Arbiter of differentiation and death: Notch signaling meets apoptosis

Arbiter of Differentiation and Death:Notch Signaling Meets Apoptosis

LUCIO MIELE1* AND BARBARA OSBORNE2

1Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, Illinois2Department of Veterinary and Animal Sciences, University of Massachusetts,

Amherst, Massachusetts

Notch-ligand interactions are a highly conserved mechanism that regulates cellfate decisions. Over the past few years, numerous observations have shown thatthis mechanism operates to regulate cell differentiation in an enormous variety ofdevelopmental and cell maturation processes. Recent studies indicate that inaddition to cell differentiation, Notch signaling has direct effects on proliferationand programmed cell death. The picture emerging from these findings suggeststhat, depending on cellular and developmental context, Notch signaling mayfunction as a general “arbiter” of cell fate, regulating differentiation potential, rateof proliferation, and apoptotic cell death. In this review, we briefly summarize thecurrent knowledge of the structure and function of Notch receptors and discussthe recent evidence that Notch signaling regulates apoptotic cell death. Thepossible mechanisms of this effect and its potential implications for develop-mental biology, immunobiology, neuropathology, and tumor biology arediscussed. J. Cell. Physiol. 181:393–409, 1999. © 1999 Wiley-Liss, Inc.

Genes of the notch family encode transmembranereceptors that control cell fate decisions during devel-opment in organisms from Drosophila to humans (Ar-tavanis-Tsakonas et al., 1995, 1999; Blaumueller andArtavanis-Tsakonas, 1997; Weinmaster, 1997; Kopanand Cagan, 1997; Greenwald, 1998; Milner and Bigas,1999). Over the past decade, studies of Drosophilanotch and its Caenorhabditis elegans counterpartslin-12 and glp-1 clearly have identified Notch signalingas an important “switch” controlling cell fate decisionsduring development in numerous tissues (Artavanis-Tsakonas et al., 1995, 1999; Greenwald, 1998; Milnerand Bigas, 1999). Notch has been aptly defined byArtavanis-Tsakonas “gatekeeper of cell fate.” Notchsignaling is believed to mediate communication be-tween neighboring cells during cell-cell contact, sinceNotch activation results from the binding of cell surfaceligands on adjacent cells. Very recently, a soluble formof the Drosophila Notch ligand Delta has been de-scribed, raising the possibility that Notch may alsomediate interactions between noncontiguous cells (Qiet al., 1999). While it is well established that Notchsignaling regulates cell differentiation under many cir-cumstances, until recently there was no direct evidenceit could affect cell death. A number of reports haveappeared in the last few months indicating that, atleast under some conditions, mammalian Notch-1 in-hibits apoptotic cell death (Jehn et al., 1999; Deftos etal., 1998; Shelly et al., 1999). These data suggest that,depending on the context, Notch signaling may act as a“master switch” controlling cell fate choices includingproliferation, differentiation, and death. The role ofNotch receptors in regulating cell death may have im-plications in developmental biology, immunology, neu-

robiology, and tumor biology. In this review, we brieflysummarize our current understanding of Notch struc-ture and function and discuss the evidence linkingNotch signaling to apoptosis regulation, focusing on itspossible biological and medical implications. A compre-hensive discussion of Notch biology is beyond the scopeof this article. The reader is referred to other recentreviews (Artavanis-Tsakonas et al., 1995, 1999; Blau-mueller and Artavanis-Tsakonas, 1997; Weinmaster,1997; Kopan and Cagan, 1997; Greenwald, 1998; Mil-ner and Bigas, 1999).

STRUCTURE OF NOTCH RECEPTORSNotch receptors have a common structural organiza-

tion that is conserved throughout evolution, with somevariation on the general theme. Vertebrate notch genesare strongly related to each other and to Drosophilanotch (Lardelli et al., 1995; Maine et al., 1995). Hu-mans and mice have four notch genes, denominatednotch-1 through 4. Similarly, multiple Notch ligandshave been identified in vertebrates (Lindsell et al.,1995; Nye and Kopan, 1995; Shawber et al., 1996a;Bettenhausen et al, 1995; Henrique et al., 1995; Myatet al., 1996; Oda et al., 1997). These are homologous tothe Drosophila ligands Delta and Serrate and the C.

Contract grant sponsor: Illinois Department of Public Health;Contract grant sponsor: NIH; Contract grant numbers:RO1CA84065 and RO1AG16690.

*Correspondence to: Lucio Miele, Cardinal Bernardin CancerCenter, Loyola University Medical Center, 2160 South First Av-enue, Maywood, IL 60153. E-mail: [email protected]

Received 7 July 1999; Accepted 7 July 1999

JOURNAL OF CELLULAR PHYSIOLOGY 181:393–409 (1999)

© 1999 WILEY-LISS, INC.

elegans ligand Lag-1, and thus, are collectively identi-fied as DSL from the initials of Delta, Serrate, andLag-1 (Tax et al., 1994; Weinmaster, 1997). Mamma-lian Delta homologs are denominated “Delta-like” andmammalian Serrate homologs are denominated“Jagged” (Lindsell et al., 1995; Shawber et al., 1996a).Notch proteins are produced as single polypeptide pre-cursors that are processed to a mature form (Fig. 1).Mature Notch receptors are heterodimers derived fromthe cleavage of Notch preproteins into an extracellularsubunit (NEC) containing multiple epidermal growth

factor (EGF)-like repeats and a transmembrane sub-unit including the intracellular region (NTM; Blaumuel-ler et al., 1997b). A furinlike convertase catalyzes thiscleavage (Logeat et al., 1998). The NEC subunit may befurther processed by an ADAM family protease (Wen etal., 1997; Pan, 1997; Sotillos et al., 1997). Such pro-teases have been shown to facilitate Notch signaling(Wen et al., 1997; Pan, 1997; Sotillos et al., 1997).However, more recently, the Drosophila ADAM familyprotease, Kuzbanian, has been found to process theNotch ligand Delta (Qi et al., 1999). Therefore, the

Fig. 1. Putative structure and processing of a Notch receptor. Notchreceptors are synthesized as single polypeptide precursors. Duringmaturation, a furinlike convertase cleaves the extracellular subunitNEC from the transmembrane subunit NTM. These two subunits arereassembled as a heterodimer in the trans-Golgi. ADAM family pro-teases may or may not be involved in further processing of NEC. Asecond cleavage that requires presenilin-1 and may be catalyzed by itcleaves the NTM subunit within or immediately distal to the mem-brane, generating the intracellular subunit NIC and a short trans-

membrane fragment. The latter cleavage appears to be necessary forNotch signaling. It is still unclear whether this cleavage is induced onligand binding or happens during receptor maturation. LP, leaderpeptide; EGF, EGF-repeat region; 11-12, EGF repeats 11 and 12,which in Drosophila are the main ligand-binding site; L/N, Lin/Notchcysteine-rich repeats; TM, single-pass transmembrane region;RAM23, high-affinity CSL-binding site; ANK, ankyrin/CDC10-likerepeats; OPA, glutamine-rich region; PEST, proline-glutamate-serine-threonine-rich region.

394 MIELE AND OSBORNE

effect of ADAM proteases on Notch signaling may beindirect.

The number of EGF repeats in the Notch preproteinsvaries from 36 repeats in Drosophila Notch and mam-malian Notch-1 and -2 to the 13 and 10 repeats of C.elegans Lin-12 and Glp-1, respectively. The NTM sub-unit includes a single-pass transmembrane region, sixankyrinlike repeats, a polyglutamine stretch (OPA),and a PEST sequence (Artavanis-Tsakonas et al.,1995). A sequence denominated RAM23, immediatelydistal to the transmembrane region, proximal to theankyrin repeats is believed to be a high-affinity-bind-ing site for transcription factors of the Suppressor ofHairless/CBF-1 group (see below; Tamura et al., 1995).Notch ligands also have multiple EGF-like domainsand an additional N-terminal cysteine-rich domain(the DSL domain) that appears to be responsible forNotch binding (Tax et al., 1994). In Drosophila Notch,EGF repeats 11 and 12 are responsible for binding bothligands Delta and Serrate (Rebay et al., 1993). Theserepeats are highly conserved in mammalian Notch re-ceptors, particularly Notch-1 and -2, and represent theputative main ligand-binding site.

