B-cell antigen receptor-induced apoptosis: looking for clues

Immunology Letters 96 (2005) 187–194

Review

B-cell antigen receptor-induced apoptosis: looking for clues

Eric Eldering∗, Rene A.W. vanLier

Department of Experimental Immunology, Academical Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

Received 31 August 2004; accepted 6 September 2004Available online 30 September 2004

Abstract

Triggering of the B cell antigen receptor (BCR) can initiate divergent reponses ranging from activation and cell division to apoptosis,depending on the differentiation stage and additional signals the cell receives. Despite considerable progress in unraveling general apoptosispathways, the route from the BCR to apoptosis execution is still quite obscure, and there is no consensus yet concerning the mechanism orthe players involved. Here, we will summarize current developments in this field and will attempt to pinpoint key questions and perspectivesfor future research.
© 2004 Elsevier B.V. All rights reserved.
Keywords:BCR; CD95; Caspase-2; Bcl-2 family; BH3-only

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

2. Apoptosis pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

3. Initiator caspases in BCR-apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

4. Caspase-2: bystander or crucial player?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

5. Piddosomes and catastrophes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

6. A TRAIL to Bid? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

7. BH3-only contenders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

8. Unusual suspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

9. Conclusions and perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

∗ Corresponding author. Tel.: +31 20 5666076; fax: +31 20 5669756.E-mail addresses:[email protected] (E. Eldering), [email protected] (R.A.W. vanLier).

0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.imlet.2004.09.003

188 E. Eldering, R.A.W. vanLier / Immunology Letters 96 (2005) 187–194

1. Introduction

Within the immune system, apoptosis is a centralmechanism in maintaining normal lymphocyte homeostasis.Dysfunctional responses to apoptotic signals may lead toautoimmune disease and predisposition to malignancy[1,2].In early development of both B and T lymphocyte, the pres-ence of cytokines such as IL-7 is required for survival. Afterrearrangement of immunoglobulin genes, the B-cell antigenreceptor (BCR) plays a central role in cell-fate decisionsat distinct stages of B-cell development. The outcome ofsignalling initiated after BCR-triggering is dependent on theB-cell maturation stage and the provision of additional extra-cellular signals and may vary from rapid cell death to massiveclonal expansion (for recent reviews[3–5]). Specifically,immature B-cells undergo apoptosis after BCR-ligationwhereas the same signal is an activation signals for B-cellsthat have progressed to the mature stage. The reason(s) forthese differential signalling outcomes appear to be complexand related to developmental regulation of expression ofkey signalling molecules (such as PKC�, NF-�B, Bcl-2)and surface receptors (CD19, CD22). Next to this, it mightbe envisaged that mature B-cells are better equipped tointeract with T-cells and in this way receive anti-apoptoticsignals transmitted through CD40 and related molecules.A one milym inF

thec our-s sig-n erea e-2 asc willb ateda

2. Apoptosis pathways

The two best-characterized pathways of apoptosis areoften referred to as extrinsic and intrinsic routes[6,7], remi-niscent of the blood coagulation system. A strict separation inapoptotic stimuli originating outside or inside the cell is dif-ficult however, because various external signals also activatethe so-called intrinsic or mitochondrial route (see below).Extrinsic apoptosis is initiated after activation of death-receptors, which are tumour necrosis factor receptor super-family proteins of which APO-1/Fas/CD95 is the prototypemember[8]. Another member, tumour necrosis factor-relatedapoptosis-inducing ligand or TRAIL has attracted great inter-est because of its potential to selectively kill cancer cells[9].Ligation of these receptors leads to recruitment of the adaptormolecule FADD, and translocation of pro-caspase-8 towardsthe plasma membrane. In this so-called death-inducing sig-nalling complex (DISC), concentration of pro-caspase-8 in-duces its proteolytic activation after which it activates effectorcaspases-3 and -7. These proteases in turn, induce degrada-tion of structural and regulatory proteins that eventually resultin demise of the cell. This direct route from death-receptorvia caspase-8 to effectors occurs in so-called type I cells[10].

