CURRENT TOPIC: To Be, or Not to Be, That is the Question. Apoptosis in Human Trophoblast

Placenta (2000), 21, 1–13 Article No. plac.1999.0450

CURRENT TOPIC To Be, or Not to Be, That is the Question. Apoptosis in Human Trophoblast R. Levy and D. M. Nelsona Department of Obstetrics and Gynecology, Washington University School of Medicine, St Louis, MO, USA Paper accepted 4 August 1999

Apoptosis, the morphology of cell suicide, may result from programmed cell death or may be a response to exogenous stimuli. Apoptosis can be induced in cultured trophoblast and can be identified in the trophoblast of placental villi. The trophoblast regulates maternal–fetal gas, nutrient and waste product exchange; therefore, the presence of apoptosis in this key cellular interface highlights the importance of understanding what controls apoptosis in the placenta. In this review, we describe the signal transduction pathways that trigger apoptosis in other systems, identify key genetic controls for the process and outline the final common pathway which effects execution in cells committed to suicide. Multiplicity, redundancy and cross talk among pathways characterize the surface membrane signals and exogenous stimuli that trigger apoptosis in other cells. As each step in the apoptotic process is discussed, we describe what is known about the step in human placental villi. Recent studies suggest that a further understanding of receptor-mediated signalling pathways, the Bcl-2 regulators and the caspases and substrates involved in placental apoptosis will surely provide insights into both normal placental development and the placental dysfunction associated with some abnormal pregnancies.  2000 Harcourt Publishers Ltd Placenta (2000), 21, 1–13

INTRODUCTION The season is fall. We drive through the countryside and note the rolling hills covered mostly by green trees of the same deciduous variety and uniform beauty; yet, isolated trees, sprinkled through the hillside forest, are different, showing leaves of glistening gold, yellow and orange [Figure 1(A)]. The soil is uniform, the temperature similar and the tree variety identical for all on the hillside. So, what controls the process that makes one tree shine with colours of fall and another retain the green of a summer’s day? The trees on this hill are like cells in many tissues, including placental villi [Figure 1(B)]. Ambient conditions seem identical yet some cells flourish while selected cells commit to apoptosis, an orderly process of cell suicide which does not disrupt the overall tissue terrain. Kerr, Wyllie and Currie (1972) coined the term apoptosis, Greek for ‘falling off’ like autumn leaves, to describe the unique morphology of tumour cells dying. The anatomy of apoptotic cell death, first described by Carl Vogt in a

To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, 4911 Barnes-Jewish Hospital, St Louis, MO 63110-1094, USA. Fax: +1 314 362 8580; E-mail: [email protected]

0143–4004/00/010001+13 $35.00/0

1842, includes loss of surface membrane specializations and cell anchorage concomitant with blebbing, chromatin condensation with nuclear fragmentation and cytoplasmic condensation with cell shrinkage. The resulting cellular fragments, called apoptotic bodies, undergo phagocytosis by resident cells with a noticeable absence of an inflammatory response. The features of apoptosis contrast with necrotic cell death where damage to groups of cells, cytoplasmic swelling and a robust inflammatory response are characteristic. Unlike necrosis, apoptosis is energy dependent and modulated by diverse environmental and genetic cues. Apoptosis is a descriptive term for the unique morphology of cell suicide. Importantly, the morphological changes characterizing this non-necrotic process of cell death may be a part of normal physiology or may be secondary to pathological insults. Apoptosis is thus not a synonym for programmed cell death. The functional phrase programmed cell death is used to describe a normal part of tissue turnover in multicellular organisms. The apoptotic morphology may thus result from programming that occurs during normal embryonic development. However, this anatomy may also be a response of mature tissues to exogenous stimuli such as ambient hypoxia or cytotoxic agents where the response is not programmed into a  2000 Harcourt Publishers Ltd

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cell’s life cycle. Acknowledging that the term apoptosis and programmed cell death have different meanings, we use the term apoptosis in this review to describe the common biochemical and morphological events that occur when either endogenous or exogenous signals lead to cell suicide. The process of apoptosis is highly conserved among multicellular organisms (Vaux and Korsmeyer, 1999), and death signals delivered to subsets of cells allow selective deletion without damage to overall tissue architecture. Multiplicity, redundancy and cross talk among signalling pathways characterize the road to apoptotic cell death [Figure 1(C)]. The death signals can be ligands for membrane receptors or environmental stimuli, transmitted independent of surface membrane receptors. The two signal routes ultimately converge on a common machinery of enzymes and substrates that promote cell execution. Although the above features are common to the process of apoptosis, unique aspects of the death pathway are often cell-type or stimulus-specific, and we are just starting to identify unique aspects of the apoptotic process in the human placenta. Apoptosis can be induced in cultured trophoblast exposed to cytokines in vitro (Yui et al., 1994). In addition, apoptosis was recently described in trophoblast of placentae from uncomplicated pregnancies (Nelson, 1996; Smith, Baker and Symonds, 1997a). Little is known of the environmental and genetic controls that regulate programmed cell death in trophoblast. Nevertheless, the incidence of apoptosis is higher in third trimester villi compared to first trimester placentae, suggesting that placental apoptosis is developmentally regulated (Smith, Baker and Symonds, 1997a). The trophoblast regulates maternal–fetal gas, nutrient and waste product exchange. Therefore, the identification of apoptosis in this key cellular interface highlights the importance of understanding what controls apoptosis in the placenta. In this review, we describe the signal transduction pathways that trigger apoptosis in other systems, identify key genetic controls for the process and outline the enzymes and substrates that effect execution in cells committed to suicide. Further information regarding selected steps in the process can be found in recent reviews (Cohen, 1997; Adams and Cory, 1998; Ashkenazi and Dixit, 1998; Thornberry and Lazebnik, 1998; Green and Reed, 1998; Devereaux and Reed, 1999). As we discuss each step, we describe its relevance to apoptosis in human placental trophoblast.

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Fas receptor, also known as CD95 or Apo-1, is a 40 kD member of the tumour necrosis factor (TNF) receptor (Baker and Reddy, 1998). Upon ligand activation Fas is oligomerized and recruits the death domains (DD) of the adaptor protein FADD (Fas-associated death domain). FADD then activates procaspase 8, also known as FLICE (FADD-homologous ICE-like protease), through an interaction between the death effector domains (DED) of FADD and procaspase 8. The cascade continues as caspase 8 causes downstream activation of other effector caspases. The ability of the adaptors to transfer the signal can be regulated by other proteins that interact directly with the adaptors. For example, FLICE-inhibitory protein (FLIP) is a recently identified FADD-binding suppressor of apoptosis, serving as an intracellular caspase 8 inhibitor (Irmler et al., 1997). Apoptosis-sensitizing agents such as oxidized low density lipoproteins decrease FLIP expression, and thereby enhance Fas induced apoptosis (Sata and Walsh, 1998). The Fas system illustrates the complexity of the signalling cascade downstream of ligand-receptor binding, with several points at which modulation of signal transduction is possible before cell suicide is irreversible. Tumour necrosis factor-alpha (TNF-
) is an apoptosis inducing ligand that can bind either a 55 kD low-affinity (TNF-R1), or a 75 kD high-affinity (TNF-R2) receptor. TNF-
binding to the 55 kD receptor activates sphingomyelin hydrolysis, generating the second messenger ceramide which induces apoptosis (Smyth, Obeid and Hannun, 1997). Ceramide works downstream of FADD and upstream of the caspases. The ceramide signal for apoptosis can be attenuated by the anti-apoptotic protein Bcl-2. In addition, the TNF receptor negatively regulates its own ability to induce apoptosis by concomitantly activating nuclear factor kappa B (NFB), which induces one or more genes to prevent cells from undergoing apoptosis (Baldwin et al., 1996). The examples of cross talk in signalling cascades highlight the modulation of upstream signals by downstream effectors. Such modulation may be a cell type specific mechanism which yields different outcomes despite shared upstream signals. In addition, cell type and tissue type selective expression of receptors also determines whether or not a cell lives. For example, Wsl-1, a member of the TNF receptor superfamily, is expressed in selected tissues, including the spleen, thymus and peripheral blood lymphocytes (Kitson et al., 1996). This selective receptor expression provides a mechanism for different cells, exposed to a given ligand, to have varying fates.