BIOLOGICAL CONSEQUENCES OFNOTCH SIGNALING

Notch signaling affects cell differentiation programsin many experimental models. The classical paradigmis Drosophila neurogenesis (Artavanis-Tsakonas et al.,1991; Artavanis-Tsakonas and Simpson, 1991; Cam-pos-Ortega, 1994). In this model, putative stochasticoscillations in the levels of expression of Notch and itsligand Delta in neuroectodermal cells identify cells des-tined to commit to the neuronal phenotype. These neu-ronal precursors predominantly express Delta. Deltaactivates Notch in the cells surrounding each neuronalprecursor and causes them to upregulate Notch expres-sion. The activation of Notch in the cells surroundingneuronal precursors prevents them from differentiat-ing toward the neuronal lineage. Subsequently, thesecells switch to an epidermal differentiation program.Thus, notch deletions or loss of function mutationsresult in an overabundance of cells that assume a neu-ronal cell fate. This is defined a “neurogenic” phenotype(Artavanis-Tsakonas et al., 1991; Artavanis-Tsakonasand Simpson, 1991; Campos-Ortega, 1994). Recently,oscillations in the levels of Notch and Delta expressionin the developing Drosophila eye have been shown tobe controlled by Wingless signaling (Fanto andMlodzik, 1999; Cooper and Bray, 1999).The C. elegansnotch homologs lin-12 and glp-1 play analogous rolesduring the development of uterine and vulvar precur-sors and of germ cells and pharyngeal epithelium, re-spectively (Yochem et al., 1988; Yochem and Green-wald, 1989; Struhl et al., 1993; Greenwald, 1998).

The function(s) of Notch in cell fate determinationappear to be conserved throughout evolution from fliesto vertebrates including mammals. Notch and its li-gands play a key role in Xenopus neurogenesis (Chitniset al., 1995; Chitnis and Kintner, 1996; Coffman et al.,1993) and mesoderm segmentation (Jen et al., 1997). Inmice, Notch-1 is necessary for embryonic developmentand targeted disruption of the notch-1 gene results inembryonic lethality at 11.5 days postconception (Swi-atek et al., 1994; Conlon et al., 1995). Notch-1 expres-

sion correlates with cell fate determination in mousedeveloping hair follicles (Kopan and Weintraub 1993)and rat tooth buds (Mitsiadis et al., 1995, 1997).

The effects of Notch signaling on cell fate decisions invertebrates have been extensively studied in tissueculture, ex vivo systems, and transgenic animals. Ex-pression of constitutively active forms of Notch recep-tors, lacking the extracellular subunit, inhibits termi-nal differentiation in vitro in murine models ofmyogenesis and granulocytopoiesis (Kopan et al., 1994;Shawber et al., 1996b; Milner et al., 1996; Kopan et al.,1996). In chick retina explants, expression of constitu-tively active Notch-1 inhibits differentiation of retinalprogenitors to ganglion cells, while notch-1 antisenseoligonucleotides increase differentiation toward a neu-ronal phenotype (Austin et al., 1995). Similarly, ligand-induced activation of Notch-1 inhibits oligodendrocytematuration in vitro (Wang et al., 1998). Overall, thesestudies suggest that in many instances, Notch activa-tion inhibits or delays differentiation along a develop-mental pathway until the cell is able to respond tosignals that determine its subsequent fate. In contrast,in some mammalian experimental models, such as CD4vs. CD8 and a/b vs. g/d lineage decisions in thymocytes(Robey et al., 1996; Washburn et al., 1997) and in vitroadipocyte differentiation of 3T3-L1 cells (Garces et al.,1997), expression of Notch-1 appears to be necessaryfor proper interpretation of differentiation stimuli.

The possible functions of Notch receptors in postna-tal animals are poorly understood. Notch-1 mRNA canbe detected in various organs, especially in lymphoidorgans, in the central nervous system, and in the lung(Ellisen et al., 1991). Notch-1 and -2 are expressed inCD341 human bone marrow stem cells and other he-matopoietic precursors and are suggested to be in-volved in the control of cell fate decisions during hema-topoiesis (Milner et al., 1994, 1996; Bigas et al., 1998;Li et al., 1998; Milner and Bigas, 1999). Recently,Notch-1 signaling has been shown to affect cell cycleprogression in HL-60 promyelocytic leukemia cells andCD34 bone marrow stem cells. Both transfection ofconstitutively active Notch-1 and stimulation by 3T3cells expressing the Notch ligand Jagged-2 acceleratedprogression through G1 (Carlesso et al., 1999). Since aG1 lag commonly precedes commitment to terminaldifferentiation in many cell types, these data suggestthat, at least in some models, Notch signaling maydirectly affect cell proliferation and that this may belinked to a delay in differentiation.

In summary, the available data indicate that inmany experimental models, Notch signaling affects theinterpretation of differentiation stimuli and regulatesthe balance between proliferation and differentiation.Recent evidence indicates that an additional role ofNotch signaling may be the regulation of cell death(Jehn et al., 1999; Deftos et al., 1998; Shelly et al.,1999). These observations suggest that, at least insome systems, Notch signaling regulates cell survivalbesides differentiation. The evidence supporting an an-tiapoptotic effect of Notch signaling and the possiblepathophysiological implications of these findings willbe discussed in detail in separate sections.

395ARBITER OF DIFFERENTIATION AND DEATH

Fig. 2. a: Schematic representation of the current working hypoth-eses for CSL-mediated and Deltex-mediated Notch signaling. Recentdata suggest that presenilin-1- mediated cleavage is necessary forNotch signaling through CSL. In the absence of Notch, CSL factorsbehave as transcriptional repressors. At least in mammalian cells,CSL forms complexes with various nuclear corepressors and HDAC-1.Notch NIC binding to CSL factors results in release of corepressor andHDAC-1 and converts CSL molecules into transcriptional activators.Whether transcriptional activation is mediated by a Notch-CSL com-plex and whether coactivators are recruited by CSL remain unclear.Deltex proteins bind to the ankyrin repeats of Notch and remaincytoplasmic after Notch activation. Recent evidence suggests thatDeltex inhibits the activity of bHLH transcription factor E47 throughinhibition of JNK-dependent activation of E47. PS-1, presenilin-1;CSL, CBF-1/Suppressor of Hairless/Lag-1family transcriptional reg-ulators; CoR, nuclear corepressor molecule; HDAC-1, histone deacety-lase-1; E(Sp), target genes of the Enhancer of Split group (or mam-malian homologs such as HES-1); JNK, c-Jun N-terminal kinase; E47,bHLH transcription factor E47. b: Top: Notch-binding proteins thatare thought to mediate Notch signaling. In addition to Deltex andCSL, direct binding of several other key signaling molecules to Notch

has been described. These include the chromatin-remodeling factorEMB-5 in C. elegans, the p50 subunit of NF-kB in human transformedT-cell lines, the c-Abl accessory protein Disabled (Dab) in neurons,and the orphan nuclear receptor Nur77 in mouse T-cell hybridomas.Whether all of these molecules mediate different functions of Notchand whether different mediators are used in different cell types re-main to be established. Bottom: Notch-binding proteins that arethought to modulate Notch signaling. The secretory protein Fringe inDrosophila and its mammalian homologs (e.g., Lunatic fringe) mod-ulate Notch-ligand interactions extracellularly. The C. elegans pro-teins Sel-1 and Sel-10 have been shown to modulate Notch proteinturnover/processing. In Drosophila, Numb downregulates Notch sig-naling by binding to the intracellular region of Notch. Numb affectscell fate by distributing asymmetrically in daughter cells during mi-tosis, and thus, causing different cells to have different levels of Notchsignaling. Disheveled is a downstream effector of Wingless, the Dro-sophila counterpart of Wnt oncogenes. It binds to the C-terminalportion of NIC and is thought to mediate crosstalk between Notch andWingless signaling. Through Disheveled binding, Notch can modulateWingless signaling and vice versa.


