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Altered Sensitivity to Retinoid-Induced Apoptosis Associated with Changes in the Subcellular Distribution of Bcl-2
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
233, 281 –287 (1997)
EX973594
Altered Sensitivity to Retinoid-Induced Apoptosis Associated with Changes in the Subcellular Distribution of Bcl-2 A. Bruel,* E. Karsenty,* M. Schmid,† T. J. McDonnell,‡ and M. Lanotte*,1 *INSERM U-301 and †INSERM U-462, Centre G. Hayem, Hoˆpital Saint-Louis, 75010 Paris, France; and ‡M. D. Anderson Cancer Research Center, Houston, Texas 77225
In the acute promyelocytic leukemia cell line NB4, Bcl-2 downregulation occurred as a late event of retinoid-induced differentiation. In the maturation-resistant NB4-R1 subclone, retinoids failed to downregulate Bcl-2 even in the situation of apoptosis massively induced by pan-agonists and RXR-selective agonists. We observed that NB4 and NB4-R1 cells differed with respect to the intracellular localization of Bcl-2 which showed a perinuclear localization in NB4-R1 cells, while Bax was broadly expressed in the cytoplasm and to only a minor extent in the perinuclear area. Therefore, the distinct intracellular localization of Bcl-2 and Bax was in general nonoverlaping. Bcl-2 remained massively expressed until cell disruption. Bax was not significantly upregulated in cells committed to death. However, Bax localization changed from a diffuse pattern to concentrate in few specific cytoplasmic area at a stage preceding the formation of apoptotic bodies. A human Bcl-2 transgene was transiently overexpressed in NB4-R1 cells which showed increased resistance to apoptosis induced by retinoids. Stably transfected clones of NB4-R1 cells showed an increased expression of Bcl-2 and a marked resistance to apoptosis. Interestingly, the overexpression of Bcl-2 restored a pattern of uniform Bcl-2 labeling in the cytoplasm and, remarkably, the colocalization of Bcl-2 with Bax. This work demonstrates that the ability of retinoid-induced cells to undergo apoptosis depends on the level of expression and the functional interaction between Bcl-2 and Bax. q 1997 Academic Press
INTRODUCTION
Retinoids have recently been shown potent therapeutic drugs for t(15;17) acute promyelocytic leukemia [1, 2] and other tumor cells (reviewed in [3 –5]. By terminal granulocytic maturation, retinoid acid (RA), partic1
To whom correspondence and reprint requests should be addressed at INSERM-U301, Centre G. Hayem, 1, rue Claude Vellefaux, Hoˆpital Saint-Louis, F-75475-Paris Cedex, France. Fax: 33 1 42 40 95 57. E-mail: [email protected].
ularly all-trans RA, induced a clonal extinction of leukemia cells after terminal maturation. However, it has been shown in vivo that maturation resistance, whose causes are not yet clarified, occurred after prolonged treatments with retinoids [6]. This has justified the use of combined therapies based on both maturation by retinoids and apoptosis induced by chemotherapeutic drugs [7]. Recent reports suggest that therapies based on retinoid-induced apoptosis could be developed [8, 9]. Apoptosis is a physiological process of cell death that is controlled by effector genes, either suppressors or activators (see ref. 10 and 11). Importantly, apoptosis suppression or a decreased susceptibility of clonogenic cells to undergo apoptosis induction might constitute an early event in leukemogenesis. This is suggested by the fact that several translocations or mutations involve genes known to regulate apoptosis (myc, Bcl-2, abl, p53). It has been proposed that the chimeric protein PML-RARa resulting from the t(15;17) translocation [12 –14] was responsible for an increased resistance to apoptosis [15]. Therefore in leukemia cells, increased resistance to apoptosis is quite often a feature acquired by means of genetic alterations. Alternatively, transient resistance to apoptosis might result from induction of maturation. Treatment of leukemia cells with retinoids committed cells to a maturation program in which they become temporarily less susceptible to apoptosis induction [16]. When this maturation program fails in its