SIGNAL TRANSDUCTION PATHWAYS Figure 1(C) illustrates the pathways that commit the cell to apoptosis. The ligand-receptor systems (Table 1) share similarities. The death signal is usually transmitted through a receptor-associated adaptor death domain which couples to one or more caspases. A prototypical surface membrane receptor is Fas [Figure 1(D)]. Fas was originally described as a mediator of cell death effected by T lymphocytes (Nagata and Goldstein, 1995). The

SIGNAL TRANSDUCTION PATHWAYS IN PLACENTA Fas–Fas ligand (Fas-L) interactions function to protect immune privileged organs such as the eye (Griffith et al., 1995) and testis (Bellgrau et al., 1995) from immunological attack, and abnormal Fas-L expression is associated with disease processes in the eye (Kaplan et al., 1999). These observations suggest that the expression of Fas-L by placental cells may

Figure 1. (A) Hillside covered with green. Isolated trees show autumn leaves despite the seemingly uniform conditions of the environment. The term apoptosis is Greek for ‘falling off’ like autumn leaves. (B) Placental villi stained by the TUNEL method. Most nuclei are TUNEL negative and counterstained by methyl green, but a few nuclei are stained brown (arrow) by the TUNEL method and are undergoing apoptosis. (C) Apoptotic pathways. Signal transduction in response to either ligand–receptor or exogenous stimuli converge on a final common pathway of caspases and substrates. (D) Fas–FasL interaction. This ligand–receptor system is a prototype of a signalling pathway that involves the death domain (DD) and death effector domains (DED) of FADD in an intermolecular activation of caspase 8. Multiple regulatory points modulate the ligand-induced signal, including the inhibitor FLIP, as described in the text, and we illustrate only one possibility here. (E) Apoptotic sequence outlined. (F) Exogenous stimulus pathway. Non-specific stimuli can alter the life-death balance controlled by the bcl-2 family of proteins located in mitochondria. Tipping the balance in favour of the pro-apoptotic Bax protein results in permeability transition pore formation in mitochondria, release of cytochrome c and AIF, and complex formation of Apaf-1-cytochrome-c-dATP in the cytosol. The latter activates caspase 9 which triggers the downstream apoptotic cascade. (G) Final common pathway. Independent of which route makes the activator caspases enzymatically active, the effector caspases and substrates downstream are common to either route inducing apoptosis. The cell ultimately implodes and the cellular residue is packaged in apoptotic bodies which are often engulfed by resident cells. (See tables for abbreviations.)

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Table 1. Ligands, receptors, and adaptor proteins Mediator

Alternate names

APO-1 APO-1L APO-2 APO-3 CLARP CRADD DAM DAP Daxx DcR-1 DcR-2 DD DED DR-3 DR-4 DR-5

Fas, CD-95 LFas L DR-4 DR-3, WSL-1, TRAMP, LARD Caspase like apoptosis regulatory protein Caspase and RIP adaptor with death domain; RAIDD Death adaptor molecules Death associated protein

FADD FAP-1 Fas Fas L FLAME I-TRAF

Fas-associated death domain; MORT-1 Fas associated phosphatase CD95, APO-1 Fas ligand; APO-1L FADD-like anti-apoptotic molecule; FLIP TANK Lymphocyte-associated receptor of death; DR-3, WSL-1, TRAMP, APO-3 Lymphocyte inhibitor of TRAIL; TRID, DcR-1, TRAIL-R3 Mitogen activated death domain Mitogen activated protein kinase kinase kinase 5; Ask1, MEKK5 Mediator of receptor-induced toxicity; FADD Nerve growth factor; NT-3, BDNF, NT-4/5 NF-kappa-B-inducing kinase

LARD LIT MADD MAPKKK5 MORT-1 NGF NIK par-4

Decoy receptor-1; TRID, LIT, TRAIL-R3 Decoy receptor-2; TRAIL-R4; TRUNDD Death domain Death effector domain Death receptor-3; APO-3, WSL-1, TRAMP, LARD Death receptor-4; TRAIL-R1; APO-2 Death receptor-5; TRAIL-R2

RAIDD

RIP associated Ich-1/CED-3 homologous protein with death domain; CRADD

RANK

Receptor activator NFB

RIP TANK TNF-R1 TNF-R2 TRADD TRAF-1 TRAF-2 TRAF-3 TRAF-4 TRAF-5 TRAF-6 TRAIL TRAMP TRANCE TRAP TRID

Receptor interacting protein TRAF associated NFB; I-TRAF Tumour necrosis factor receptor 1; CD120a Tumour necrosis factor receptor 2; CD120b TNF-R1 associated death domain TNF receptor associated factor 1 TNF receptor associated factor 2 TNF receptor associated factor 3 TNF receptor associated factor 4 TNF receptor associated factor 5 TNF receptor associated factor 6 APO-2L; DR-4 TNF receptor apoptosis-mediating protein TNF related activation induced cytokine TNF-related activation protein; CD40, CD154 TRAIL receptor without an intracellular domain; DcR-1, TRAIL-R3, LIT

TRIP TRUNDD WSL-1

TRAF interacting protein TRAIL receptor with truncated death domain APO-3, DR-3, TRAMP, LARD

Function TNF receptor family membrane protein that induces apoptosis when activated by ligand Ligand for Apo-1 or Fas receptor Receptor for TRAIL/APO-2 ligand in TNF receptor family Receptor for APO-3 ligand and contains death domain Protein with death effector domain that interacts with caspase 8 Signaling molecule in response to TNF-R1 stimulation Adaptor molecules that activate caspases, e.g. FADD Family of proteins mediating -interferon induced cell death Fas-binding protein that enhances apoptosis distinct from FADD Receptor for TRAIL that inhibits TRAIL signaling Inactive receptor for TRAIL with a truncated death domain Apoptotic adaptor Apoptotic adaptor Member of TNF receptor family Receptor for TRAIL, APO-2 ligand Receptor for TRAIL, APO-2 ligand Apoptotic adaptor molecule that recruits caspases 8 and 10 to activated Fas or TNF-R1 receptors Protein involved in Fas mediated apoptosis TNF receptor family membrane protein for Fas ligand Ligand for Fas, also called APO-1 Inhibitor of Fas/TNF-R1 induced apoptosis TRAF interacting protein Receptor for Apo-3L Lymphocyte inhibitor of TRAIL TNF-R1 associated death domain protein Kinase that induces apoptosis Apoptotic adaptor molecule (see FADD) TNF receptor superfamily member Activator of NFB Intracellular effector associated with protein kinase C isoform Death adaptor molecule Interacts with TNF receptor-associated factors, NFB and c-junterminal kinase Fas binding protein with a death domain crucial for TNF-R1 mediated NFB activation TRAF family member associated with NFB activation 55 kD membrane receptor for TNF 75 kD membrane receptor for TNF Involved in TNF induced apoptosis and activation of NFB Signal transducer for TNF-R2 receptor cytoplasmic domain Mediates CD-30 induced NFB activation Interacts with CD-30 cytoplasmic domain Interacts with cytosolic domain of the lymphotoxin receptor Mediates CD-40 signaling Mediates CD-40 signaling Ligand activating apoptosis in transformed cell lines Apoptosis mediating receptor homologous to TNF-R1 and Fas TNF family member expressed mainly in T cells TNF family member Antagonist decoy receptor Involved in TNF-R and CD-30 TRAF signaling that inhibits TRAF-mediated NFB activation Protein with inhibitory role in apoptosis Receptor with death domain mediating apoptosis

Levy and Nelson: Apoptosis in the Human Placenta

contribute to the immune privileged status of the products of conception. Huppertz et al. (1998) localized the expression of Fas-L to the villous cytotrophoblast in first trimester placentae, whereas the Fas receptor localized to the microvillous surface of syncytiotrophoblast. Other authors identified Fas-L in both layers of the trophoblast throughout gestation (Bamberger et al., 1997; Runic et al., 1996; Uckan et al., 1997; Zorzi et al., 1998). Mice with a mutated Fas-L gene express a dysfunctional Fas-L protein, and the placentae of these animals exhibit extensive leukocyte infiltration and necrosis at the decidual–placental interface (Hunt et al., 1997). The immune privilege bestowed by Fas-L at the maternal–fetal interface is postulated to protect the placenta against a maternal leukocytic influx that reduces fertility. Furthermore, Fas-L is implicated in the immune privileged status of tumour cells including choriocarcinoma cells (Bamberger et al., 1997; Mor et al., 1998). Payne et al. (1999) demonstrated that Fas-mediated apoptosis is blocked in cultured trophoblast from the first and third trimester villi despite the presence of Fas-L expression on cytotrophoblast and syncytiotrophoblast throughout gestation. Future studies will likely clarify specific roles for Fas and Fas-L in allowing the placenta to be an immune privileged organ. TNF-
(Chen et al., 1991; Eades, Cornelius and Pekala, 1988) and gamma interferon (IFN-; Bulmer et al., 1990), are expressed in the human placenta. In vitro studies indicate that cytotrophoblasts undergo apoptosis after TNF-
exposure, and the process is enhanced by IFN- and blocked by epidermal growth factor (Garcia-Lloret et al., 1996). The TNF-
effect is mediated through the low affinity TNF-R1 receptors, and antibodies against this receptor abrogate the response, while antibodies to TNF-R2 receptors have no effect (Yui et al., 1996). In addition, the apoptosis induced in trophoblast by TNF-
is not mediated by reactive oxygen or nitrogen intermediates such as superoxide anion, nitric oxide, or peroxynitrite (Smith et al., 1999), an hypothesis suggested from studies in other cell systems (Kroemer, 1997). Other receptors may also be important for placental apoptotic signalling (Table 1). For example, the death receptor-6 is expressed in placenta and is thus available to mediate death signals (Pan et al., 1998). Collectively, these studies suggest that multiple pro-apoptotic signalling cascades are present in placental villi, but we are in the very early stages of knowing how the receptor cascades regulate apoptosis in the trophoblast and other cell types that comprise the villus.