In the intrinsic pathway of apoptosis, various sig-nals converge at the level of the mitochondria (e.g.,r aticr ion)[ re-l omeC . Int s co-f 9. Itf l-likem se-9 -s bjectt olic

F uction -ar homa is regua and IAP ac/P or this as domain lumn: IAPm red, on cantlyu ere plo le apopta

n illustration of the opposing effects of BCR-triggeringxpression of either protective or apoptogenic Bcl-2 faembers in a Burkitt lymphoma cell line is presentedig. 1.

Here, we will focus on BCR-mediated apoptosis inontext of contemporary apoptosis research, limitingelves predominantly to human studies while mentioningificant similarities or deviations from murine studies whppropriate. The recent excitement concerning caspasrucial initiator of intrinsic or stress-induced apoptosise reviewed with respect to the likelihood that BCR-medipoptosis utilizes this pathway as well.

ig. 1. Opposing effect of BCR-triggering on apoptosis regulators: indegulators can be induced upon BCR-stimulation. Ramos Burkitt lympt the indicated time points after BCR-stimulation. The entire Bcl-2I-9/serpin B9) were monitored in multiplex PCR-like procedure[102]. Fubset of the 34 target genes are shown. (Legend left column: multiembers, Flip and housekeeping�2-microglobulin). Of the genes monitopregulated with a maximum at 2–4 h after stimulation. Responses wnd gene expression values are no longer reproducible.

adiation- and DNA damage, cell cycle arrest, endoplasmeticulum(ER)-stress, and cytokine or nutrient deprivat4]. Central in this mitochondrial apoptosis pathway is theease of pro-apoptotic factors, most prominently cytochr

(cytC), from the mitochondrial intermembrane spacehe cytoplasm, cytC adopts a remarkable new function aactor together with cytosolic Apaf-1 and pro-caspase-orms the so-called apoptosome. This heptameric, wheeultiprotein complex[11] causes the activation of capaand downstream effector caspases[12]. Notably, apopto

ome and more specifically caspase-9 activation is suo further regulation by several additional factors: cytos

of A1/Bfl-1 and Bik. This example demonstrates that both anti- and propoptoticcells were assayed for the relative expression of a variety of apoptoslators

families plus various miscellaneous regulators (e.g., Flip, AIF, SmDiablo,example, the relative expression att= 0 was set at 1, and for clarity onlyBcl-2 family members, middle column: BH3-only members, right coly the Bcl-2-like A1/Bfl1 and the BH3-only member Bik become signifitted up to 16 h. After that time point, Ramos cells undergo considerabosis

E. Eldering, R.A.W. vanLier / Immunology Letters 96 (2005) 187–194 189

XIAP (and additional IAP family members), the released mi-tochondrial protein SMAC/DIABLO, and the mitochondrialHtrA2 (Omi) is a SMAC/DIABLO-like inhibitor of XIAPthat induces cell death in a serine-protease-dependent fashion[13,14]. These complex interactions illustrate that apoptoticprocesses are tightly regulated at several levels.

A variety of pro- and anti-apoptotic members of the Bcl-2 family can interact in as yet incompletely resolved waysto regulate release the apoptogenic factors from mitochon-dria [4,15]. The prototype anti-apoptotic member Bcl-2 wasdiscovered as a result oft (14:18) translocation in follicu-lar lymphoma[16]. As a proto-oncogene it was unique sinceit antagonized cell death, instead of promoting proliferation[17]. The exact function of Bcl-2 remained enigmatic until itwas uncovered as a factor that prevents release of cytochromeC [18,19]. Structurally, members of the Bcl-2 family con-tain up to four conserved homology domains (BH-domains),and optionally a transmembrane segment[20]. Close homo-logues of Bcl-2 are pro-apoptotic Bax and Bak which togetherare indispensable for mitochondrial damage and subsequentapoptosis in response to various stimuli: staurosporine, UVradiation, growth factor deprivation and ER-stress[21,22].