THE BIOCHEMISTRY OF NOTCH SIGNALINGNotch signaling involves several putative pathways

and remains incompletely understood (Fig. 2), espe-cially in mammalian cells (Artavanis-Tsakonas et al.,1995, 1999; Jarriault et al., 1995; Shawber et al.,1996b; Kopan et al., 1996; Ordentlich et al., 1998; Os-wald et al., 1998). For clarity, we describe under sepa-rate subsections the “classical” signaling pathway me-diated by Suppressor of Hairless [Su(H)] and relatedtranscription factors of the CSL family, putative CSL-independent signaling, and proteins that may modu-late Notch signaling.

Signaling mediated by CSL familytranscriptional regulators

The transcription factor Su(H) has been known forseveral years to mediate many effects of DrosophilaNotch (Fortini and Artavanis-Tsakonas, 1994). Dro-sophila Su(H) is the prototype of a family that includesC. elegans LAG-1 and mammalian CBF-1(also knownas RBP-Jk). These transcription factors are sometimescollectively referred to as the CSL family, from theinitials CBF-1, Su(H), and LAG-1 (Weinmaster, 1997).Ligand-induced release and nuclear access of the intra-cellular portion of Notch (NIC) accompanied by activa-tion of CSL-dependent transcription have been demon-strated in Drosophila (Struhl and Adachi, 1998) andmammalian cells (Schroeter et al., 1998). Release ofNIC requires a proteolytic cleavage within or near themembrane. Recently, presenilin-1 has been shown to

be necessary for Notch cleavage and CSL-mediatedsignaling (Ye et al., 1999; Struhl and Greenwald, 1999;De et al., 1999). Presenilins are multiple-pass trans-membrane proteins whose function is still unclear(Karran et al., 1998; Mattson and Guo, 1997). Muta-tions of human presenilin-1 and -2 are believed tocause most cases of familial, early-onset Alzheimer’sdisease (Kovacs and Tanzi, 1998; Renbaum and Levy-Lahad, 1998). These proteins have been proposed toregulate neuronal cell death in mammals by affectingthe function of the endoplasmic reticulum (Mattson etal., 1998). Presenilin family proteins had been previ-ously found to facilitate Notch processing and intracel-lular trafficking in C. elegans (Levitan and Greenwald,1995, 1998). A direct physical interaction between pre-senilin-1 and Notch, occurring early during Notch pro-cessing and prior to cleavage of Notch preprotein, hasbeen demonstrated in cell culture (Ray et al., 1999).Whether presenilin-1 directly cleaves Notch in re-sponse to ligand binding and whether this is their onlyfunction in Notch maturation remains to be estab-lished.

The cleaved intracellular domain of Notch is believedto enter the nucleus and interact with transcriptionfactors of the CSL family. The main CSL interactionsite has been mapped to the RAM23 region (Tamura etal., 1995), with a lower-affinity-binding site in theankyrin region (Aster et al., 1997). The mammalianmember of the CSL family, CBF-1/RBP-Jk, is a ubiq-uitous transcriptional regulator that acts as a repres-

Fig. 2. (Continued.)


sor in the absence of Notch (Hsieh and Hayward, 1995;Oswald et al., 1998). This repressor activity may insome cases be mediated by a complex including CBF-1,the SMRT corepressor (silencing mediator of retinoidand thyroid hormone receptors), and histone deacety-lase HDAC-1 (Kao et al., 1998). Notch binding disruptsthis complex, releasing SMRT and HDAC-1. CBF-1,either alone or as a complex with Notch, is then be-lieved to become a transcriptional activator. Accordingto this model, the Notch/CBF-1 complex (or SMRT-freeCBF-1) upregulates the expression of Notch targetgenes. In addition to SMRT, other transcriptional core-pressors such as n-CoR were also bound by CBF-1 andreleased by activated Notch (Kao et al., 1998). Morerecently, a CBF-1-specific corepressor, CIR, has beenidentified (Hsieh et al., 1999). Thus, it appears that, atleast in cultured mammalian cells, several corepres-sors can bind CBF-1 and mediate its transcriptionalrepressor activity. According to this model, a generalmechanism of Notch activation would involve bindingof NIC subunits to CSL/corepressor/HDAC-1 com-plexes, release of corepressors/HDAC-1, and conver-sion of CSL molecules to transcriptional activators.

Notch activation leads to CSL-mediated transcrip-tional activation of target genes. These include bHLHtranscription factors of the “Enhancer of Split” group inDrosophila (Bailey and Posakony, 1995) or their ho-mologs “Hairy Enhancer of Split” (HES) in mammaliancells (Jarriault et al., 1995; Stifani et al., 1992). Thelatter are thought to mediate Notch effects by inhibit-ing the activity and/or the expression of differentiation-inducing, cell type-specific bHLH transcription factors,such as Achaete-Scute in Drosophila or its mammalianhomolog Mash-1 (Goodbourn, 1995; Lewis, 1998). Anadditional target of Notch-mediated transcriptionalregulation in Drosophila is the groucho gene. The lat-ter encodes a corepressor that interacts directly withbHLH proteins and may affect chromatin structure(Fisher and Caudy, 1998; Grbavec et al., 1998; Jimenezet al., 1997; Paroush et al., 1994; Palaparti et al., 1997).Additionally, the p100/NF-kB2 promoter has been re-cently shown to be a target of CBF-1-mediated Notchsignaling (Oswald et al., 1998). CBF-1 normally re-presses transcription of the NF-kB2 promoter and ac-tivates it after binding NIC. Finally, the transcriptionalrepressor Mastermind also mediates several Notch ef-fects in Drosophila (Smoller et al., 1990; Bettler et al.,1996; Schuldt and Brand, 1999). It is not clear whetherMastermind is regulated through a CSL-mediatedmechanism. Genetic interactions between the Su(H)and mastermind loci suggest that it may be. In sum-mary, it appears that Notch activation results in CSL-dependent transcriptional activation of a set of effectorgenes, including the “Enhancer of Split” group andothers. These Notch-effector genes encode transcrip-tional regulators that in turn affect the function oftissue-specific bHLH transcription factors (e.g., En-hancer of Split, HES) or modulate cell fate throughother molecular targets (e.g., NF-kB). This pathwayalone can plausibly explain complex cascades of generegulatory events. Context specificity may depend onthe set of bHLH factors expressed in individual celltypes.

Signaling mediated by CSL-independentmechanisms

Not all the effects of Notch signaling appear be me-diated by CSL family transcriptional regulators (Shaw-ber et al., 1996b). In mammalian cells, a CBF-1-inde-pendent pathway has been recently described, whichinvolves the intracellular Notch-binding protein Deltexand possibly c-Jun N-terminal kinase (JNK, a memberof the MAP kinase family; Ordentlich et al., 1998).Deltex is an evolutionarily conserved cytoplasmic pro-tein that binds to the ankyrin repeats of Notch anddoes not appear to localize to the nucleus after Notchactivation (Matsuno et al., 1998). Through Deltex-me-diated effects on JNK, Notch signaling may crosstalkwith Ras signaling (Matsuno et al., 1998). Additionally,Guan et al. (1996) have shown that a human Notch-1construct including the ankyrin repeats and contiguoussequences interacts with p50-containing NF-kB com-plexes. When this construct was cotransfected with p50and p65 NF-kB subunits, its effects on NF-kB-depen-dent reporter gene expression depended on the stoichi-ometry of the system. The authors suggest that in vivoNotch-1 may stimulate NF-kB-dependent transcriptionby interacting with inhibitory p50 homodimers. Sincethe construct used did not contain the main CBF-1binding RAM23 region, this effect is most likely CBF-1independent. A role for the protein kinase c-Abl hasbeen proposed in Notch-1 signaling in neurons (Gini-ger, 1998). This effect is suggested to be mediatedby the Abl-accessory protein Disabled, which binds NIC

(Giniger, 1998). Direct interaction of constitutively ac-tive Notch-1 constructs with the orphan nuclear recep-tor nur77, resulting in inhibition of nur77-dependenttranscription, has been recently demonstrated (Jehn etal., 1999). In C. elegans, the transcriptional regulatorEMB-5 directly interacts with the intracellular domainof Lin-12 and is necessary for signaling (Hubbard et al.,1996). In summary, several different proteins in addi-tion to CSL family transcriptional regulators have beenfound to physically associate with Notch receptors invarious models and are putative mediators of Notchsignaling. Which of these putative signaling mecha-nisms are physiologically relevant and how they inter-act with CSL-mediated effects remain to be estab-lished.