GENETIC REGULATION OF APOPTOSIS Rather than a single stimulus resulting in a predictable result, the multiplicity of apoptotic signalling cascades results in a well-controlled balance between activating and inhibiting cell death signals. Indeed, some cell death signals are reversible, allowing cells to avoid self-destruction and to recover differentiated functions (Hetts, 1998). Modification of a death signal through modulation of the signal transduction pathways is

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illustrated by the ligand-receptor pathways described above. In addition, a variety of exogenous stimuli, such as hypoxia and radiation, communicate a death signal through modulation of genetically programmed proteins [Figure 1(F); Table 2], many of which reside in the inner and outer membranes of the mitochondria. The significance of this localization is discussed below. Bcl-2 was the first proto-oncogene identified which inhibits apoptosis in a variety of cells (Hockenbery et al., 1990; Hockenbery et al., 1991). This protein is the prototype of a family of pro- and anti-apoptotic proteins which share structural homology in four peptide sequences called Bcl-2 homology (BH) domains. Specific characteristics of the four BH domains determine the function of these proteins and their ability to dimerize with self and non-self family members. The ratio of pro-versus anti-apoptotic proteins is an important life and death rheostat for cells (Adams and Cory, 1998) and this rheostat is influenced by competitive dimerization between selective pairs of the Bcl-2 protein family, as listed in Table 2. For example, Bax homodimerizes or forms hetero-dimers with Bcl-2 in hematopoietic cell lines (Oltvai et al., 1993). When there is an excess level of Bax, hetero-dimerization of Bax and Bcl-2 inhibits the action of Bcl-2 while homo-dimerization of Bax triggers the death signal cascade in the mitochondria. Death signals cause a conformational change in Bax manifested by translocation of the protein into the mitochondrial membrane where homodimerization occurs (Gross et al., 1998). Bax homodimers destabilize the lipid bilayer structure of the outer mitochondrial membrane, promoting formation of a pore large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol (Basanez et al., 1999). The transmembrane domain present in many Bcl-2 members accounts for the localization of these proteins to the outer mitochondrial membrane (Adams and Cory, 1998). Absence of the transmembrane domain results in a cytosolic location for some proteins (e.g. Bid and Bad). This sub-cellular localization allows these proteins to shuttle between mitochondria and surface membrane receptors that are involved in apoptotic regulation. The amino acid sequence of the BH1 and BH2 domains is important in mitochondrial pore formation during apoptosis. The BH3 domain is a key interaction site between pro-apoptotic and anti-apoptotic members when these proteins form hetero- and homodimers. Several pro-apoptotic members, for example Bik, Bim, Blk, Hrk, Bid and Bad, have sequence homology to Bcl-2 only at BH3. These proteins cannot form homodimers and function by hetero-dimerization to anti-apoptotic proteins, thereby enhancing apoptosis. Finally, the BH4 domain provides docking sites for other proteins that modulate the Bcl-2 family life-death rheostat (Adams and Cory, 1998), including the proteins Bag-1, Raf-1 and Ced-4. In addition, the BH4 site mediates the deathpromoting effect of some chemotherapeutic agents by inducing serine phosphorylation of Bcl-2, neutralizing the anti-apoptotic effects of this protein (Kroemer, 1997). Hetero- and homodimerization are not the only mechanism that regulates the function of the Bcl-2 family proteins. The pro-apoptotic protein Bad, hetero-dimerizes with Bcl-2 and

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Table 2. Bcl-2 family of proteins and associated regulators Mammalian anti-apoptotic proteins A1 Bag-1 Bcl-2 Bcl-XL Bcl-W Mcl-1

BFL-1 Bcl-2 associated athanogene-1 B-cell lymphoma 2 Myeloid cell leukemia-1

Mammalian pro-apoptotic proteins Bad Bcl-XL/Bcl-2 associated death protein Bak Bax Bcl-Xs Bid Bik Bim Blk

Bcl-2 antagonist killer protein Bcl-2 associated X protein Splice variant of Bcl-X BH-3 interacting domain Bcl-2 interacting killer (GP4, Bip-1, NBk) Bcl-2 interacting motif Bik-like killer protein

Bod Bok HrK

Bcl-2 related ovarian death gene Bcl-2 ovarian killer Harakiri

Non mammalian anti-apoptotic proteins BHRF-1 BH reading frame CED-9 NR-13 ORF-16 19 kDa E1B

C. elegans death gene Neuroretina RSV-13 Open reading frame-16

Non-mammalian pro-apoptotic proteins BNIP3 Bcl-2/adenovirus E1B-19B interacting protein Associated regulator proteins AIF Apoptosis inducing factor BRAG-1 Brain related apoptosis gene Apaf-1 Apoptosis protease activating factor-1 CED-3

C. elegans death gene-3

CED-4 cyt c Diva NFB

C. elegans death gene-4 cytochrome C; Apaf-2 Death inducing vBcl-2 activator Nuclear factor kappa B

Promotes cell survival in hematopoietic tissues Binds Bcl-2 and reduces apoptosis Promotes cell survival in different tissues Functions as dominant inhibitor of apoptosis, splice variant of Bcl-X Promotes cell survival in lymphoid and myeloid tissues Promotes cell survival in myeloid tissues

Hetero-dimerizes with Bcl-XL and Bcl-2 displacing Bax and promoting cell death, BH3-only Pro-apoptotic protein Pro-apoptotic protein with multiple isoforms (a, b, g) Pro-apoptotic protein Death agonist for ICE-like protease induction of apoptosis, BH3-only Pro-apoptotic protein, BH3-only Pro-apoptotic, neutralizes Bcl-2, Bcl-XL and Bcl-W, BH3-only BH-3 containing mouse protein that interacts with Bcl-2 and Bcl-XL to induce apoptosis Pro-apoptotic with restrictive expression in reproductive tissues Pro-apoptotic with restrictive expression in reproductive tissues Member of Bcl-2 family and activates apoptosis, BH3-only Immediate EBV early antigen, homologue to Bcl-2, delay cell death in EBV infected cells Homologue of Bcl-2 Homologue to Bcl-2, increases life span of RSV infected cells Homologue to Bcl-2, in Kaposi’s sarcoma herpes virus Homologue to bcl-2, in adenovirus Proapoptotic protein, suppresses E1B-19E and Bcl-XL Released by the mitochondria and induces apoptosis in the nucleus Expressed in glioma cells Human homologue to CED-4 that interacts with caspase 9 and cytochrome C to cause apoptosis Homologue of Apaf-3/caspase 9 and promotes apoptosis and cell death activator Homologue of Apaf-1 Released by the mitochondria to induce apoptosis Bcl-2 homologue, promote apoptosis by direct binding to Apaf-1 Inhibits TNF induced apoptosis

Bcl-XL, neutralizing the protective effect of these antiapoptotic proteins and thereby promoting cell death (Gajeweski and Thompson, 1996). A variable region between BH3 and BH4 has multiple phosphorylation sites, and serine phosphorylation of Bad in response to growth factors sequesters this protein in the cytosol (Datta et al., 1997). The phosphorylated Bad is thereby unavailable to induce apoptosis through interaction with anti-apoptotic Bcl-2 family members in the mitochondria (Zha et al., 1996). Caspase 8 cleavage of the pro-apoptotic protein Bid, which has only the BH3 domain, causes translocation of Bid to the mitochondria where the protein triggers cytochrome c release and the mitochon-

drial damage induced by caspase 8 (Li et al., 1998; Chou et al., 1999; McDonnell et al., 1999). Collectively, these examples illustrate the important and specific roles played by the four BH domains in different Bcl-2 family members, and this structural diversity allows cells to adapt to numerous endogenous and exogenous stimuli. Proteins such as growth factors and p53, which are not members of the Bcl-2 family, also influence cell life and death. For example, the absence of growth factors enhances apoptosis, essentially causing death by neglect. Bag-1 is an apoptosis inhibitory protein that interacts with Bcl-2 (Takayama et al., 1995). Lacking the transmembrane domain, Bag-1 is cytosolic