The third subgroup of the Bcl-2 family is formed by thepro-apoptotic BH3-domain-only proteins. They have in com-mon only a short hydrophobic amino acid stretch which isr ter-a -t eptorp them irecti ,B hon-d tr lacei m,B d oru asht sB ro-t er-i fora test howt ito-c zzlea mostB bers,t

3

aftera hisi ultsf u-

tocrine or paracrine cell death via CD95 and caspase-8 acti-vation [31,32]. In analogy with T-cells, apoptosis occurringvia BCR-ligation has sometimes also been called AICD[33].Various reports agree however that in contrast to T-cells, inB-cells this occurs independently of upstream components ofthe CD95/death-receptor pathway[34,35]. Moreover, block-ing CD95 via antagonistic antibodies does not interfere withBCR-mediated apoptosis[36] (and our own observations),which seems to exclude a role for CD95-CD95L interactions.Most recent investigations that have addressed the mecha-nism of BCR-mediated apoptosis agree on involvement ofthe mitochondrial/intrinsic pathway, ultimately resulting incaspase-3 activation[37–39].

Caspases involved in apoptotic processes can be dividedin initiators and effectors, dependent on the presence ofprodomains that mediate protein–protein interactions andare involved in activation of the protease[40]. The longprodomain-containing caspase-2, -8 and -9, all three been as-signed initiator status in BCR-mediated apoptosis[36,41,42].The case for caspase-8 is relatively weak though, as vari-ous inhibitors such as a dominant negative form of FADD[37,43], the viral caspase-8 inhibitor crmA[34], and alsothe caspase-8 surrogate Flip (Lens and van Lier; unpublishedobservation) cannot prevent BCR-mediated apoptosis. Acti-vation of caspase-8 can also occur via caspase-3, and thisa pro-c po-t t rolei

se oft aseda irest t alson pase-9 ase-9 ta cellsh deedt thec oku oneo

4

upt sug-g spe-c pto-s du pop-t ells.H ase-3a orC spase

equired for apoptosis induction and which mediates inction with other family members[23,24]. Bid is a notewor

hy BH3-only protein because it connects the death-recathway to the mitochondrial route of apoptosis. It is alsoember for which the strongest evidence exists for a d

nteraction with Bax and Bak[25,26]. Upon CD95-ligationid is cleaved by caspase-8 and migrates to the mitocria where it effectuates release of cytC[27,28]. This indirecoute from death-receptor to mitochondria via Bid takes pn type II cells[10]. Other BH3-only proteins such as Bimf, Puma and Noxa are either transcriptionally inducendergo changes in localization or conformation to unle

heir apoptogenic potential[29]. Apart from Bid and perhapim, BH3-only proteins appear to interact only with p

ective Bcl-2 family members, yet activation and oligomzation of Bax and/or Bak at the mitochondria is requiredpoptosis induction[22,30]. This apparent paradox illustra

hat although the basic principles may be known, exactlyhe Bcl2-like, Bax-like and BH3-only proteins achieve mhondrial damage and cytC release is in fact still a pund a matter of hot debate. What seems clear is thatH3-only proteins are sequester protective Bcl-2 mem

hereby shifting the equilibrium towards apoptosis.

. Initiator caspases in BCR-apoptosis

Antigen-experienced T-cells can undergo apoptosissecond wave of stimulation via the T cell receptor. T

s called activation-induced cell death (AICD) and resrom upregulation of CD95-ligand (FasL) followed by a

mplification loop can then feed into ongoing apoptoticesses[44,45]. Depending on the cell-type studied, thisential loop via caspase-8 may play a major or redundann BCR-mediated apoptosis[36].

Caspase-9 is generally viewed as the initiator caspahe intrinsic route, activated once cytochrome C is relend the apoptosome is formed. So, if BCR-triggering requ

his route, caspase-9 involvement is not only expected buecessary. The use of pharmacological inhibitors of cas[38,39], as well as dominant negative forms of casp[46,47] and over-expression of Bcl-2[43,48] to preven

poptosis after BCR-triggering in human and mouseave together provided strong evidence that this is in

he case. The implication is that BCR-triggering falls inategory of ‘intrinsic’ apoptotic stimuli, and we need to lopstream in the signalling pathway for crucial players,f which appears to be Bax[47].

