Programmed cell death (PCD) or apoptosis is a common form of cellular demise ... RNA and protein, and induced apoptosis in WEHI-231. B cells. This effect .... endogenous bcl-2 RNA in WEHI-23 1 B cells was important for their apoptotic ...
The EMBO Journal vol.12 no.4 pp.1555 - 1560, 1993
Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells
Eduardo Cuende2, Jose Enrique Ales-Martinez, Liyun Ding1, Maribel Gonzalez-Garcial, Carlos Martinez-A and Gabriel Nunez' Centro de Biologfa Molecular (CSIC), Universidad Aut6noma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain and 'Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA 2Corresponding author Communicated by C.Martfnez-A
Programmed cell death (PCD) or apoptosis is a common form of cellular demise during embryogenesis, tumorigenesis and clonal selection in the immune system. The bcl-2 proto-oncogene has been recently implicated as a potential physiological regulator of the PCD pathway. Gene transfer studies have shown that overexpression of bcl-2 blocks apoptosis mediated by several stimuli in cultured cell lines and promotes the survival of B and T lymphocytes in transgenic mice. However, it remains unclear whether under normal conditions bcl-2 is responsible for controlling cell death. We have investigated the role of bcl-2 in the antimembrane IgM (mIgM)-induced apoptotic death of WEHII-231 B cell lymphoma, a model that mimics clonal deletion of immature B cells by antigen. Signalling of mIgM receptors triggered downregulation of both bcl-2 RNA and protein, and induced apoptosis in WEHI-231 B cells. This effect appeared to be specific since (i) the levels of 32-microglobulin and ,B-actin RNA remain unchanged and (ii) signalling of the apoptosis-resistant B cell lymphoma line BAL-17 with anti-,u was not associated with downregulation of bcl-2 RNA. However, stable expression of bcl-2 by transfection did not rescue WEHI-231 B cells from apoptosis, yet WEHI-231 cells overexpressing bcl-2 were more resistant to programmed cell death induced by heat-shock. These results suggest that depending on the cell lineage, both bcl-2-sensitive and -resistant mechanisms of cell death leading to apoptosis are present and we conclude therefore that in addition to bcl-2, other proteins are important in the apoptosis of WEHI-231 B lymphoma cells. Key words: apoptosis/bcl-2/B lymphoma cells/programmed cell death/proto-oncogene
levels of Ca2+/Mg2+-dependent endonucleases (Cohen, 1991). However, only in immature B cells is there a link between signalling through the B cell receptor for antigen (BCR) and the activation of the cell death program (PCD) (Ales-Martfnez et al., 1992). This developmental feature is thought to be at the heart of the deletion process that eliminates autoreactive B cells (Basten et al., 1991; Nemazee et al., 1991). Although it is clear that the deletion mechanism depends on the interaction of the surface Ig receptor with antigen, the intracellular signals that control PCD in B cells are unknown. The bcl-2 proto-oncogene product, a protein expressed in the inner mitochondrial membrane (Hockenbery et al., 1990), as well as nuclear membrane associated (Alnemri et al., 1992), originally isolated from the t(l4; 18) of human follicular B cell lymphoma (Tsujimoto et al., 1984; Cleary et al., 1986) has been shown to interfere with apoptotic cell death in several experimental models (Tsujimoto, 1989; Hockenbery et al., 1990; Nuniez et al., 1990; Strasser et al., 1990; Vaux et al., 1990; Nuiiez et al., 1991; Strasser et al., 1991a; Siegel et al., 1992). Its expression in mouse B cell differentiation is stage-specific and topographically restricted to tissues characterized by apoptotic death (Garfinkel et al., 1987; Hockenbery et al., 1991). Transgenic mice expressing bcl-2 within the B cell lineage, are characterized by long-lived B cells (T.J.McDonnel et al., 1989; T.G.McDonnel et al., 1990), prolonged B cell responses, production of auto-antibodies (Strasser et al., 1991b) and prolonged B cell memory (Nufnez et al., 1990). The WEHI-231 B cell lymphoma is a model of an immature B cell that behaves like a nascent B cell in terms of its sensitivity to activation-induced cell death (De Franco et al., 1987; Hasbold and Klaus, 1990). Signalling in WEHI-231 cells via their surface IgM receptors leads to irreversible cell cycle arrest in GI/Go within 24-48 h (Scott et al., 1986), a process closely linked to activation of PCD in these cells. Given the protective effects of bcl-2 against apoptosis in several models, regulation of bcl-2 may be a physiological mechanism controlling the survival or death of developing B cells. In this study, we examine the role of bct-2 in the PCD of WEHI-231 induced by several stimuli and show that while bcl-2 prevents the PCD induced by heat-shock, it does not affect mIgM cross-linking mediated apoptosis.