Levy and Nelson: Apoptosis in the Human Placenta

and is thereby available to interact with the cytoplasmic domains of the receptors for hepatocyte growth factor and platelet-derived growth factor. In the absence of growth factors, Bag-1 binds these receptors and is unavailable to promote cell life by binding Bcl-2 in mitochondria. Cell death results from this tip in the balance toward apoptosis. The tumour suppressor gene p53 encodes a 53 kD phosphoprotein important in DNA repair (Ko and Prives, 1996; Oren, 1994; Gottlieb and Oren, 1998). Cells exposed to radiation may undergo mitotic arrest, allowing DNA repair by a p53 driven mechanism (Di Leonardo, 1994). However, p53 also promotes apoptosis by transcriptional induction of redox-related genes (Polyak et al., 1997) and activation of the pro-apoptotic gene Bax (Miyashita and Reed, 1995). A mutated form of p53 is unable to perform these functions and is associated with neoplastic transformation in a variety of human malignancies (Harris, 1990). The above examples show that the level of protein expression, competitive dimerization, post translational modification, sub-cellular localization and cross talk between surface growth factors and mitochondrial-based regulators are all involved in determining whether a cell will survive or commit suicide. Importantly, members of the Bcl-2 family play different roles in different tissues. This is illustrated by single gene disruptions in mouse models. For example, bcl-2 deletion in mice allows offspring to complete embryonic development. However, the fetuses display growth retardation and the newborns experience early mortality associated with severe polycystic kidney disease, hypopigmentation and thymic hypoplasia (Veis et al., 1993). Deletion of the bcl-xL gene in mice causes fetal death at day 13 of gestation and the fetuses exhibit extensive brain damage (Motoyama et al., 1995). In contrast, deletion of the bax gene results in normal development but infertility (Knudson et al., 1995). A common manifestation of apoptosis irrespective of the inducing stimulus is a disruption of mitochondrial function. Permeability transition pores are multiprotein complexes formed at contact sites between the mitochondrial inner and outer membranes. The Bcl-2 protein family is one of several factors, among which are hypoglycemia, hypoxia and free radical producing agents (Kroemer, 1997), that influence opening and closing of the permeability transition pores. Induction of permeability transition favours pore opening, and a self-amplification process acts as an all-or-none switch for apoptosis. The altered mitochondrial permeability allows release of cytochrome c and apoptosis inducing factor (AIF) into the cytosol [Reed, 1997; Figure 1(F)]. In the cytosol, cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1) in the presence of dATP. Caspase 9 is then activated by binding of the caspase recruitment domain (CARD) in its prodomain to the CARD in Apaf-1 (Hofmann, Bucher and Tschopp, 1997). Apaf-1 itself is a key mediator of some, but not all, apoptotic processes (Cecconi et al., 1998). For example, Diva, a Bcl-2 homologue lacking critical residues in the BH3 domain, can induce apoptosis by binding directly to Apaf-1 independent of the BH3 domain (Inohara et al., 1998). As

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expected, an Apaf-1 deficient mouse shows defects in most tissues that depend on cell death, yet Fas mediated apoptosis is normal in T cells from these mice (Yoshida et al., 1998). As stated before proteins from the Bcl-2 family may effect the death signal induced by the ligand-receptor system. In the hematopoietic cell line it was shown that Bcl-XL inhibits Fas-mediated apoptosis via an unknown mechanism that is downstream of caspase 8 (Medema et al., 1998). This example again highlights the cross talk that occurs among the signalling cascades involved in apoptosis.

Genetic regulation of apopotosis in placenta Bcl-2 is primarily expressed in the trophoblast layer of placental villi. The higher expression of Bcl-2 in syncytiotrophoblast, compared to cytotrophoblast and choriocarcinoma cells, points to a differentiation-dependent regulation of Bcl-2 expression (Sakuragi et al., 1994; Marzioni et al., 1998). A role for Bcl-2 in trophoblast survival was suggested by Marzioni et al. (1998) who found a higher expression of this protein in trophoblast adjacent to injuries on the villous surface, compared to intact villi. A high expression of Bcl-2 in syncytiotrophoblast would protect this key layer of placental villi from apoptosis. However, the role of Bcl-2 in trophoblast associated with fibrin containing fibrinoid deposits is controversial (Marzioni et al., 1998; Toki et al., 1999) and Smith et al. (1997a) showed ultrastructural evidence of apoptosis in syncytiotrophoblast suggesting Bcl-2 does not completely protect the syncytium from self destruction. Both cytotrophoblast and syncytiotrophoblast express the anti-apoptotic protein Mcl-1 (Huppertz et al., 1998), and the pro-apoptotic protein p53 is expressed in normal cytotrophoblasts and intermediate trophoblasts while syncytiotrophoblast shows only rare immunolocalization of the protein (Qiao et al., 1998). The enhanced expression of p53 in undifferentiated trophoblasts could be a mechanism for controlling trophoblast proliferation in normal placenta. Unexpectedly, the wild type gene for p53, instead of a mutated p53 gene, shows enhanced expression in gestational trophoblastic disease, and these tissues display a high rate of apoptosis (Yasuda et al., 1995; Cheung et al., 1994; Shi et al., 1996; Cheville, Robinson and Benda, 1996; Fulop et al., 1998). Speculations about the role of the inhibitory members of the Bcl-2 family in villous trophoblast can be found in the recent work of Huppertz and colleagues (Huppertz et al., 1998).

THE FINAL COMMON PATHWAY OF CELL EXECUTION Irrespective of the stimulus that triggers cell suicide, the execution phase of apoptosis uses a common pathway of enzymes and substrates [Figure 1(G); Tables 3 and 4]. The enzymes are an evolutionarily conserved, constitutively expressed group of cytosolic cysteine proteases (caspase) with

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Table 3. The caspases and their regulators Mediator Group A Caspase Caspase Caspase Caspase Caspase Caspase Caspase Group B Caspase Caspase Caspase Caspase Caspase Group C Caspase Caspase

1 4 5 11 12 13

Alternate name

Function

ICE, CED-3 homologue ICErel-II, TX, ICH-2 ICErel-III, TY

Pro-inflammatory, Pro-inflammatory, Pro-inflammatory, Pro-inflammatory, Pro-inflammatory Pro-inflammatory,

activate IL-1, IL-18 cytokine activator cytokine activator activated by cathepsin B

14

ERICE (Evolutionarily Related Interleukin-1beta Converting Enzyme) Mini-ICE, (MICE)

2 3 6 7 10

ICH-1, Nedd2 CPP32, Yama, SCA-1, LICE; apopain Mch2 Mch3, ICE-LAP3, CMH-I, SCA-2 FLICE2, Mch4

Effector Effector Effector Effector Effector

8 9

Mach-1, FLICE, Mch5 ICE-LAP6, Mch6, Apaf3

Activator caspase in Fas receptor and TNF-R1 mediated cell death Activator caspase upstream in mitochondrial induced apoptosis

BIR CARD Caspase CPAN CRADD CrmA DFF DISC E1B55 E6 FLICE FLIP Granzymes

Regulators Baculovirus IAP repeat, p35 Caspase activation and recruitment domain Cysteinyl aspartic acid protease Caspase activated nuclease Caspase and RIP adaptor with death domain Cytokine response modifier A DNA fragmentation factor Death inducing signaling complex FADD-like I C E protease; MACH FLICE inhibitory protein; FLAME, I-FLICE

HIAP-1 HILP IAP

Human inhibitor of apoptosis protein 1; cIAP Human IAP-like protein Inhibitors of apoptosis proteins

ICE LMW5-HL

Interleukin-1
converting enzyme

MACH ORF16

MORT-1 associated CED-3 homologue; FLICE Open reading frame 16

p35 PFP

BIR Pore forming protein; perforin

XIAP

X-linked inhibitor of apoptosis; HILP

specificity for aspartic acid residues in their substrates (Salvesen and Dixit, 1997). These enzymes were first implicated in apoptosis by genetic analysis in the nematode C. elegans (Yuan and Horvitz, 1990), and fourteen caspases have subsequently been identified (Thornberry and Lazebnik, 1998; Hu et al., 1998). The caspases reside in the cytosol as single chain pro-enzymes which aggregate as a step in enzyme activation. This aggregation allows the relatively inactive pro-caspases to work synergistically to generate active caspases,

can be activated by caspase 8

Highly expressed in embryonic tissues but absent in adult tissues caspase caspase caspase caspase caspase

for apoptosis execution for apoptosis execution, mainly in brain and thymocytes for apoptosis execution, cleaves lamins for apoptosis execution for apoptosis execution