Modulation of Notch signalingYet another group of molecules that affect Notch

signaling includes proteins that modulate the activityor processing of Notch receptors. In Drosophila, at leasttwo such proteins have been identified: the membraneprotein Numb, which negatively regulates Notch sig-naling by directly binding to Notch (Spana and Doe,1996; Guo et al., 1996), and Disheveled, which medi-ates crosstalk between Wingless (the Drosophila ho-molog of wnt oncogenes) and Notch signaling by bind-ing to Notch (Axelrod et al., 1996; Blair, 1996). In C.elegans, there is evidence that accessory proteins Sel-1and Sel-10 negatively regulate Lin-12/Notch signalingby affecting the processing of Notch proteins (Grantand Greenwald, 1996; Hubbard et al., 1997). Finally,secretory proteins have been identified that modulateNotch-ligand interactions in vivo. These include Dro-sophila Fringe (Fleming et al., 1997; Panin et al., 1997)


and its mammalian homologs (Zhang and Gridley,1998; Evrard et al., 1998; Aulehla and Johnson, 1999;Barrantes et al., 1999).

Given the number of proteins that can interact withNotch receptors, the complexity of the possible signal-ing pathways, and the different experimental modelsin which information has been gathered, it is still quitedifficult to determine what constitutes “physiologic”Notch signaling. It is entirely possible that Notch sig-naling may vary in different cells under different con-ditions. The level of Notch expression in a given cell islikely to be an important factor. Prominent gene dosageeffects have been demonstrated in Drosophila (Heitzlerand Simpson, 1991). This may suggest that the amountof Notch proteins can influence which signaling path-way(s) are triggered. The physical state of wild-typeNotch molecules in the cell is still poorly understood.Most of the available data in mammalian systems havebeen obtained studying Notch-1 or Notch-2, which ap-pear to be functionally interchangeable. However, it isstill unclear whether individual forms of Notch have“private” signaling pathways and whether variousforms of Notch can functionally interact in the samecell. Whether Notch receptors exist physiologically asmonomers, dimers, multimers, or supramolecular com-plexes containing several Notch-binding proteins hasimportant implications for the mechanisms of Notchsignaling. Whether several Notch-binding proteins(e.g., CSL, Deltex, Disabled, p50, EMB-5) can be boundsimultaneously to a single Notch molecule or there are“pools” of Notch molecules bound to each or a subgroupof the mediator proteins is also unclear. Finally, Notchreceptors exposed at the cell surface appear to be afraction of the total cellular pool. This suggests thatredistribution from intracellular pools to the cell sur-face and/or receptor internalization may be used tomodulate Notch signaling. The context- and gene dos-age-dependent nature of Notch activities are mostlikely functional manifestations of this biochemicalcomplexity. The intracellular concentrations, subcellu-lar localization, time course of expression, and post-translational modifications of Notch receptors, media-tors (CSL, Deltex, others), targets (bHLH, JNK, nur77,others), and modulators (Numb, Fringe, Sel-1, others)may all contribute to determining the ultimate biolog-ical effects of Notch activation in a specific cell type.Crosstalk with other signaling pathways, such as thosetriggered by Wingless/Wnt (Axelrod et al., 1996; Blair,1996; Fanto and Mlodzik, 1999; Cooper and Bray,1999), cytokines (Bigas et al., 1998), or steroid hor-mones (Deftos et al., 1998), adds a further dimension tothe range of possible Notch effects in vivo.

NOTCH AND THYMIC DEVELOPMENT:SELECTION BY APOPTOSIS?

T lymphocytes begin life in the bone marrow as plu-ripotent stem cells. These cells migrate to the thymusand the process of T-cell receptor (TCR) rearrangementbegins, committing the cells to the T-cell lineage. Tcells possess two different types of TCR known as theabTCR or the gd TCR. These earliest of thymic pre-Tcells are thought to commit to either the ab or the gdlineage by the commencement of rearrangement of theappropriate TCR genes. Presumably, if no TCR rear-

rangements take place, the cell will die from lack ofgrowth factor stimulation (von Boehmer et al., 1998).

During the acquisition of mature TCR expression,these cells begin another developmental process. Ma-ture T cells express either the CD4 or the CD8 corecep-tor in addition to the TCR. However, immature T cellsdo not begin to express either CD4 or CD8 until TCRrearrangement commences. This population of very im-mature thymic T cells is called CD4-,CD8- or double-negative T cells. Once TCR rearrangements begin,these cells rapidly progress to CD41, CD81 or double-positive T cells. At the conclusion of TCR rearrange-ments, double-positive T cells are capable of expressinga mature TCR as well as both CD4 and CD8 corecep-tors (reviewed in Ellmeier et al., 1999). These cells arethen competent to bind to MHC and undergo eitherpositive or negative selection. The CD8 coreceptorbinds to MHC class I while the CD4 coreceptor binds toMHC class II. In a process that is not entirely under-stood, some double-positive T cells bind MHC class Iwhile others bind MHC class II. Those that bind classI become CD8 single-positive T cells while those thatbind class II become CD4 single-positive T cells. Thesecells are then functionally competent and exit the thy-mus. This process is known as positive selection. Somedeveloping T cells can bind self-MHC with high affinityand are capable of inducing autoimmune responses inthe periphery. It is thought that high- affinity bindingof self-MHC causes negative selection or the inductionof apoptosis. Thus, CD41, CD81 T cells can either bepositively selected for further development and exportto the peripheral immune system or undergo negativeselection and death within the thymus (reviewed inGrossman and Singer, 1996; Amsen and Kruisbeek,1998).

However, the majority of double-positive thymocytesnever encounter MHC, most probably because theirTCRs are not able to recognize self-MHC. Since antigenis presented to T cells by self-MHC, it is critical that allmature T cells are able to recognize and bind MHC.The majority, probably upward of 95% of double-posi-tive thymocytes, cannot recognize self-MHC and henceare never signaled to continue development. Thesecells die by neglect because they never receive theappropriate growth factor stimulation. Death by ne-glect is thought to be mediated, at least in part, byglucocorticoids endogenously produced in the thymus(reviewed in Vacchio et al., 1998).

Therefore, thymic development is the result of pro-gressive differentiation mediated by TCR rearrange-ment and expression as well as expression of the CD4and CD8 coreceptors. This is coupled with the induc-tion of apoptosis in various populations of developingthymocytes. Thus, differentiation and death are twocritical components of thymic development.

Notch-1 expression in the thymus is developmentallyregulated with expression highest in immature, dou-ble-negative cells, decreased in double-positive cells,and increased in the mature single-positive population(Hasserjian et al., 1996). Since Notch has been impli-cated in both differentiation and cell death and Notchexpression is developmentally regulated in the thymus,it is reasonable to suggest that Notch may play a crit-ical role in thymic development.

A number of recent reports suggest an important role


for Notch in both differentiation and death in develop-ing T cells. First of all, Robey et al. (1996) have shownin a transgenic mouse model that expression of anactivated intracellular form of Notch-1 in immaturethymic T cells results in the development of larger thannormal numbers of CD8 single-positive T cells. Thesedata suggest a role for Notch-1 in CD4/CD8 lineagedecisions. Using this transgenic model in addition to achimeric stem cell model, the same group subsequentlydemonstrated an effect of Notch-1 on ab vs. gd TCRratios in developing thymocytes (Washburn et al.,1997). Taken together, these data suggest that Notch-1can exert significant influences on both the CD4 vs.CD8 lineage commitment as well as ab vs. gd TCRexpression.