Results
Introduction B cells are prone to apoptosis throughout most stages of their maturation. For example, a 60-70% cell loss has been found during the pre-B to B cell transition in bone marrow (Osmond, 1991). Also, mature spleen B cells rapidly undergo oligonucleosomal DNA fragmentation upon removal from their natural environment and their nuclei contain high dC Oxford University Press
WEHI-231 B cell lymphoma stimulated with anti-,l die by apoptosis The biological consequences of mIgM signalling in B cells differs according to their stage of maturation (Raff et al., 1975), while signalling of B lymphocytes with a mature phenotype through mIgM results in an activation response, in immature B cells it leads to unresponsiveness. First we investigated the induction of apoptosis in WEHI-231 B cell 1 555
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lymphoma line, a model of clonal deletion for B lymphocytes (Boyd et al., 1981). As shown in Figure 1, treatment of WEHI-231 with cycloheximide (an inhibitor of protein synthesis) or actinomycin D (an RNA synthesis inhibitor) induces DNA degradation in an oligonucleosomal pattern, which is characteristic of apoptosis. This effect is promptly detected (8 h after treatment) suggesting that maintenance of WEHI-231 cell viability is a sensitive process in that disruption of RNA and/or protein synthesis leads to a rapid onset of the apoptotic process. On the other hand, triggering of apoptosis by BCR signalling is a more protracted process, requiring > 8 h to take effect in a non-reversible manner, being clearly observed at 24 h after stimulation (Figure 1). The length of the process suggests the need for a significant and successive number of metabolic steps to occur before nucleosomal degradation is initiated. ., .)1 ,
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Fig 1. Induction of apoptosis in WEHI-231 at 8 and 20 h after treatment with medium (lane 1), 10 Ag/ml actinomycin D (lane 2), 10 Ag/ml Bet-2 mAb (lane 3), 10 Agg/ml cycloheximide (lane 4), actinomycin D plus Bet-2 (lane 5) and cycloheximide plus Bet-2 (lane 6).
Stage-specific regulation of bcl-2 RNA in B cell lymphoma lines following migM signalling We reasoned that bcl-2 may be involved in modulating the differential response to anti-A observed in mature and immature B lymphocytes. To investigate this possibility, immature WEHI-231 cells and mature BAL-17 B cell lymphoma cells were stimulated with 10 Ag/ml of affinity purified F(ab)'2 goat anti-mouse IgM and the steady-state levels of bcl-2 RNA were assessed by a semi-quantitative S1 nuclease assay. Signalling through mIgM of WEHI-231 B cells resulted in strong downregulation of bcl-2 RNA by 12 h (Figure 2A). Incubation of WEHI-23 1 B cells with 10 ,ug/ml purified F(ab)' goat IgG, 100 U/ml recombinant IL-4 or 50 U/ml IL-5 did not result in any alteration of bcl-2 RNA levels. In each lane, hybridization with a control 32-microglobulin SI probe was included to ensure that equivalent amounts of intact cellular RNA were being compared. Downregulation of bcl-2 RNA by anti-, appeared specific, as the steady-state levels of 32-microglobulin (Figure 2) and ,B-actin RNA (data not shown) remained unchanged under the same culture conditions. In contrast to WEHI-23 1, signalling of the mature B cell line BAL- 17, which is resistant to apoptosis, resulted in no variation of bcl-2 RNA levels (Figure 2B). Overexpression of bc1-2 fails to block anti-l-induced apoptosis in WEHI-231 B cells To investigate whether the decrease in the level of endogenous bcl-2 RNA in WEHI-23 1 B cells was important for their apoptotic response to mIgM signalling, WEHI-23 1 was transfected with a human bcl-2 expression vector driven by the LTR promoter of the spleen focus-forming (SSF) virus. We generated stable clones by limiting dilution and selected for bcl-2 expression by growth in medium containing G418. Three independent clones of WEHI-23 1, which expressed high levels of the transfected 25 kDa human bcl-2 protein, were identified by analysis of cellular lysates by Western blot analysis using 6C8, a hamster anti-human bcl-2 monoclonal antibody (Figure 3B). Analysis of WEHI-231
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Fig. 2. A. SI RNA analysis of WEHI-231 immature cells stimulated with anti 1gM. P1 and P2 are the murine bcl-2 and (32-microglobulin probes, respectively. Control tRNA (lane 1) or total cellular RNA (- 10 isg) from B cells cultured in the presence of medium alone (lane 2), 10 Ag/ml F(ab)'2 goat anti-mouse IgM (lane 3), 100 jg/ml recombinant IL-4 (lane 4), 50 tg/ml recombinant IL-5 (lane 5) or control goat immunoglobulin (lane 6) for 12 h at 37°C. RNAs were hybridized simultaneously with probes P1 and P2. Protected fragments of 600 (bcl-2) and 202 ((2-microglobulin) nucleotides are shown. B. SI RNA analysis of BAL-17 mature cells stimulated with anti IgM. Probes were murine bcl-2 (P1) and ,B2-microglobulin (P2). Control tRNA (lane 1) or total cellular RNA (10 rig) from B cells cultured in the presence of medium alone (lane 2), control goat immunoglobulin (lane 3), 10 /tg/ml F(ab)'2 goat anti-mouse IgM (lane 4), 10 14g/ml goat anti-mouse IgD (lane 5) and 100 jig/ml recombinant IL-4 (lane 6) for 12 h at 37°C. RNAs were hybridized simultaneously with P1 and P2. Protected fragments are shown.
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