completion, then apoptosis is delayed. It has been shown in non-APL (acute promyelocytic leukemia) HL60 cells [17] and in NB4 cells [18] that retinoid-induced maturation was accompanied by a marked downregulation of Bcl-2 which did not result in apoptosis [8, 9]. Finally, the retinoid-Xreceptor (RXR)-specific retinoids were highly efficient inducers of apoptosis in cells independent of cell maturation and, in the case of NB4-R1 cells [19, 20], this occurred despite high levels of Bcl-2 protein [8]. Bcl-2 belongs to an ever-expanding family of genes implicated in the control of apoptosis [10, 11]. These proteins show diverse cytoplasmic localization, membranous, mitochondrial, or nuclear membranes, or adopt a diffuse cytoplasmic pattern. Functionally these
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0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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proteins belong to two distinct categories:suppressors of apoptosis (Bcl-2, Bcl-xL) and inducers of apoptosis (Bax, Bad, Bik, BclxS). Bcl-2 family members are able to physically interact, resulting in homo- or heterodimer formation [21]. Interestingly, the function of these proteins might be modulated by their subcellular localization and the finding of dimerization partners in these particular cell compartments [22]. Therefore, apoptosis is exquisitely tuned by the balanced expression of these regulators in a single cell. The biological response can be altered by (1) imbalanced expression, (2) ectopic expression which modifies dimerization, (3) posttranslational modification of the proteins, or (4) alterations in subcellular localization. In NB4-R1 cells, in addition to the unusually high cell sensitivity to retinoid-induced apoptosis previously reported [8], we show here a specific localization of Bcl2 in the perinuclear area. We have analyzed the distinct patterns of expression of endogenous Bcl-2 and Bax, in relation to the susceptibility of cells to undergoing apoptosis. The effects of enforced expression of Bcl2 after gene transfer are reported. MATERIALS AND METHODS Reagents. Anti-human Bcl-2 mouse monoclonal antibody and goat anti-mouse polyclonal antibody conjugated to peroxidase were furnished by DAKO A/S (Glostrup, Denmark). An anti-human Bax mouse monoclonal antibody was previously described [21]. A 7.2-kb expression vector was constructed in the splenic focus-forming virus (SFFV) using a human Bcl-2 cDNA expression plasmid, as previously reported [23, 24]. All-trans retinoic acid was purchased from Sigma. The 9-cis retinoic acid was a generous gift from Dr. R. Heyman (Ligand Pharmaceutical Inc., CA). Stock solutions (1002 M) were made by dissolving the compounds in either ethanol or DMSO, stored in the dark at 0307C, and routinely used at 1 mM. Cell lines and cell cultures. The human promyelocytic leukemia cell line NB4 [18] and the resistant promyelocytic leukemia cell line NB4-R1 [8, 19, 20] were cultured as previously described. Briefly, cultures were initiated by seeding 2r105 cells/ml of fresh RMPI 1640 medium (Gibco-BRL) supplemented with L-glutamine (2 mM), antibiotics, and 10% (v/v) of selected batches of fetal calf serum (Boehringer). Cultures were maintained at 3 to 5 1 105 cell/ml by a daily adjustment of the cell concentration, adding fresh culture medium with supplements when necessary. Cells were incubated at 377C in an air/5% CO2 atmosphere. Differentiation was evaluated by morphology with May– Grunewald –Giemsa and by nitroblue tetrazolium (NBT, Sigma) reduction assay. Apoptosis was assessed by morphological criteria, DNA fragmentation, in situ TdT assay, and flow cytometry, as previously described [8]. Cell proliferation and/or viability were evaluated by the WST-1 colorimetric assay (Boehringer), using a Pasteur Diagnostic (Paris, France) double-wavelength automated spectrophotometer. All assays were carried out in triplicate. Cell transfection and selection of stable transfectants. NB4-R1 cells were transfected by electroporation as described by Mu et al. [25]. Briefly, NB4-R1 cells growing exponentially were washed twice in phosphate-buffered saline (PBS). Cells at a concentration of 5 1 106 cells in 500 ml of ice-cold PBS were mixed with 25 mg of expression plasmid and 250 mg of denatured salmon DNA was kept on ice for 5 min. Cells were then electroporated twice with a GenePulser (BioRad Laboratories, Richmond, CA) at a capacitor setting of 500 mF and 200 mV, with a pulse length of 15 to 20 ms. Cells were immediately