Suppressor of apoptosis Activation of apoptotic process Proteases important in apoptosis Also known as DFF40, CAD Novel 70a.a. domain Cowpox viral serpin caspase inhibitor Trigger DNA fragmentation Signal transduction systems involved in apoptosis 55 kD adenovirus protein that inactivates p53 Human papillomavirus 16 inhibitor of p53 Caspase 8 Caspase inhibitor of Fas/TNF-R1 induced apoptosis Serine proteases in granules of cytotoxic T cells and NK cells involved in induction of target cell apoptosis An inhibitor of apoptosis Regulates apoptosis downstream of Bcl-XL and cytochrome C Family of proteins inhibiting apoptosis including NAIP, cIAP-1, cIAP-2, X-IAP, and survivin Caspase 1 African swine fever virus Bcl-2 homologue that inhibits cell death of cells infected by the virus Caspase 8 Herpes virus samiri Bcl-2 homologue that protects cells from virus induced apoptosis Baculovirus caspase inhibitor Protein secreted by cytotoxic T cells or NK cells that punctures a pore in cell membranes Inhibits apoptosis

amplifying the pro-apoptotic downstream signal. The procaspase aggregation is mediated by interaction domains in the enzymes, including caspase recruitment domains (CARDs) and death effector domains (DEDs). Autocatalytic activation occurs through two enzymatic cuts at asparagine sites within the procaspase molecule releasing p]20 and p]10 subunits. A subsequent cleavage releases the N-terminal domain which is involved in the regulation of activation. The mature caspase is a hetero-dimer with high sequence conservation in regions

Levy and Nelson: Apoptosis in the Human Placenta

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Table 4. Substrates Nucleus Cytosol v v v v v v v v v v v v v v v v v v v v v v v

Caspase 1 Caspase 3 Caspase 6 Caspase 7 G-Actin CAD DNA -PKcs D4-GDI DFF45 FAK
-Fodrin Gelsolin Gas2 HD hnRNP-c MDM2 MEKK1 Lamin A,B NUMA PAK2 PARP PS SP-1,2 PKC-, PRK2 Pro-IL1 Rabaptin Rb SP-1,2 SREBP-1 and 2 Topo II U1-70 kDsnRNP

Cytoskeletal component Caspase activated deoxyribonuclease DNA protein kinase catalytic site GDP dissociation inhibitor type 4 of small G-proteins DNA fragmentation factor (inhibit CAD) Focal adhesion kinase Membrane associated cytoskeletal protein Actin-severing protein Growth arrest specific protein 2 cytoskeletal component Huntington disease protein Heterogeneous nuclear ribonucleoprotein C Murine double minute-2 Inhibitor of p53; HDM2 MAP/Erk kinase kinase 1 Nuclear cytoskeleton component Nuclear mitotic apparatus protein p21-activated kinase 2 Poly (ADP-ribose) polymerase Phosphatidyl serine Presenilins, familial Alzheimer Protein kinase C-,  Protein kinase C related kinase 2 Pro-interleukin 1  Membrane fusion Retinoblastoma protein General transcription factor Sterol regulatory element binding proteins 1 and 2 Topoisomerase II U1 small nuclear ribonucleoprotein

important for substrate binding and catalysis (Nicholson and Thornberry, 1997). The caspases of human origin can be divided into three groups based on their function (Table 3). Group A includes caspases 1, 4 and 5, as well as the recently identify caspases 11–14, all of which play a role in inflammation. For example, caspase 1 cleaves pro-interleukin 1 to its active form. Nevertheless, the normal phenotype of mice with a caspase 1 deficiency suggests this enzyme is not crucial for most apoptotic processes (Li et al., 1995). Group B includes caspases 2, 3, 6, 7 and 10 which are effectors in the execution process, cleaving aspartic acid residues in key substrates, like lamins and focal adhesion kinase, to yield the apoptotic phenotype. Caspases in this group exhibit a tissue specific function. This is exemplified by the dramatic abnormalities in neural development associated with the caspase 3 gene knockout in the mouse, leaving other tissues with normal function (Kuida et al., 1996). Group C caspases 8 and 9 are key activators of the caspase cascade, and adaptor proteins facilitate aggregation in the transduction step, enhancing the autoactivation described above (Muzio et al., 1998). For example, there is reduced apoptosis and cytochrome c mediated caspase 3 activation in mice lacking caspase 9, indicating that caspase 9 is a critical upstream activator of this apoptotic cascade in vivo (Kuida et al., 1998). Caspase 10 may be an activator, as well as effector (Thornberry and Lazebnik, 1998). Caspases are not the only

  

   





 





enzymes that participate in cell execution although they are absolutely required to effect the specific proteolytic events that lead to the apoptotic morphology. Several endogenous inhibitors of apoptosis (IAP) regulate adventitious proteolysis (Deveraux and Reed, 1999). Numerous viruses produce proteins that inhibit caspases, thereby preventing death of the host cell. In addition, synthetic caspase inhibitors have been developed (Nicholson and Thornberry, 1997), and early studies suggest they may provide neuroprotection in acute bacterial meningitis (Braun et al., 1999) and in the tissue damage in a rat model of hypoxic-ischemic brain injury (Cheng et al., 1998). Furthermore, the synthetic inhibitors may also be useful in rescuing apoptosis that follows the acute ischemia associated with stroke and myocardial infarction (Hetts, 1998). Taken together these studies suggest therapeutic interventions might be available to avoid the sequelae of harmful apoptotic insults to vital organs, even in differentiated tissues. The proteolytic activity of caspases targets several subsets of proteins that are involved in homeostasis, repair and structure of cells (Hale et al., 1996). What is unclear is whether or not one or many substrates must be cleaved for the cell to be irreversibly committed to apoptosis. Cleavage of these substrates yields different functional consequences. In some cases proteolysis may abolish critical structures and functions while in other instances substrate cleavage may activate a previously

10

suppressed function. For example, caspases activate several kinases such as protein activated kinase (PAK) by removing an amino-terminal regulatory domain. The activated kinase induces cytoplasmic and nuclear condensation, cellular detachment and phosphatidylserine externalization, which are all characteristic of the apoptotic process (Lee et al., 1997). The apoptotic substrates can be divided into groups according to their site of origin and presumed function. A few examples are described, and others can be found in Table 4. (1) Enzymes involved in genome repair: Poly (ADP-ribose) polymerase (PARP) was the first protein identified as a substrate for caspases. PARP catalyzes the synthesis of poly (ADP-ribose) from nicotinamide adenine dinucleotide (NAD+) and PARP also binds DNA strand breaks and modifies nuclear proteins by attaching poly ADP-ribose chains (Satoh and Lindahl, 1992). PARP is cleaved by caspase 3, and this occurs prior to the degradation of nuclear DNA into the internucleosomal fragments that characterize apoptosis. Enhanced PARP turnover is a marker of caspase 3 activity and of apoptosis in general (Kaufmann et al., 1993). (2) Enzymes involved in cell replication and cell cycle progression: DNA topoisomerase II (topo II) is a nuclear enzyme essential for DNA replication and transcription, and dysregulation of this enzyme results in DNA damage (McPherson and Goldenberg, 1998; Nakajima et al., 1996). Thus, caspase inactivation of topo II clearly interferes with DNA function. (3) DNA as a substrate: The characteristic internucleosomal fragmentation of DNA associated with apoptosis (Wyllie, 1980) is triggered by CAD (caspase activated deoxyribonuclease). This enzyme is inhibited by DNA fragmentation factor (DFF45). Cleavage of DFF45 by caspase 3 releases CAD to function as a nuclease and results in DNA fragmentation (Liu et al., 1997; Sakahira, Enari and Nagata, 1998). (4) Nuclear and cytoskeletal proteins: The lamins are intranuclear proteins that maintain nuclear shape and mediate chromatin–nuclear membrane interactions. Caspase 6 degrades lamins, and this degradation results in the chromatin condensation and nuclear fragmentation characteristic of the apoptotic morphology (Rao, Perez and White, 1996). Gelsoline is a cytoskeletal protein that organizes the actin filament network. In the cytoplasm, cleavage of gelsoline by caspase 3 results in an amino-terminal cleavage fragment that disrupts actin filaments leading to an altered cytoplasmic architecture (Kothakota et al., 1997). (5) Cell membranes and matrix attachment: Cell attachment to extracellular matrix is mediated by integrins and by focal adhesion kinase (FAK). The FAK is a tyrosine kinase that stimulates cell spreading by promoting formation of focal adhesion contact sites between cells and matrix. An early event in apoptosis is detachment of the cell from the substratum and loss of cell–cell interactions. During this process, FAK is cleaved at two sites by caspases 3 and 6 (Gervais et al., 1998; Wen et al., 1998). This disrupts cell adhesion irreversibly and interferes with transmission of matrix derived cell survival signals. Cells undergoing apoptosis also have altered expres-

Placenta (2000), Vol. 21

sion of surface membrane components, such as phosphatidylserine, thrombospondin and integrin carbohydrate moieties, which identify dying cells and target them for phagocytosis (Savill et al., 1993; Hale et al., 1996). For example, phosphatidylserine is known to translocate from the inner leaflet of the surface membrane lipid bilayer to the external leaflet as an early event in apoptosis (Martin et al., 1995) and in this new location, phosphatidylserine binds annexin V. Localization of phosphatidylserine and annexin V to cell surfaces is used as an experimental marker to identify cells undergoing apoptotic cell death, and expression of these molecules may be identified by resident phagocytic cells as a signal for phagocytosis of apoptotic bodies (Savill et al., 1993). However, trophoblast phosphatidylserine expression may not be a good marker of apoptosis, as described below.