. Caspase-2: bystander or crucial player?

A potential role for caspase-2 in the events leadingo mitochondrial triggering after BCR engagement wasested by Chen et al. who already in 1999 proclaimed aific role for this initiator caspase in BCR-mediated apois in B104 lymphoma cells[41]. These findings promptes to investigate involvement of caspase-2 in various a

osis pathways operative in Ramos Burkitt lymphoma cere, caspase-2 was processed concomitant with caspnd apoptosis induction after triggering of CD95, BCRD20. These events were prevented by the general ca


inhibitor zVAD and dominant negative caspase-9, but not bya dominant negative version of caspase-2[43,47] (and ourunpublished observations). Since caspase-2 itself is poorlyinhibited by zVAD[49], this would at first-sight suggest thatits activation is: (A) not specific for BCR-stimulation and (B)results from – instead of leads to – cytochrome C release andcaspase-3 activation. This would fit with reports that placecaspase-2 activity downstream of caspase-9[50,51] and as-sign a potential function in amplification of ongoing apopto-sis. It would also be compatible with two independent studiesin mice, where caspase-2 ablation had no effects on T or B-cell apoptosis[52,53], except maybe for a slight decreasein experimental granzym B-mediated cell death. A potentialcaveat in these studies is that in knock-out mice caspases maycompensate for loss of each other[54].

On the other hand, a number of findings have suggestedan important role for caspase-2 in upstream steps leading tomitochondrial activation. Various reports already alluded toa cytochrome C-releasing activity of caspase-2[55–57]. Thisculminated in a study using the modern tool of RNA interfer-ence to show that multiple stress stimuli required caspase-2for mitochondrial permeabiliation[58]. This effect was ob-served only in three out of six malignant cell lines tested, butnevertheless lended authority to a leading role for caspase-2 inthe intrinsic pathway. What needs to be determined now is tow ant’c t kindo CR-i ase-2i ia. Ift howc portet age[ er-i

5

DD-l yc -ts lledPt pon-t lly itr rhapsn them eed,v ns int t itm pop-t yondc in-t via a

cell cycle check point. Maybe some cells (such as B104 cellsmentioned above) undergo mitotic catastrophe followed bycaspase-2 engagement after BCR-triggering while other cellsgo into G1 arrest. This would provide two routes to apopto-sis, one of which goes via caspase-2. Other cell types suchas Burkitt Ramos cells display a G1-arrest prior to BCR-mediated apoptosis, and no specific activation of caspase-2[67,68](and unpublished observations). It would certainly beof interest to extend these findings from cell lines to normaland malignant B-cells.

6. A TRAIL to Bid?

The possible roles for caspase-2 seem to grow even largerwith recent information that it may in fact be involved inTRAIL-induced apoptosis, either upstream[69] or down-stream[70] of Bid cleavage. Recent findings show that inits ‘upstream capacity’ caspase-2 is itself not processed[69]or stranger still is not even catalytically active[71]. In thatcase, the caspase-2 processing observed by us and others asa result of various apoptotic stimuli may be a late, down-stream event serving an amplification purpose. The unex-pected connection to TRAIL may be linked with the knownbut ill-understood synergy between TRAIL sensitivity ands ni iousl ase-2aa uchi ety-l ict-i lvesdB lus,i inv a-t mt andT lde

7

ousc n areB smsn riants[a ot toos apa-b driaa rec-o berB ated

hat extent these findings hold true for other ‘non-malignell-types, how caspase-2 becomes activated and whaf message is send to the mitochondria. Concerning B

nduced apoptosis, it remains to be settled whether casps activated upstream or downstream of the mitochondrhe former possibility is true it remains to be determinedaspase-2 becomes active. Since it has recently been rehat initial activation requires dimerization but not cleav59], this boils down to the question of: what drives dimzation of caspase-2?