Other recent evidence suggests that Notch-1 mayinfluence T-cell differentiation by leading to selectivesurvival of particular cell populations (von Boehmer,1997, 1999). Two groups have independently discov-ered that Notch-1 has antiapoptotic properties in T-cellsystems. The observations reported suggest thatNotch-1 may regulate “death by neglect” and/or nega-tive selection. Deftos et al. (1998) have reported thatretrovirally transduced, constitutively active Notch-1inhibits glucocorticoid-induced cell cycle arrest and ap-optosis in a thymic lymphoma line (AKR1010) and aT-cell hybridoma (2B4.11). Glucocorticoid receptor lev-els or known downstream effector genes were not af-fected. The RAM23 region was shown to be necessaryfor this effect, suggesting that it is CBF-1 dependent.However, Deltex expression was upregulated by con-stitutively active Notch-1, suggesting that Deltex-me-diated events may also participate in this effect. Inter-estingly, TCR expression was upregulated by Notch-1.The antiapoptotic protein Bcl-2 was upregulated inAKR1010 cells but not in 2B4.11 cells transfected withconstituitively active Notch-1. Finally, thymocytesfrom transgenic mice expressing a truncated, constitu-itively active Notch-1 under the lck promoter showedsome resistance to dexamethasone-induced apoptosis.These data indicate that Notch-1 signaling has com-plex effects on molecules involved in T-cell develop-ment and that activation of Notch-1 may protectthymocytes from “death by neglect” mediated by glu-cocorticoids.

Jehn et al. (1999) have shown that in a T-cell hybrid-oma (DO11.10), constituitively activated forms ofNotch-1 inhibit Nur-77-dependent apoptosis. Nur-77 isa zinc finger transcription factor of the NGFI-B family,which includes Nur77/NGFI-B, Nurr1, and NOR-1.Nur-77 and Nor1 are upregulated during apoptosisinduced by TCR engagement and are required for TCR-mediated apoptosis during negative selection (Liu etal., 1994; Winoto, 1997). These transcription factorshave structural features similar to steroid receptorsand other ligand-activated nuclear receptors (Ma-ruyama et al., 1998). Since specific ligands for thesemolecules have not been identified, they are oftencalled orphan nuclear receptors. Jehn et al.(1999) iden-tified the intracellular region of Notch-1 as a candidateNur-77-binding protein in yeast two-hybrid experi-ments and confirmed the interaction in affinity captureexperiments. Expression of activated Notch-1 con-structs also reduced Nur77 expression triggered byPMA and Ca21 ionophore or TCR ligation. Since Nur77

is known to positively regulate its own expressionthrough interaction with a Nur77 site in its promoter,these data suggest a possible mechanism for the anti-apoptotic activity of Notch-1 may be a direct interac-tion between the intracellular region of Notch-1 andNur77/NGFI-B family members (B.A.O, unpublished).This interaction may reduce Nur77 expression by in-terfering with the autoregulatory loop through whichNur77 induces its own expression. Additionally, thisgroup, using luciferase reporter constructs, demon-strated that Notch-1 expression results in the repres-sion of Nur77-induced transcription. Taken together,data from Deftos et al. (1998) and Jehn et al. (1999)suggest that Notch signaling may regulate apoptosisduring thymocyte maturation by preventing death byneglect and/or negative selection in cells destined todie.

Thus, the collected data support a role for Notch inboth differentiation and death in thymic T cells. It isentirely possible that Notch signaling in the thymusmay regulate both differentiation programs and apo-ptosis or one might speculate that an antiapoptoticfunction of Notch signaling promotes the survival ofcertain populations of thymic T cells. The expressionpattern of Notch-1 in the thymus (Hasserjian et al.,1996; see Fig. 3) may support such a model. Whetherthis happens predominantly in those thymocyteswhose cell phenotype is thought to be induced by in-creased Notch signaling, i.e., ab TCR (Washburn et al.,1997) and CD8 cells (Robey et al., 1996), remains to beestablished. In human peripheral blood T cells, expres-sion of Notch-1 mRNA and protein can be detected inboth CD4 and CD8 cells (L. Shelly and L.M., unpub-lished). This raises the separate question of whetherNotch signaling has a role in regulating apoptosis dur-ing the immune response, for example, during the es-tablishment of immunological memory.

Another attractive model is suggested by the recentreports that demonstrate a role for HES-1 in thymocytedevelopment (Tomita et al., 1999). HES-1 is a bHLHprotein known to be a direct downstream target ofNotch/CBF-1 signaling in mammals, homologous toDrosophila Enhancer of Split proteins. The data showthat HES-1 is essential for the expansion of double-negative T-cell precursors. This report suggests thatNotch signaling is a critical component of progressionfrom the double-negative stage to double-positive stageof thymic development. HES-1 also was recently impli-cated in CD4 commitment (Kim and Siu, 1998). Here,the authors show that HES-1 can silence CD4 expres-sion through specific binding to the CD4 silencer foundin the CD4 promoter. These data provide evidence thatNotch signaling can result in the silencing of CD4expression through interaction with the silencer andfurther suggest one possible mechanism that explainsthe data of Robey et al. (1996).

In a series of experiments using mice that can spe-cifically inactivate Notch via Cre/lox excision, Notch-1expression was shown to be required for the progres-sion of double-negative T cells to the double-positivestage (Radtke et al., 1999). These data lend support tothe conclusions that HES-1 is critical to thymocytedevelopment and further suggest that HES-1 may be adirect target of Notch-1 signaling in T-cell develop-ment.


Interestingly, in the studies showing a role forNotch-1 in apoptosis (Deftos et al., 1998; Jehn et al.,1999), Notch-1 modulated the function of a member ofthe steroid/retinoid/thyroid/orphan receptor superfam-ily. This raises the intriguing possibility that Notchand nuclear receptors of the steroid/retinoid/thyroid/orphan superfamily may functionally interact in vivo.Support for this hypothesis is provided by the observa-tion that in Drosophila, expression of constituitivelyactive Notch, but not full-length Notch, protects sali-vary glands from the ecdysone-induced histolysis thatnormally occurs following pupation (T. Palaga, S. Cum-berledge, and B.A.O., unpublished). Whether Notch-1physically interacts with the glucocorticoid receptor asit does with Nur77 is still unclear. An alternative pos-sibility may be that Notch-1 indirectly regulates theactivity of some members of the nuclear receptor su-perfamily. Since these proteins are known to regulatemany different cellular functions, including cell death

and differentiation, the regulation of these proteinsprovides an attractive model to address much of thedata described above.

A number of caveats and unanswered questions re-main. First, it should be pointed out that in the studiesshowing antiapoptotic effects of Notch-1 in T-cell linesand effects of Notch-1 on lineage commitment, consti-tuitively active Notch constructs were used. These con-structs encode forms of Notch-1 that lack all or most ofthe extracellular subunit NEC. Additionally, the con-struct used by Robey et al. (1996) lacked the OPA andPEST sequences found in the C-terminal end of Notch-1NIC. Such forms of Notch proteins function as consti-tutively active receptors in many systems. However,they have biological properties, such as transformingactivity (Capobianco et al., 1997), which have not beenobserved with intact Notch receptors. Additionally,these truncated forms of Notch proteins localize to thenucleus in readily detectable amounts (Capobianco et

Fig. 3. Notch expression patterns and possible functions during thy-mocyte development. Immature, double-negative thymocytes residingin the cortical area of the thymus express the highest levels of Notch.Notch expression is significantly downregulated in double-positivecells, the population of thymocytes that undergoes selection (see text).This population also can die from lack of growth factor stimulation, aprocess known as death by neglect. This is thought to be regulated by

glucocorticoids. Notch has been shown to inhibit apoptosis induced byglucocorticoids as well as apoptosis induced by TCR activation inT-cell hybridomas. This may suggest that cells destined to surviveeither negative selection or death by neglect do so by upregulatingNotch. Indeed, the expression of Notch is upregulated in single-posi-tive thymocytes.