CASPASES AND SUBSTRATES IN PLACENTAL APOPTOSIS Procaspase 3 and caspase 3 are the only caspases identified to date in the placenta. Both enzymes are expressed in cytotrophoblast and syncytiotrophoblast at higher levels in first, compared to third trimester villi (Huppertz et al., 1998). Third trimester villi have localized areas of syncytiotrophoblast that express phosphatidylserine and annexin V, with or without apoptotic nuclei in the subjacent cytoplasm (Huppertz et al., 1998). Transglutaminase II, TIAR, PARP, lamin B and topo II have been localized to cytotrophoblast of first trimester villi with limited expression in first trimester syncytiotrophoblast and lowest expression in trophoblast of term villi (Huppertz et al., 1998). The apoptotic morphology is commonly found in trophoblast where fibrin containing fibrinoid deposits mark injury of the trophoblast layer (Nelson, 1996; Marzioni et al., 1998; Huppertz et al., 1998; Chan, Lao and Cheung, 1999). Recent morphological studies of villous trophoblast suggest that nuclear differentiation in syncytiotrophoblast proceeds through two phases (Mayhew et al., 1999). On entering the syncytium, nuclei commit to a long programmed pre-apoptotic phase which is followed by a short apoptotic execution phase. Mayhew and colleagues (Mayhew et al., 1999) suggest that the clustered nuclei (pre-apoptotic and apoptotic) in syncytial knots may reflect this sequence and extruding nuclei in these knots may be a component of normal continuous epithelial turnover. In addition, surface expression of phosphatidylserine in localized areas of trophoblast, notably at villous tips and syncytial knots and sprouts, is associated with nuclei showing condensed chromatin typical of the apoptotic morphology (Huppertz et al., 1998). However, phosphatidylserine expression may not be a good marker of apoptosis in trophoblast since a role for phosphatidylserine expression separate from apoptosis has been proposed for cytotrophoblast differentiation in the BeWo cell model (Lyden, Ng and Rote, 1993) and in cytotrophoblast on placental villi (Huppertz et al., 1998). Collectively, these observations suggest that localized areas of syncytiotrophoblast undergo apoptosis. These studies also

Levy and Nelson: Apoptosis in the Human Placenta

suggest that selected caspases and numerous substrates are differentially expressed by trophoblast during gestation. What regulates this change during development, what controls the apoptotic process in the syncytium and whether or not dysregulation leads to placental dysfunction remain key questions for future studies.

APOPTOSIS AND PREGNANCY PATHOLOGY Dysregulation of the apoptotic process probably causes a variety of diseases (Rudin and Thompson, 1997; Hetts, 1998), contributing to the proliferation associated with neoplasia or the premature cell loss characteristic of degenerative and autoimmune diseases. Preliminary studies suggested that enhanced apoptosis is associated with abnormal pregnancies such as first trimester abortions and ectopic pregnancies (Kokawa, Shikone and Nakano, 1998a,b). Hypoxia is a known trigger of apoptosis in different tissues (Linnik, Zobrist and Hatfield, 1993; Graeber et al., 1996) and hypoxia may trigger apoptosis in the placenta as a possible mechanism of pregnancy related complications. A recent study has demonstrated wide spread apoptosis and a decrease in Bc1-2 expression in cytotrophoblasts obtained from placental bed of pregnancies complicated by pre-eclampsia compared to little or no apoptosis in similar specimens of non pre-eclamptic women (DiFederico, Genbacev and Fisher, 1999). Under-perfusion of the placenta and chronic hypoxia in clinical conditions such as maternal anemia, smoking and pre-eclampsia has been implicated as a mechanism for fetal growth restriction (Kingdom and Kaufmann, 1997). Placentae of pregnancies complicated by fetal growth restriction tend to be small with a decreased surface area exposed to maternal circulation. A higher level of apoptosis has been found in both animal and human placentae

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from pregnancies complicated by fetal growth restriction, compared to placentae from pregnancies with normal growth (Miller et al., 1996; Smith, Baker and Symonds, 1997b). These two examples implicate a role for apoptosis in the placenta response to hypoxic stress. Apoptosis may eliminate abnormal cells that are exposed to exogenous stress. Finally, observational studies implicating enhanced apoptosis in trophoblast neoplasia are described in previous sections of this review. Mechanistic studies are likely to provide important insights into what mediates trophoblastic disease. Collectively, these preliminary observations in pregnancy pathologies suggest that detailed study of placental apoptosis most certainly will enhance our understanding of why some pregnancies fail to proceed to delivery of a normal newborn.

SUMMARY Signals that trigger cells to undergo apoptosis utilize shared pathways, whether the initiating event is receptor mediated or a response to an exogenous stimulus. The pathway is summarized in Figure 1(E). The duration of time from signal to completed execution varies among cells, limiting our ability to estimate apoptosis in given tissues over time. Indeed, apoptosis may take up to three days with only the last 1–3 h of the process reflected by the characteristic apoptotic morphology (Messam and Pittman, 1998). Most of the placenta-related studies cited above have focused on measuring nuclear changes that occur in the apoptotic process. However, understanding the details of receptor-mediated signalling pathways, the Bcl-2 regulators, and the caspases and substrates involved in placental apoptosis will surely provide insights into both normal placental development and placental dysfunction associated with abnormal pregnancy.

ACKNOWLEDGEMENTS We thank Dr Jon Tilly and Dr Yoel Sadovsky for their helpful editing, and Ms Lori Rideout and Ms Veronica Mulherin for figure and manuscript preparation, respectively.

REFERENCES Adams JM & Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science, 281, 1322–1326. Ashkenazi A & Dixit VM (1998) Death receptors: signaling and modulation. Science, 281, 1305–1308. Baker SJ & Reddy EP (1998) Modulation of life and death by the TNF receptor superfamily. Oncogene, 17, 3261–3270. Baldwin AS Jr (1996) The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol, 14, 649–683. Bamberger A-M, Schulte HM, Thuneke I, Erdmann I, Bamberger CM & Asa SL (1997) Expression of the apoptosis-inducing Fas ligand (FasL) in human first and third trimester placenta and choriocarcinoma cells. J Clin Endocrinol Metab, 82, 3173–3175. Basanez G, Nechushtan A, Drozhinin O, Chanturiya A, Choe E, Tutt S, Wood KA, Hsu Y-T, Zimmerberg J & Youle RJ (1999) Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc Natl Acad Sci USA, 96, 5492–5497. Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A & Duke RC (1995) A role for CD95 ligand in preventing graft rejection. Nature, 377, 630–632.

