nature 412, 198–202.

Renehan, A. G., Booth, C., and Potten, C. S. (2001). What is apoptosis, and whyis it important? Bmj 322, 1536–8.

Rubio-Moscardo, F., Blesa, D., Mestre, C., Siebert, R., Balasas, T., Benito, A.,Rosenwald, A., Climent, J., Martinez, J. I., Schilhabel, M., Karran, E. L.,Gesk, S., Esteller, M., deLeeuw, R., Staudt, L. M., Fernandez-Luna, J. L.,Pinkel, D., Dyer, M. J., and Martinez-Climent, J. A. (2005). Characteri-zation of 8p21.3 chromosomal deletions in B-cell lymphoma: TRAIL-R1and TRAIL-R2 as candidate dosage-dependent tumor suppressor genes.Blood 106, 3214–22.

Russell, J. H., and Ley, T. J. (2002). Lymphocyte-mediated cytotoxicity. AnnuRev Immunol 20, 323–70.

Saelens, X., Festjens, N., Vande Walle, L., van Gurp, M., van Loo, G., andVandenabeele, P. (2004). Toxic proteins released from mitochondria incell death. Oncogene 23, 2861–74.

Sakahira, H., Enari, M., and Nagata, S. (1998). Cleavage of CAD inhibitor inCAD activation and DNA degradation during apoptosis. Nature 391, 96–9.

Savill, J., and Fadok, V. (2000). Corpse clearance defines the meaning of celldeath. Nature 407, 784–8.

Scaffidi, C., Schmitz, I., Krammer, P. H., and Peter, M. E. (1999). The roleof c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem 274,1541–8.

Schimmer, A. D. (2004). Inhibitor of apoptosis proteins: translating basic knowl-edge into clinical practice. Cancer Res 64, 7183–90.

Schuler, M., and Green, D. R. (2001). Mechanisms of p53-dependent apoptosis.Biochem Soc Trans 29, 684–8.

Schwartz, L. M., Smith, S. W., Jones, M. E., and Osborne, B. A. (1993). Do allprogrammed cell deaths occur via apoptosis? Proc Natl Acad Sci USA 90,980–4.

Scorrano, L., Ashiya, M., Buttle, K., Weiler, S., Oakes, S. A., Mannella, C. A.,and Korsmeyer, S. J. (2002). A distinct pathway remodels mitochondrialcristae and mobilizes cytochrome c during apoptosis. Dev Cell 2, 55–67.

Silke, J., Hawkins, C. J., Ekert, P. G., Chew, J., Day, C. L., Pakusch, M., Verhagen,A. M., and Vaux, D. L. (2002). The anti-apoptotic activity of XIAP isretained upon mutation of both the caspase-3- and caspase-9-interactingsites. J Cell Biol 157, 115–24.




Slee, E. A., Adrain, C., and Martin, S. J. (2001). Executioner caspase-3, -6, and-7 perform distinct, non-redundant roles during the demolition phase ofapoptosis. J Biol Chem 276, 7320–6.

Smyth, M. J., Godfrey, D. I., and Trapani, J. A. (2001). A fresh look at tumorimmunosurveillance and immunotherapy. Nat Immunol 2, 293–9.

Sperandio, S., de Belle, I., and Bredesen, D. E. (2000). An alternative, non-apoptotic form of programmed cell death. Proc Natl Acad Sci USA 97,14376–81.

Suliman, A., Lam, A., Datta, R., and Srivastava, R. K. (2001). Intracellularmechanisms of TRAIL: apoptosis through mitochondrial-dependent and-independent pathways. Oncogene 20, 2122–33.

Susin, S. A., Daugas, E., Ravagnan, L., Samejima, K., Zamzami, N., Loeffler,M., Costantini, P., Ferri, K. F., Irinopoulou, T., Prevost, M. C., Brothers, G.,Mak, T. W., Penninger, J., Earnshaw, W. C., and Kroemer, G. (2000). Twodistinct pathways leading to nuclear apoptosis. J Exp Med 192, 571–80.

Talasz, H., Helliger, W., Sarg, B., Debbage, P. L., Puschendorf, B., and Lindner,H. (2002). Hyperphosphorylation of histone H2A.X and dephosphoryla-tion of histone H1 subtypes in the course of apoptosis. Cell Death Differ9, 27–39.

Tilly, J. L., Kowalski, K. I., Johnson, A. L., and Hsueh, A. J. (1991). Involvementof apoptosis in ovarian follicular atresia and postovulatory regression. En-docrinology 129, 2799–801.

Trapani, J. A., and Smyth, M. J. (2002). Functional significance of the per-forin/granzyme cell death pathway. Nat Rev Immunol 2, 735–47.

Trump, B. F., Berezesky, I. K., Chang, S. H., and Phelps, P. C. (1997). Thepathways of cell death: oncosis, apoptosis, and necrosis. Toxicol Pathol25, 82–8.