al., 1997). This is different than what is observed withintact Notch receptors. In the latter case, the amount ofNIC that reaches the nucleus and is sufficient to triggerCBF-1- dependent transcription is extremely small andcan be detected only indirectly through its effects(Struhl and Adachi, 1998; Schroeter et al., 1998). Con-siderable dosage effects are observed in Drosophila(Heitzler and Simpson, 1991), suggesting that levels ofNotch expression strongly affect the biochemical andbiological consequences of Notch activation. Thus, foreach experimental system, it is probably prudent to askto what extent unregulated, high-level expression of atruncated Notch protein models ligand-induced activa-tion of wild-type Notch under physiological conditions.These caveats apply to cellular systems and transgenicanimals. Given the complexity of Notch signaling andthe possible crosstalk with other pathways, it shouldnot be surprising that the effects of Notch activationare context dependent. This is illustrated by the find-ings that dexamethasone protection was not observedin DO11.10 cells (Jehn et al., 1999) while Notch-1-induced Bcl-2 upregulation was observed in AKR1010cells but not in 2B4.11 cells (Deftos et al., 1998), al-though apoptosis protection was observed in both. Allin all, the data obtained thus far clearly show thatconstituitively active Notch-1 prevents apoptosis in T-cell-derived cell lines and in mouse thymocytes. Theremay be several, nonmutually exclusive biochemicalmechanisms for this effect. Whether inhibition of apo-ptosis is a physiological function of Notch signalingduring thymocyte maturation and which thymocyteselection process(es) are regulated by Notch-1 throughits antiapoptotic activity remain to be established. Ex-perimental models that do not rely on constituitivelyactive Notch-1 constructs may be useful in answeringthese questions.

NOTCH, CANCER, AND APOPTOSIS: NOTCHSIGNALING AS A POTENTIAL TARGET

FOR ANTINEOPLASTIC TREATMENTThe inability to undergo apoptosis through physio-

logical mechanisms and resistance to therapeuticallyinduced apoptosis are well recognized features of thetransformed phenotype in many human malignancies(Jaattela, 1999; Kliche and Hoffken, 1999; Zhivotovskyet al., 1999). Thus, if Notch signaling is involved inregulating apoptosis in malignant cells, this may iden-tify a group of novel potential therapeutic targets, in-cluding Notch receptors, ligands, and mediators. Sev-eral lines of evidence suggest that alterations of Notchsignaling or expression contribute to tumorigenesis. Itis firmly established that Notch expression and signal-ing are altered in spontaneous human tumors and intumor models. Two distinct phenomena have been de-scribed in the literature: First, strikingly increasedexpression of apparently intact Notch-1 and -2 hasbeen demonstrated in cervical carcinomas and otherepithelial malignancies, as well as in preneoplastic le-sions of the cervical epithelium (Zagouras et al., 1995;Daniel et al., 1997). In the cervix, the subcellular dis-tribution of Notch-1 changes dramatically during theprogression from a preneoplastic CIN 3 lesion to micro-invasive carcinoma, with strong nuclear immunoreac-tivity observed only in carcinomas (Daniel et al., 1997).Notch overexpression was present in 100% of the cer-

vical cancer specimens studied so far (Zagouras et al.,1995; Daniel et al., 1997). Similarly, increased expres-sion of Notch-1 has been described in colon adenocar-cinomas and lung squamous carcinomas (Zagouras etal., 1995). Moreover, Notch ligands Jagged-1 and Del-ta-1 are also increased in cervical carcinomas concom-itantly with Notch-1 and -2 overexpression (Gray et al.,1999). This supports the hypothesis that Notch signal-ing is chronically upregulated during cervical cancerprogression. Abundant expression of apparently intactNotch-1 (the most studied member of the family) can bedetected in transformed cell lines of many differentlineages, from cervical cancer to T-cell acute lympho-blastic leukemia (T-ALL), acute promyelocytic leuke-mia, erythroleukemia, neuroblastoma, and medullo-blastoma to pleural mesothelioma (L.M., unpublished).This suggests that increased expression of Notch recep-tors and ligands is a common molecular consequence oftransformation, regardless of cell type. Interestingly,the human notch-1, -2, and -3 genes are in chromo-somal regions associated with hematopoietic malignan-cies of lymphoid, myeloid, and erythroid lineages (Lars-son et al., 1994).

Second, constitutively active forms of Notch havetransforming activity. Such forms of Notch-1, resultingfrom deletions of the extracellular subunit NEC, areassociated with approximately 10% of the cases of T-ALL (Ellisen et al., 1991; Aster et al., 1994) and areoncogenic in mouse T cells in vivo (Pear et al., 1996).Similarly, constitutively active forms of Notch-4 causebreast cancer in mice (Robbins et al., 1992; Jhappan etal., 1992; Smith et al., 1995; Gallahan et al., 1996).Constitutively active Notch-1 and Notch-2 transformrat kidney cells in vitro in association with adenovirusoncogene E1A (Capobianco et al., 1997), with Notch-1slightly more potent than Notch-2. Notch-1-activatingmutations frequently cooperate with c-myc in thepathogenesis of thymomas in MMTVD/c-myc trans-genic mice (Girard et al., 1996). Epstein-Barr virus(EBV) immortalizing protein EBNA-2, which is neces-sary for EBV-induced transformation, mimics Notch-1and -2 signaling. EBNA-2 binds CBF-1 and converts itinto a transcriptional activator, by masking its repres-sor domain (Hsieh and Hayward, 1995; Hsieh et al.,1996, 1997).

These observations support the conclusion thatchronically active Notch signaling is associated withthe transformed phenotype in many cell types. In mostcases, including several common epithelial malignan-cies and tumor cell lines, overexpression of apparentlyfull-length Notch receptor(s) and ligand(s) is observed.In a small number of cases, so far limited to 10% of thecases of T-ALL, activated Notch signaling results fromdeletions that produce constitutively active Notch re-ceptors. The expression of Notch in invertebrate mod-els is increased by a positive feedback loop triggered byNotch-ligand binding (for review, see Greenwald,1998). Similarly, constitutively active Notch-1 upregu-lates wild-type Notch-1 in mouse T-cell hybridomas(Deftos et al., 1998). Thus, constitutive activation ofNotch signaling may itself lead to Notch overexpres-sion. The mechanism(s) responsible for the shift fromsimple overxpression to the heavy nuclear localizationthat accompanies malignant transformation are un-known. It should be noted that at least in one experi-


mental model, simple overexpression of intact Notchproduces a phenotype similar to the one produced byexpressing a constitutively active, truncated form (Guoet al., 1996).

Until recently, it was unclear whether Notch overex-pression in transformed cells has any functional con-sequences in controlling cell fate determination. Shellyet al. (1999) investigated whether Notch signaling par-ticipates in pharmacologically induced differentiationof murine erythroleukemia (MEL) cells. These authorsused hexamethylene-bisacetamide (HMBA), a proto-type hybrid polar drug agent that induces differentia-tion in transformed cells of many different embryoniclineages (Marks et al., 1996) and apoptosis in others(Siegel et al., 1998). Like many other tumor lines, MELcells were found to express readily detectable amountsof Notch-1 protein and mRNA, which are lowest imme-diately after passaging and progressively increase withincreasing cell density (Shelly et al., 1999). In the pres-ence of HMBA, Notch-1 was rapidly and transientlyupregulated and disappeared with commitment to ter-minal differentiation. This is consistent with the par-adigm of Notch-1’s role in delaying differentiation. In-deed, enforced expression of constitutively activeNotch-1 prevents differentiation of MEL cells (M.S.Jang and L.M, unpublished). However, when the up-surge in Notch-1 expression was blunted with threedifferent Notch-1 antisense S-oligonucleotides, MELcell differentiation was inhibited and apoptosis wasinduced (Shelly et al., 1999). MEL clones stably trans-

fected with an 1,100-bp antisense notch-1 constructshowed drastically inhibited differentiation and astriking loss of viability due to apoptosis upon HMBAtreatment, compared to clones transfected with anempty vector (Shelly et al., 1999). Antisense-trans-fected clones showed a specific reduction in Notch-1protein steady-state levels and earlier disappearance ofNotch-1 during HMBA treatment compared to vector-transfected controls. The onset of apoptosis in theseclones during HMBA treatment coincided with the dis-appearance of Notch protein. Even in the absence ofHMBA, Notch-1 antisense-transfected MEL clonesshowed increased apoptosis as cell density increased,possibly as a result of growth factor deprivation. Thiscould be rescued by resuspending the cells in freshmedium. Expression of Notch-1 antisense mRNA inMEL cells did not reduce cell proliferation, nor did itaffect the G1 lag induced by HMBA treatment. All inall, these data suggest that Notch-1 prevents apoptosisin MEL cells and that the rapid increase in Notch-1expression induced by pharmacological differentiationis necessary to maintain survival in precommittedcells. Figure 4 shows our working hypothesis of thefunction of Notch-1 during HMBA-induced differentia-tion. It should be noted that rapid increases in Notch-1expression have been seen with differentiation-induc-ing drugs of several classes in human transformed celllines of various lineages (L.L.S., et al., unpublished).Retinoic acid, for example, induces terminal differenti-ation and apoptosis in cells of various lineages and has