Braun JS, Kovak R, Herzog K-H, Bodner SM, Cleveland JL & Tuomanen EI (1999) Neuroprotection by a caspase inhibitor in acute bacterial meningitis. Nature Med, 5, 298–302. Bulmer JN, Morrison L, Johnson PM & Meager A (1990) Immunohistochemical localization of interferons in human placental tissues in normal, ectopic and molar pregnancies. Am J Reprod Immunol, 22, 109–116. Cecconi F, Alvarez-Bolado G, Meyer BI, Roth KA & Gruss P (1998) Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell, 94, 727–737. Chan CCW, Lao TT & Cheung ANY (1999) Apoptotic and proliferative activities in first trimester placentae. Placenta, 20, 223–227. Chen H-L, Yang Y, Hu X-L, Yelavarthi KK, Fishback JL & Hunt JS (1991) Tumor necrosis factor alpha mRNA and protein are present in human placental and uterine cells at early and late stages of gestation. Am J Pathol, 139, 327–335. Cheng Y, Deshmukh M, D’Costa A, Demaro JA, Gidday JM, Shah A, Sun Y, Jacquin MF, Johnson, EM Jr & Holtzman DM (1998) Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J Clin Invest, 101, 1992–1999. Cheung ANY, Srivastava G, Chung LP, Ngan HYS, Man TK, Liu YT, Chen WZ, Collins RJ, Wong LC & Ma HK (1994) Expression of the p-53

12 gene in trophoblastic cells in hydatiform moles and normal human placentas. J Reprod Med, 39, 223–227. Cheville JC, Robinson RA & Benda JA (1996) P53 expression in placentas with hydropic change and hydatidiform moles. Mod Pathol, 9, 392–396. Chou JJ, Li H, Salvesen GS, Yuan J & Wagner G (1999) Solution structure of BID, an intracellular amplifier of apoptotic signaling. Cell, 96, 615–624. Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J, 326, 1–16. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y & Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cellintrinsic death machinery. Cell, 91, 231–241. Deveraux QL & Reed JC (1999) IAP family proteins—suppressors of apoptosis. Genes Dev, 13, 239–252. DiFederico E, Genbacev O & Fisher SJ (1999) Preeclampsia is associated with widespread apoptosis of placental cytotrophoblasts within the uterine wall. Am J Pathol, 155, 293–301. Di Leonardo A, Linke SP, Clarkin K & Wahl GM (1994) DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev, 8, 2540–2551. Eades DK, Cornelius P & Pekala PH (1988) Characterization of the tumor necrosis factor receptor in human placenta. Placenta, 9, 247–251. Fulop V, Mok SC, Genest DR, Gati I, Doszpod J & Berkowitz RS (1998) p53, p21, Rb and mdm2 oncoproteins: expression in normal placenta, partial and complete mole, and choriocarcinoma. J Reprod Med, 43, 119–127. Gajewski TF & Thompson CB (1996) Apoptosis meets signal transduction: elimination of a BAD influence. Cell, 87, 589–592. Garcia-Lloret MI, Yui J, Winkler-Lowen B & Guilbert LJ (1996) Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts. J Cell Phys, 167, 324–332. Gervais FG, Thornberry NA, Ruffolo SC, Nicholson DW & Roy S (1998) Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide. J Biol Chem, 273, 17 102–17 108. Gottlieb TM & Oren M (1998) P53 and apoptosis. Semin Cancer Biol, 8, 359–368. Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW & Giaccia AJ (1996) Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature, 379, 88–91. Green DR & Reed JC (1998) Mitochondria and apoptosis. Science, 281, 1309–1312. Griffith TS, Brunner T, Fletcher SM, Green DR & Ferguson TA (1995) Fas ligand-induced apoptosis as a mechanism of immune privilege. Science, 270, 1189–1192. Gross A, Jockel J, Wei MC & Korsmeyer SJ (1998) Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J, 17, 3878–3885. Hale AJ, Smith CA, Sutherland LC, Stoneman VEA, Longthorne VL, Culhane AC & Williams GT (1996) Apoptosis: molecular regulation of cell death. Eur J Biochem, 236, 1–26. Hammer A, Blaschitz A, Daxbock C, Walcher W & Dohr G (1999) Fas and Fas-ligand are expressed in the uteroplacental unit of first-trimester pregnancy. Am J Reprod Immunol, 41, 41–51. Harris AL (1990) Mutant p53: the commonest genetic abnormality in human cancer? J Pathol, 162, 5–6. Hetts SW (1998) To die or not to die. An overview of apoptosis and its role in disease. JAMA, 279, 300–307. Hockenbery D, Nun˜ ez G, Milliman C, Schrieber RD & Korsmeyer SJ (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature, 348, 334–336. Hockenbery DM, Zutter M, Hickey W, Nahm M & Korsmeyer SJ (1991) Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA, 88, 6961–6965. Hofmann K, Bucher P & Tschopp J (1997) The CARD domain: a new apoptotic signalling motif. Trends Biochem Sci, 22, 155–156. Hu S, Snipas SJ, Vincenz C, Salvesen G & Dixit VM (1998) Caspase-14 is a novel developmentally regulated protease. J Biol Chem, 273, 29 648– 29 653. Hunt JS, Vassmer D, Ferguson TA & Miller L (1997) Fas ligand is positioned in mouse uterus and placenta to prevent trafficking of activated leukocytes between the mother and the conceptus. J Immunol, 158, 4122–4128. Huppertz B, Frank H-G, Kingdom JCP, Reister F & Kaufmann P (1998) Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. Histochem Cell Biology, 110, 495–508.

Placenta (2000), Vol. 21 Inohara N, Gourley TS, Carrio R, Mun˜ iz M, Merino J, Garcia I, Koseki T, Hu Y, Chen S & Nun˜ ez G (1998) Diva, a Bcl-2 homologue that binds directly to Apaf-1 and induces BH3-independent cell death. J Biol Chem, 273, 32 479–32 486. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer JL, Schroter M, Burns K, Mattmann C, Rimoldi D, French LE & Tschopp J (1997) Inhibition of death receptor signals by cellular FLIP. Nature, 388, 190–195. Kaplan HJ, Leibole MA, Tezel T & Ferguson TA (1999) Fas ligand (CD95 ligand) controls angiogenesis beneath the retina. Nature Med, 5, 292–297. Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE & Poirier G (1993) Specific proteolytic cleavage of poly (ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res, 53, 3976– 3985. Kerr JFR, Wyllie AH & Currie AR (1972) Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer, 26, 239–257. Kingdom JCP & Kaufmann P (1997) Oxygen and placental villous development: origins of fetal hypoxia. Placenta, 18, 613–621. Kitson J, Raven T, Jiang Y-P, Goeddel DV, Giles KM, Pun K-T, Grinham CJ, Brown R & Farrow SN (1996) A death-domain-containing receptor that mediates apoptosis. Nature, 384, 372–375. Knudson CM, Tung K, Tourtelloffe WG, Brown G & Korsmeyer SJ (1995) Bax deficient mice with lymphoid hyperplasia and male germ cell death. Science, 270, 96–99. Ko LJ & Prives C (1996) p53: puzzle and paradigm. Genes Dev, 10, 1054–1072. Kokawa K, Shikone T & Nakano R (1998a) Apoptosis in human chorionic villi and decidua during normal embryonic development and spontaneous abortion in the first trimester. Placenta, 19, 21–26. Kokawa K, Shikone T & Nakano R (1998b) Apoptosis in human chorionic villi and decidua in normal and ectopic pregnancy. Molecular Hum Reprod, 4, 87–91. Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ & Williams LT (1997) Caspase 3-generated fragment of gelsolin: Effector of morphological change in apoptosis. Science, 278, 294–298. Kroemer G (1997) The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nature Med, 3, 614–620. Kuida K, Zheng TS, Na S, Kuan C, Yang D, Arasuyama H, Rakic P & Flavell RA (1996) Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature, 384, 368–372. Kuida K, Haydar TF, Kuan C-Y, Gu Y, Taya C, Karasuyama H, Su MS-S, Rakic P & Flavell RA (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell, 94, 325–337. Lee N, MacDonald H, Reinhard C, Halenbeck R, Roulston A, Shi T & Williams LT (1997) Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis. Proc Natl Acad Sci USA, 94, 13 642–13 647. Li H, Zhu H, Xu C-J & Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell, 94, 491–501. Li P, Allen H, Banerjee S, Franklin S, Herzog L, Johnston C, McDowell J, Paskind M, Rodman L & Salfeld J (1995) Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell, 80, 401–411. Liu X, Zou H, Slaughter C & Wang X (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell, 89, 175–184. Linnik MD, Zobrist RH & Hatfield MD (1993) Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke, 24, 2002–2009. Lyden TW, NG A-K & Rote NS (1993) Modulation of phosphatidylserine epitope expression by BeWo cells during forskolin treatment. Placenta, 14, 177–186. Martin SJ, Reutelingsperger CPM, McGahon AJ, Rader JA, van Schie RCCA, LaFace DM & Green DR (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Ab1. J Exp Med, 182, 1545–1556. Marzioni D, Mu¨hlhauser J, Crescimanno C, Banita M, Pierleoni C & Castellucci M (1998) Bcl-2 expression in the human placenta and its correlation with fibrin deposits. Human Reprod, 13, 1717–1722.