Tsien, R. Y. (1998). The green fluorescent protein. Annu Rev Biochem 67, 509–44.

Turmaine, M., Raza, A., Mahal, A., Mangiarini, L., Bates, G. P., and Davies, S.W. (2000). Nonapoptotic neurodegeneration in a transgenic mouse modelof Huntington’s disease. Proc Natl Acad Sci USA 97, 8093–7.

Vallat, L., Magdelenat, H., Merle-Beral, H., Masdehors, P., Potocki de Montalk,G., Davi, F., Kruhoffer, M., Sabatier, L., Orntoft, T. F., and Delic, J. (2003).The resistance of B-CLL cells to DNA damage-induced apoptosis definedby DNA microarrays. Blood 101, 4598–606.

van Loo, G., van Gurp, M., Depuydt, B., Srinivasula, S. M., Rodriguez, I.,Alnemri, E. S., Gevaert, K., Vandekerckhove, J., Declercq, W., and Van-denabeele, P. (2002a). The serine protease Omi/HtrA2 is released frommitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAPand induces enhanced caspase activity. Cell Death Differ 9, 20–6.

van Loo, G., Saelens, X., van Gurp, M., MacFarlane, M., Martin, S. J., andVandenabeele, P. (2002b). The role of mitochondrial factors in apoptosis:a Russian roulette with more than one bullet. Cell Death Differ 9, 1031–42.

Varley, J. M., Evans, D. G., and Birch, J. M. (1997). Li-Fraumeni syndrome—amolecular and clinical review. Br J Cancer 76, 1–14.

Vaux, D. L. (2002). Apoptosis and toxicology—what relevance? Toxicology181–182, 3–7.

Vaux, D. L., Cory, S., and Adams, J. M. (1988). Bcl-2 gene promotes haemopoi-etic cell survival and cooperates with c-myc to immortalize pre-B cells.Nature 335, 440–2.

Vivanco, I., and Sawyers, C. L. (2002). The phosphatidylinositol 3-Kinase AKTpathway in human cancer. Nat Rev Cancer 2, 489–501.

Volbracht, C., Leist, M., Kolb, S. A., and Nicotera, P. (2001). Apoptosis incaspase-inhibited neurons. Mol Med 7, 36–48.

Wajant, H. (2002). The Fas signaling pathway: more than a paradigm. Science296, 1635–6.

Wang, X. W., and Harris, C. C. (1997). p53 tumor-suppressor gene: clues tomolecular carcinogenesis. J Cell Physiol 173, 247–55.

Watanabe, M., Hitomi, M., van der Wee, K., Rothenberg, F., Fisher, S. A., Zucker,R., Svoboda, K. K., Goldsmith, E. C., Heiskanen, K. M., and Nieminen,A. L. (2002). The pros and cons of apoptosis assays for use in the study ofcells, tissues, and organs. Microsc Microanal 8, 375–91.

White, M. K., and Cinti, C. (2004). A morphologic approach to detect apoptosisbased on electron microscopy. Methods Mol Biol 285, 105–11.

Worth, A., Thrasher, A. J., and Gaspar, H. B. (2006). Autoimmune lympho-proliferative syndrome: molecular basis of disease and clinical phenotype.Br J Haematol 133, 124–40.

Wyllie, A. H. (1980). Glucocorticoid-induced thymocyte apoptosis is associatedwith endogenous endonuclease activation. Nature 284, 555–6.

Yang, E., Zha, J., Jockel, J., Boise, L. H., Thompson, C. B., and Korsmeyer, S.J. (1995). Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displacesBax and promotes cell death. Cell 80, 285–91.

Zeiss, C. J. (2003). The apoptosis-necrosis continuum: insights from geneticallyaltered mice. Vet Pathol 40, 481–95.

Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996). Serinephosphorylation of death agonist BAD in response to survival factor resultsin binding to 14-3-3 not BCL-X(L). Cell 87, 619–28.

Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002). Creatingnew fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3, 906–18.

Zhou, H. Z., Swanson, R. A., Simonis, U., Ma, X., Cecchini, G., and Gray, M. O.(2006). Poly(ADP-ribose) polymerase-1 hyperactivation and impairmentof mitochondrial respiratory chain complex I function in reperfused mousehearts. Am J Physiol Heart Circ Physiol 291, H714–23.

Zhu, P., Zhang, D., Chowdhury, D., Martinvalet, D., Keefe, D., Shi, L., andLieberman, J. (2006). Granzyme A, which causes single-stranded DNAdamage, targets the double-strand break repair protein Ku70. EMBO Rep7, 431–7.

Zucker, R. M., Hunter, E. S., 3rd, and Rogers, J. M. (2000). Confocal laserscanning microscopy of morphology and apoptosis in organogenesis-stagemouse embryos. Methods Mol Biol 135, 191–202.



Apoptosis: A Review of Programmed Cell...

Documents

Transcript of Apoptosis: A Review of Programmed Cell...