Fig. 4. Working hypothesis of the role of Notch-1 in apoptosis regu-lation in MEL cells. Data obtained with several complementary anti-sense approaches show that spontaneously expressed, full-lengthNotch-1 inhibits apoptosis in MEL cells during both spontaneousgrowth and pharmacologically induced differentiation. Treatmentwith HMBA induces a G1 lag in MEL cells, which is not inhibited incells expressing antisense Notch-1. This is necessary but not sufficientto induce terminal differentiation. Subsequently, at each cell cycle, afraction of the cells commit to terminal differentiation. The remainingcells either continue replicating or die. MEL cell lines expressingantisense Notch-1 mRNA undergo massive apoptosis at this stage,

rather than progressing to terminal differentiation or continuinggrowth. “Committed” cells spontaneously downregulate Notch-1 ex-pression, differentiate, and then die. This suggests that maintainingthe time course of Notch-1 expression is necessary to prevent apopto-sis of precommitted cells during pharmacologically induced differen-tiation. Since many transformed cells express high levels of Notch-1and -2, this also suggests that Notch overexpression may be advan-tageous to transformed cells and that treatments that inhibit Notchexpression or signaling may be therapeutically useful in enhancingapoptosis in cancer cells.


been shown to modulate Notch-1 expression (Del Amoet al., 1992; Mitsiadis et al., 1995).

The mechanism of the antiapoptotic effect of Notchsignaling in MEL cells is being investigated. Severalbiochemical pathways may contribute to it. As of thiswriting, Notch-mediated effects on NF-kB2 expression(Oswald et al., 1998), JNK activity (Ordentlich et al.,1998), and Nur77 expression (Jehn et al., 1999) havebeen confirmed (M.S. Jang and L.M., unpublished).These data are complementary to those of Deftos et al.(1998) and Jehn et al. (1999), since Shelly et al. (1999)documented an antiapoptotic effect by downregulatingspontaneously expressed, apparently full-length Notch-1,rather than transfecting constitutively active Notchconstructs. Taken together, these observations stronglysuggest that in some transformed cells, both intactNotch-1 and constitutively active Notch-1 possess an-tiapoptotic activity. This may indicate that increasedNotch signaling is advantageous for transformed cells,similar to what has been observed with other antiapop-totic genes such as Bcl-2 (Dragovich et al., 1998; Haqand Zanke, 1998; Adams and Cory, 1998; Zhang et al.,1998). This hypothesis does not imply that the trans-forming activity of constitutively active Notch recep-tors is always or entirely due to apoptosis protection.However, the hypothesis that Notch overexpressionand/or increased signaling has survival value for hu-man malignant cells may explain the widespread oc-currence of Notch overexpression in transformed cellsin vivo and in vitro. Whether this is the case will beestablished by studying apoptosis in other transformedcell types. However, the findings described above raisethe possibility that downregulating Notch expressionor signaling may be used to enhance apoptosis in hu-man malignant cells, in conjunction with differentia-tion-inducing antineoplastic drugs or conventional che-motherapy or radiotherapy. One possible use for suchstrategies may be the local treatment of unresectablelesions. Given the widespread and nonorgan- specificoccurrence of Notch overexpression in human malig-nancies, such therapeutic strategies may potentiallyhave vast applications in clinical oncology.

NOTCH IN THE BRAIN:AN ALZHEIMER’S CONNECTION?

The fact that Notch signaling is a central element inthe embryogenesis of the central nervous system inorganisms from Drosophila to vertebrates has beenrecognized for many years (for reviews see Artavanis-Tsakonas et al., 1991, 1999; Campos-Ortega, 1994;Greenwald, 1998). More recently, Notch signaling hasbeen implicated in neuropathology. Mutations in thenotch-3 gene are associated with CADASIL, a syn-drome characterized by multiple subcortical strokesand leukoencephalopathy with progressive dementia(Joutel et al., 1996). Ever since the C. elegans preseni-linlike molecule Sel-12 was found to facilitate Notchsignaling, presumably by regulating Notch processing(Levitan and Greenwald, 1995, 1998), there has beengrowing interest in a possible connection betweenNotch signaling and neurodegenerative disorders, par-ticularly Alzheimer’s disease. Two lines of evidencesupport this possibility. First, there is a functional andbiochemical interaction between presenilins and Notchreceptors. Presenilins 1 and 2 are mutated in the ma-

jority of cases of familial, early-onset Alzheimer’s dis-ease (Kovacs and Tanzi, 1998; Renbaum and Levy-Lahad, 1998). Thus, it is generally thought thatdysfunctional presenilin molecules directly or indi-rectly increase neuronal death (Mattson and Guo,1997; Mattson et al., 1998; Guo et al., 1997, 1999). It isnow established that presenilins are necessary forproper Notch processing and signaling (Ye et al., 1999;Struhl and Greenwald, 1999; De et al., 1999; Ray et al.,1999), although the detailed mechanism(s) remainsomewhat unclear (see above). Moreover, presenilin-1mutants derived from familial Alzheimer’s diseasecases appear to be unable to facilitate C. elegans Notchsignaling (Baumeister et al., 1997). Mice carrying atargeted disruption of the presenilin-1 gene show phe-notypes similar to Notch-1 knockout mice and havegreatly reduced expression of Notch-1 and Deltalike-1mRNA (Wong et al., 1997). This lower mRNA expres-sion may result from reduced Notch signaling, since insome systems Notch expression is upregulated by afeedback loop induced by Notch activation (for review,see Greenwald, 1998; Deftos et al., 1998). Second, thecentral nervous system is possibly the only anatomicalsite where Notch receptors are expressed in postmitoticcells, pointing to a biological function of Notch signal-ing in mature neurons. Expression of Notch-1, -2, and-3 has been observed in mature neurons of variousspecies (Higuchi et al., 1995; Sullivan et al., 1997;Berezovska et al., 1998). Interestingly, in patients withsporadic (nonfamilial) Alzheimer’s disease, Notch-1protein expression was shown to be significantly in-creased in the hippocampus (Berezovska et al., 1998),suggesting that altered Notch expression may be afeature of this disorder. Whether the overexpressedNotch protein is correctly processed and functional re-mains to be established. This may represent accumu-lation of incorrectly processed or targeted protein, or acompensatory phenomenon.

The physiological functions of Notch signaling in ma-ture neurons are unknown. It has been suggested thatNotch activation may be involved in neuronal plasticity(Ahmad et al., 1995). The presence of Notch in axonalmembranes (Giniger, 1998) may support this hypothe-sis. In light of the recent findings that Notch can pre-vent cell death in some systems and the interactionbetween Notch and presenilins, it is tempting to spec-ulate that Notch expression may protect mature neu-rons from apoptotic cell death. In this case, defectiveNotch signaling may contribute to neuronal death in-duced by a beta amyloid peptide in familial Alzheimer’sdisease associated with presenilin mutations. WhetherNotch receptors have antiapoptotic properties in neu-rons will have to be experimentally established in ap-propriate ex vivo and in vivo models. Should that be thecase, one might envision a possible use for syntheticdrugs that pass the blood-brain barrier and activateNotch signaling in reducing neuronal loss associatedwith Alzheimer’s disease and other neurodegenerativedisorders.