Levy and Nelson: Apoptosis in the Human Placenta Mayhew TM, Leach L, McGee R, Ismail WW, Myklebust R & Lammiman MJ (1999) Proliferation, differentiation and apoptosis in villous trophoblast at 13–41 weeks of gestation (including observations on annulate lamellae and nuclear pore complexes). Placenta, 20, 407–422. McDonnell JM, Fushman D, Milliman CL, Korsmeyer SJ & Cowburn D (1999) Solution structure of the proapoptotic molecule BID: A structural basis for apoptotic agonists and antagonists. Cell, 96, 625–634. McPherson JP & Goldenberg GJ (1998) Induction of apoptosis by deregulated expression of DNA topoisomerase II alpha. Cancer Res, 58, 4519–4524. Medema JP, Scaffidi C, Krammer PH & Peter ME (1998) Bcl-XL acts downstream of Caspase-8 activation by the CD95 death-inducing signaling complex. J Biol Chem, 273, 3388–3398. Messam CA & Pittman RN (1998) Asynchrony and commitment to die during apoptosis. Exp Cell Res, 238, 389–398. Miller MJ, Voelker CA, Olister S, Thompson JH, Zhang XJ, Rivera D, Eloby-Childress S, Liu X, Clark DA & Pierce MR (1996) Fetal growth retardation in rats may result from apoptosis: role of peroxynitrite. Free Radic Biol Med, 21, 619–629. Miyashita T & Reed JC (1995) Tumor suppressor p53 is a direct transcriptional activator of the human Bax gene. Cell, 80, 293–299. Mor G, Gutierrez LS, Eliza M, Kahyaoglu F & Arici A (1998) Fas-Fas ligand system-induced apoptosis in human placenta and gestational trophoblastic disease. Am J Reprod Immunol, 402, 89–94. Motoyama N, Wang FP, Roth KA, Sawa H, Nakayama K, Nakayama K, Negishi I, Senju S, Zhang Q, Fujii S & Loh DY (1995) Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science, 267, 1506–1510. Muzio M, Stockwell BR, Stennicke HR, Salvesen GS & Dixit VM (1998) An induced proximity model for caspase-8 activation. J Biol Chem, 273, 2926–2930. Nagata S (1997) Apoptosis by death factor. Cell, 88, 355–365. Nagata S & Goldstein P (1995) The Fas death factor. Science, 267, 1449–1456. Nakajima T, Morita K, Ohi N, Arai T, Nozaki N, Kikuchi N, Osaka F, Yamao F & Oda K (1996) Degradation of topoisomerase II alpha during adenovirus EIA-induced apoptosis is mediated by the activation of the ubiquitin proteolysis system. J Biol Chem, 271, 24 842–24 849. Nelson DM (1996) Apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrin type fibrinoid is deposited at discontinuities in the villous trophoblast. Placenta, 17, 387–391. Nicholson DW & Thornberry NA (1997) Caspases: killer proteases. Trends Biochem Sci, 8, 299–306. Oltvai ZN, Milliman CL & Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell, 74, 609–619. Oren M (1994) Relationship of p53 to the control of apoptotic cell death. Semin Cancer Biol, 5, 221–227. Pan G, Bauer JH, Haridas V, Wang S, Liu D, Yu G, Vincenz C, Aggarwal BB, Ni J & Dixit VM (1998) Identification and functional characterization of DR6, a novel death domain-containing TNF receptor. FEBS Lett, 431, 351–356. Payne SG, Smith SC, Davidge ST, Baker PN & Guilbert LJ (1999) Death receptor Fas/Apo-1/CD95 expressed by human placental cytotrophoblasts does not mediate apoptosis. Biol Reprod, 60, 1144–1150. Polyak K, Xia Y, Zweier JL, Kinzler KW & Vogelstein B (1997) A model for p53-induced apoptosis. Nature, 389, 300–305. Qiao S, Nagasaka T, Harada T & Nakashima N (1998) p53, Bax and Bcl-2 expression, and apoptosis in gestational trophoblast of complete hydatidiform mole. Placenta, 19, 361–369. Rao L, Perez D & White E (1996) Lamin proteolysis facilitates nuclear events during apoptosis. J Cell Biol, 135, 1441–1455. Reed JC (1997) Cytochrome c: Can’t live with it—can’t live without it (comment). Cell, 91, 559. Rudin CM & Thompson CB (1997) Apoptosis and disease: regulation and clinical relevance of programmed cell death. Annu Rev Med, 48, 267–281. Runic R, Lockwood CJ, Ma Y, Dipasquale B & Guller S (1996) Expression of Fas ligand by human cytotrophoblasts: implications in placentation and fetal survival. J Clin Endocrinol Metab, 81, 3119–3122. Sakahira H, Enari M & Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degredation during apoptosis. Nature, 392, 96–99.

13 Sakuragi N, Matsuo H, Coukos G, Furth EE, Bronner MP, VanArsdale CM, Krajewsky S, Reed JC & Strauss III JF (1994) Differentiationdependent expression of the BCL-2 proto-oncogene in the human trophoblast lineage. J Soc Gynecol Invest, 1, 164–172. Salvesen GS & Dixit VM (1997) Caspases: Intracellular signaling by proteolysis. Cell, 91, 443–446. Sata M & Walsh K (1998) Endothelial cell apoptosis induced by oxidized LDL is associated with the down-regulation of the cellular caspase inhibitor FLIP. J Biol Chem, 273, 33 103–33 106. Satoh MS & Lindahl T (1992) Role of poly(ADP-ribose) formation in DNA repair. Nature, 26, 356–358. Shi Y-F, Xie X, Zhao C-L, Ye D-F, Lu S-M, Hor JJ & Pao CC (1996) Lack of mutation in tumour-suppressor gene p53 in gestational trophoblastic tumors. Br J Cancer, 73, 1216–1219. Savill J, Fadok V, Henson P & Haslett C (1993) Phagocyte recognition of cells undergoing apoptosis. Immunol Today, 14, 131–136. Smith SC, Baker PN & Symonds EM (1997a) Placental apoptosis in normal human pregnancy. Am J Obstet Gynecol, 177, 57–65. Smith SC, Baker PN & Symonds EM (1997b) Increased placental apoptosis in intrauterine growth restriction. Am J Obstet Gynecol, 177, 1395–1401. Smith SC, Guilbert LJ, Yui J, Baker PN & Davidge ST (1999) The role of reactive nitrogen/oxygen intermediates in cytokine-induced trophoblast apoptosis. Placenta, 20, 309–315. Smyth MJ, Obeid LM & Hannun YA (1997) Ceramide: A novel lipid mediator of apoptosis. Adv Pharmacol, 41, 133–153. Takayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA & Reed JC (1995) Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell, 80, 279–284. Thornberry NA & Lazebnik Y (1998) Caspases: enemies within. Science, 281, 1312–1316. Toki T, Horiuchi A, Ichikawa N, Mori A, Nikaido T & Fujii S (1999) Inverse relationship between apoptosis and Bcl-2 expression in syncytiotrophoblast and fibrin-type fibrinoid in early gestation. Mol Human Reprod, 5, 246–251. Uckan D, Steele A, Wang B-Y, Chamizo W, Koutsonikolis A, GilbertBarness E & Good RA (1997) Trophoblasts express Fas ligand: a proposed mechanism for immune privilege in placenta and maternal invasion. Mol Hum Reprod, 3, 655–662. Vaux DL & Korsmeyer SJ (1999) Cell death in development. Cell, 96, 245–254. Veis DV, Sorenson CM, Shutter JR & Korsmeyer SJ (1993) Bcl-2 deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell, 75, 229–240. Wen L-P, Fahrni JA, Troie S, Guan J-L, Orth K & Rosen GD (1997) Cleavage of focal adhesion kinase by caspases during apoptosis. J Biol Chem, 10, 26 056–26 061. Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature, 284, 555–556. Yasuda M, Kawai K, Serizawa A, Tang X & Osamura RY (1995a) Immunohistochemical analysis of expression of p53 protein in normal placentas and trophoblastic diseases. Appl Immunohistochem, 3, 132–136. Yoshida H, Kong Y-Y, Yoshida R, Elia AJ, Hakem A, Hakem R, Penninger JM & Mak TW (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell, 94, 739–750. Yuan J & Horvitz HR (1990) The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death. Dev Biol, 138, 33–41. Yui J, Garcia-Lloret MI, Wegmann TG & Guilbert LJ (1994) Cytotoxicity of tumor necrosis factor alpha and gamma-interferon against primary human placental trophoblasts. Placenta, 15, 819–825. Yui J, Hemmings D, Garcia-Lloret M & Guilbert LJ (1996) Expression of the human p55 and p75 tumor necrosis factor receptors in primary villous trophoblasts and their role in cytotoxic signal transduction. Biol Reprod, 55, 400–409. Zha J, Harada H, Yang E, Jockel J & Korsmeyer SJ (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-XL. Cell, 87, 619–628. Zorzi W, Thellin O, Coumans B, Melot F, Hennen G, Lakaye B, Igout A & Heinen E (1998) Demonstration of the expression of CD95 ligand transcript and protein in human placenta. Placenta, 19, 269–277.