. Piddosomes and catastrophes

Caspase-2 associates via its CARD domain with a FAike adaptor protein called RAIDD[60], and together thean form discrete structures in a cell[61]. Also, a large muliprotein complex has been described for caspase-2[62], andimilar to the apoptosome it contains a third protein caIDD (p53-induced protein with a death domain)[63]. Al-

hough this complex dubbed the ‘piddosome’ arised saneously in cell lysates, it was suggested that naturaequires genotoxic stress and to initiate apoptosis it peeeds a second signal. If so, could this signal arrive fromachinery that controls the cell cycle and mitosis? Ind

arious reports stated that caspase-2 resides or functiohe nucleus[55,61,64], and recently it was put forward thaay be involved in mitotic catastrophe, a special case of a

osis triggered when aberrant cell division proceeds beell cycle arrest[65,66]. Together these findings provideriguing hints to connect BCR engagement to apoptosis

d

tress- or cytotoxic stimuli[72]. This co-operation betweentrinsic and extrinsic apoptosis could take place at varevels: death-receptor upregulation, Bid cleavage, caspctivation, down-regulation of protective Flip or XIAP[73]nd also kinase activity (see below). There is now of m

nterest in the combined use of TRAIL and histone deacase (HDAC) inhibitors in cancer treatment, and conflng reports appeared whether HDAC inhibitors themseirectly employ Bid cleavage[74,75]. Since we now viewCR-triggering as a potential intrinsic apoptosis stimu

t is certainly worthwhile to explore this connection, alsoiew of the claim that TRAIL might be involved in eliminion of autoreactive T-cells[76]. These various clues seeo indicate BCR-signals going to cell cycle regulationRAIL activation of Bid, the combination of which couvoke caspase-2 action.

. BH3-only contenders

Switching perspective now to the mitochondria, obviandidates to induce cytC release upon BCR stimulatioH3-only proteins. Their number in mammalian organiow approaches 12, and there are numerous splice va

4,15]. New candidates keep appearing (e.g.[77,78]), maybelso because the criteria to become a member are ntringent: a short BH3-only amino acid stretch and the cility to induce apoptosis. Localization at the mitochonnd interaction with multidomain Bcl-2 members aremmended qualifications though. The certified BH3-memik was already proposed to be involved in BCR-medi


apoptosis in B104 cells[79]. Apoptosis mediated by Bik re-quires Bax but not Bak, and occurs indirectly via seques-tration of Bcl-2/Bcl-Xl as there is no physical interactionbetween Bik and Bax[80]. Bik can be transcriptionally in-duced by BCR-triggering as demonstrated inFig. 1, but thisapparently has a relatively modest effect on the actual apop-tosis process[79]. How it becomes activated is not exactlyknown, but Bik’s apoptogenic potential is apparently regu-lated by an as yet unidentified kinase[81]. Again however,in mice, Bik is redundant in hematopoietic development andB-cell apoptosis[82]. Maybe the kinase that can regulate Bikis not expressed in normal B lymphocytes.

In contrast to the negligible effect of Bik ablation, ex-tensive studies on Bim in KO-mice by the Strasser grouphave demonstrated that it has a huge impact on controllinglymphocyte survival, including apoptosis via in vitro BCR-ligation and elimination of autoreactive B-cells[83,84]. BimKO-mice have various autoimmune manifestations. Possi-bly, apart from the obvious explanation that BCR-mediatedapoptosis in mice and men follow different rules, the experi-mental situation using murine primary cells cannot be easilycompared to the predominantly malignant B-cell lines usedin human studies. Also, multiple BH3-only proteins mightvery well collaborate such that the perceived contribution ofa single member depends on the experimental system used.

8

ptors,B rs ina acti-van andss on-f re-p ulars overb imt es ofs esc n (B)l , andm e tom eed,d BimaT e ki-n andB inm

pli-c CR-t ain

can cleave caspase-3 and -7, this in fact generated proteolyt-ically inactive caspase fragments[93,94]. A role for calpainin initiation or relay of apoptosis may lie in its reported ca-pacity to cleave upstream mediators Bid and Bax to generatepro-apoptotic fragments[95,96].