CONCLUSIONS AND FUTURE DIRECTIONSThree independent observations have provided com-

pelling evidence to suggest that regulation of pro-grammed cell death should be added to the long list ofbiological effects elicited by Notch signaling. These


findings have potentially provocative implications indevelopmental biology, immunology, neurobiology, andtumor biology.

Together with recent data from other groups (e.g.,Carlesso et al., 1999), these observations suggest thatNotch signaling can regulate each of the three mainfate choices a cell can make: differentiation, prolifera-tion, or death. This involvement in all aspects of cellfate determination is a recurring theme with many keysignaling molecules, such as p53, the retinoblastomaproteins, Ras and Myc family members, and others.The picture emerging from the last decade of studies oncell fate determination suggests that the same signal-ing cascades regulate proliferation, differentiation, anddeath, depending on the cellular context. The finaloutcome of cell fate determination seems to result fromthe integration of numerous intracellular and extracel-lular inputs by a central network of signaling mole-cules with extensive crosstalk. The relative intensitiesand/or timing of activation of the various componentsof this complex cell-fate determining network deter-mine whether the cell will proliferate, differentiate, ordie.

The general aspects of Notch-ligand interaction areextraordinarily conserved throughout evolution (Arta-vanis-Tsakonas et al., 1999) and are used in manydifferent situations to modulate cell fate determina-tion. Yet, the biological consequences of Notch activa-tion are strongly dependent on cellular and develop-mental context (Artavanis-Tsakonas et al., 1999). Thiscomplexity of biological effects, which defies simplegeneralizations, reflects the variety of biochemicalpathways that can be affected as a result of Notchactivation.

The possible function of Notch signaling in the reg-ulation of cell death deserves further investigation.Numerous questions remain open about the possiblerole of this effect in physiological and pathological pro-cesses. The data collected so far do not necessarilyprove that the regulation of apoptosis is a physiologicalfunction of Notch in nontransformed cells. Two possi-bilities exist: either Notch signaling does regulate ap-optosis physiologically, at least in some cells or undersome circumstances, or the antiapoptotic effect is dueto altered Notch signaling, only observable in trans-formed cells or in transfected cells that overexpressconstitutively active Notch-1.

Physiological antiapoptotic effects of Notch signalingmay be consistent with the multiple developmentalroles of notch genes, since conditional apoptosis is usedduring many developmental processes to select cellspermitted to progress to the next stage. Thymocytedevelopment is a prime example of such a process.Additionally, during postnatal life, Notch signalingmay be used to regulate apoptosis in certain cell types,such as neurons or mature T cells. Studies in notch-1knockout mice may provide indirect evidence thatNotch-1 affects cell survival during development. Intwo separate studies, notch-1 homozygous knockoutmice did not survive past the second stage of gestationand the embryos exhibited widespread cell death (Swi-atek et al., 1994; Conlon et al., 1995). However, celldeath in this context was interpreted as a secondaryconsequence of failure to differentiate and apoptosiswas not the primary cause of embryonic cell death,

although apoptosis did increase with developmentalage in mutant mice (Conlon et al., 1995). This raisesthe additional question of whether Notch regulatesother forms of cell death besides “classical” apoptosis.In Drosophila, expression of constitutively activeNotch, but not full-length Notch, protects salivaryglands from the histolysis that normally occurs follow-ing pupation (T. Palaga, S. Cumberledge, and B.A.O.,unpublished). Further investigations in appropriateanimal models will likely determine whether and un-der what circumstances Notch signaling regulates celldeath physiologically. Transgenic mice carrying knock-outs of genes involved in Notch signaling are eitherlethal prenatally or perinatally (Swiatek et al., 1994;Conlon et al., 1995; Jiang et al., 1998; Xue et al., 1999;Zhang and Gridley, 1998) or have no discernible phe-notype, possibly as a result of redundancy amongNotch homologs. Organ-specific, antisense, or inducibledominant negative models may be more useful to studythe possible role of Notch signaling in cell death regu-lation during postnatal life. Thymocyte development,hematopoiesis, immunological memory, and neuronalapoptosis are among the physiological processes thatmay involve this novel function of Notch. The clinicalimplications would be far-reaching, from autoimmunedisorders to Alzheimer’s disease.

In the context of tumor biology and pharmacology,the possible role of Notch in preventing cell death intransformed cells should be further investigated. Thismay or may not represent physiological Notch signal-ing mechanisms, for various reasons. First, Notch pro-teins are significantly overexpressed in many trans-formed cells compared to normal cells, including cellsthat normally express Notch receptors, such as bonemarrow CD34 “stem” cells. The level of expression ofNotch receptors may affect their interactions with themany putative intracellular targets and accessory mol-ecules (see above). Second, Notch processing, intracel-lular targeting, and/or half-life may be affected intransformed cells. Third, the amount and/or activity ofNotch targets or accessory molecules may be differentin such cells. Indications that such effects may occur invivo exist in the literature. Guo et al. (1996) haveshown that simple overexpression of intact DrosophilaNotch produces a phenotype similar to the one pro-duced by expressing a constitutively active form. Vir-tually undetectable amounts of NIC are sufficient toactivate CBF-1-dependent transcription after nucleartranslocation (Struhl and Adachi, 1998; Schroeter etal., 1998). Yet, intense nuclear staining for Notch-1 isdetected in cervical carcinomas, but not CIN preneo-plastic lesions, that overexpress Notch-1 (Daniel et al.,1997; Zagouras et al., 1995). This suggests that theprogression from preneoplastic lesion to carcinoma, thein vivo correlate of transformation, is accompanied bychanges in the processing or intracellular distributionof Notch-1. As a consequence, much higher amounts ofNotch-1 are present in the nucleus of transformed cellsthan under physiological conditions. This may lead ei-ther to exaggerated signaling through physiologicalpathways or to aberrant signaling via interaction withnonphysiological targets. For example, by virtue ofits ankyrin repeats, NIC at high intracellular concen-tration may interact with nonphysiological targets,such as Rel family proteins (Guan et al., 1996) and


possibly other ankyrin-repeat containing molecules,such as the INK family cyclin-dependent protein ki-nase inhibitors (Chellappan et al., 1998). From a clin-ical standpoint, the widespread occurrence of Notchoverexpression in human cancers and cancer cell linessuggests that Notch signaling may be a very promisingtarget for novel therapeutic agents, such antisensedrugs (Shelly et al., 1999), or recombinant antagonists(Garces et al., 1997).

The biochemical mechanism(s) through which Notchactivation affects apoptotic cascades are still unclear.Not surprisingly, numerous, equally plausible mecha-nisms can be hypothesized: interaction with nuclearreceptors of the steroid/thyroid/retinoid/orphan super-family (Jehn et al., 1999); regulation of the expressionof antiapoptotic members of the Bcl family (Deftos etal., 1998); regulation of NF-kB family members expres-sion (Oswald et al., 1998) or function (Guan et al.1996); and regulation of JNK activity (Ordentlich et al.,1998). Moreover, the possible role of nuclear corepres-sor complexes (Kao et al., 1998) will have to be clari-fied. It is entirely possible that several or all of thesemechanisms may be used by a Notch receptor, simul-taneously or alternatively depending on the cellularcontext. This underscores the recurrent theme of theinherent complexity of Notch signaling, which cannotbe described by simple off/on switch linear models (Ar-tavanis-Tsakonas et al., 1999). A fitting analogy forNotch signaling may be the interaction between a con-ductor and the orchestra. A conductor (Notch) simulta-neously directs different sections of the orchestra (sig-naling pathways), each playing its part of the score.The ultimate musical output results from the integra-tion of these separate sounds, their relative intensities,and temporal sequence. Depending on the musicalscore (cellular and developmental context), the sameconductor and players can generate a seemingly end-less variety of sounds. Finding out which “musicalscores” code for proliferation, differentiation, or cellsurvival symphonies, and how Notch and its “players”interpret those scores is the challenge that lies aheadof us.

ACKNOWLEDGMENTSPart of the work described in this manuscript is

supported by grants from the Illinois Department ofPublic Health to L.M.; by NIH grants RO1CA84065 toL.M. and RO1AG16690 to B.A.O.

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Arbiter of differentiation and death: Notch signaling meets apoptosis