This aspect touches on the familiar chicken and egg ques-tion, and such ‘cause and effect’ cases certainly have appearedin apoptosis investigations. For example, an initiating role forceramide[97], once a prominent theme in apoptosis research,now seems unlikely as formation of this lipid has convinc-ingly been shown to be predominantly an effect of apoptosis,and not a cause[98]. Once a cell crosses the threshold forapoptosis induction, various feedback loops may exits to ter-minate basic processes. This is reminiscent of amplificationloops mentioned earlier involving caspase-8, Bid and perhapsalso caspase-2. In general, it might be said that if a factor orprotein is affected subsequent to effector caspase activation,this cannot constitute a crucial step in apoptosis induction.With respect to BCR-mediated apoptosis, it is important todetermine early steps that lead to mitochondrial activation,independent from downstream caspases. Maybe we shouldlook for a combination of effects taking place upon BCR-signaling, such as activation of a BH3-only protein in combi-nation with stress kinases, calcium signaling or reactive oxy-gen species (ROS) production. There are certainly indicationst can bei edt ateda

9

plexa risonw wayf rmlyv lackb f mi-t thatd pop-t tteri morer

ys-t ove,m froms inedw ly top ith-o an-n pto-s s ore pect,t emicc s to

. Unusual suspects

Apart from the usual suspects such as death-rececl-2 family members and caspases as crucial playepoptosis, other studies have also implicated specification of stress kinase pathways[85,86] in BCR-mediatedpoptosis. In fact, demonstrations that Jun NH2-terminal ki-ase (Jnk) can be responsible for phosphorylation of Bimubsequent apoptosis in neuronal cells and fibroblasts[87,88]eem significant in view of findings mentioned above. Cusingly however, other studies on phosphorylation of Bimorted protection from apoptosis or implicated extra-cellignal-regulated kinases (ERK1/2) but not Jnk in its turny the proteasome[89,90]. There is also at least one cla

hat Jnk can collaborate with caspase-2 in certain typtress-induced apoptosis[91]. Admittedly, these sometimontrasting effects were observed in other cell-types thaymphocytes, but the same proteins keep popping up

aybe only a few missing pieces of the puzzle sufficake the connection with BCR-mediated apoptosis. Independent on the degree of BCR cross-linking humanppears to be subject to regulation by phosphorylation[48].hus, it seems a matter of time before the responsiblase is identified, and a potential link between the BCRim phosphorylation can hopefully be verified, not onlyalignant but also in primary cells.The Ca2+-activated protease calpain has been also im

ated to play a role in effector caspase-7 activation upon Briggering[92]. Although it has been confirmed that calp

hat second messengers such as gluthathione and ROSnvolved in B-cell apoptosis[99,100], and we have observhat ROS scavenging affects BCR- but not CD95-medipoptosis[47].

. Conclusions and perspectives

The program initiated after BCR engagement is comnd may activate opposing cellular responses. In compaith the CD95 route, the assignment of a signalling path

rom antigen receptor to effector caspase that is unifoalid or accepted has proved difficult. Nevertheless, the box seems to be narrowed down to events upstream o

ochondrial activation via Bax. Possibly, the crucial stepetermines whether a cell undergoes BCR-mediated a

osis is taken in the nucleus. An important current mas to determine whether caspase-2 plays a general orestricted role in apoptosis initiation.

A complicating factor is that different experimental sems may yield disparate answers. As mentioned abouse gene knock-out studies can diverge considerably

tudies with human cells. Furthermore, findings obtaith malignant or transformed cells do not translate easirimary B-cells. Tonsil cells undergo apoptosis already wut added stimuli in a reportedly CD95/Flip-responsive mer[101], complicating the dissection of the various apois pathways. It seems worthwhile to identify key factorvents valid in diverse experimental systems. In this reshe aberrant expression of apoptotic regulators in leukells is a well-known phenomenon and could yield clue


key apoptosis decisions in normal lymphocyte differentia-tion. This may hold true for protective as well as apoptogeneicabnormalities.

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B-cell antigen receptor-induced apoptosis: looking for clues