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transferred in 10 ml RPMI 1640 containing 20% fetal calf serum (Gibco-BRL) and incubated for 24 h at 377C in an air/CO2 incubator; then the cultures were replenished with fresh medium supplemented with serum. The efficacy of transfection was controlled before antibiotic selection by evaluating transient expression by Western blots in cellular extracts from aliquots of cultures. Two days after electroporation was performed, G418 (500 mg/ml) was added in cultures. Selection was carried out for 3 weeks, until cultures grew rapidly. Pools of transfected cells were frozen and stored at 0807C. Clones selected by limiting dilution in medium supplemented with G418 and expanded for 2 weeks in antibiotic-free medium. Plasmid insertion was controlled by Southern blot and stable expression confirmed using a neo probe on Northern blot. Immunohistological labeling and confocal analyses. Histological slides were prepared by cytocentrifugation, as previously described [8], and fixed. They were then incubated with normal goat serum (2% in PBS) for 15 min to reduce nonspecific antibody binding. Each incubation, performed in a humidified chamber, was followed by 5min washes in PBS. The slides were then successively incubated with the monoclonal mouse anti-human Bcl-2 immunoglobulin (1 mg/ ml; DAKO A/S) for 24 h at 47C and then with the bridging goat antibody anti-mouse immunoglobulin (10 mg/ml) and with alkaline phosphatase-coupled goat anti-mouse (0.5 mg/ml) for 30 min each at room temperature. After a final rinsing the Bax and Bcl-2 proteins were revealed with FITC-conjugated (Dako-France) or anti-mouse IgG CY3-labeled (Sigma) fluorescent second anti-rabbit antibodies, respectively. Fluorescent preparations were examined by confocal laser scanning microscopy using a Bio-Rad MRC-600 confocal imaging system (Bio-Rad Microscience Ltd., Hertfordshire, UK) and an inverted Diaphot 300 Nikon microscope. Images were collected using an oil immersion lens (60X, NA I.4 plan Apochromat). For fluorescein and CY3 excitation a krypton/argon ion laser (Ion Laser Technology Inc., Salt Lake City, UT) operating with the 488-nm line was used. For DAPI excitation, an Entreprise ion laser (Coherent Laser Group, Santa Clara, CA) operating with a 353 nonline was used. Images of FITC, CY3, and DAPI were merged and pseudo-colored green for FITC, red for CY3, and blue for DAPI. Each image represents a single section for which the confocal system was adjusted to allow a field depth of about 0.8 mm. Preparation of subcellular fractions. Cells were harvested by centrifugation at 800g for 10 min at 47C, washed with cold PBS, and resuspended at 2r107 cell/ml. The following steps were performed at 47C, as previously described [26]. Briefly, cells were disrupted with a type B Dounce homogenizer in a 10 mM KCl, 1.5 mM MgCl2, 1 mM PMSF, 10 mM Tris– HCl buffer at pH 7.2. The nuclei were separated by centrifugation at 1000g for 30 min at 47C and then immediately stored frozen at 0807C. The supernatant was centrifuged for 30 min at 105,000g at 47C (TL100, Beckman, Palo Alto, CA). The pellet constituted the membrane fraction and the supernatant was the cytosol. All fractions were stored frozen at 0807C. For SDS–PAGE electrophoresis, the protein content in subcellular fractions was determined by the Sigma ‘‘Protein Assay’’ (Sigma-Chimie, France). Western blot analysis. Nuclear and cytoplasmic protein extracts (100 mg) were boiled in the presence of 5% b-mercaptoethanol and loaded (25 ml/slot) on 12% sodium dodecyl sulfate (SDS)– polyacrylamide gels, electrophoresed, and blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After transfer to nitrocellulose membranes, proteins were visualized with Ponceau S (Sigma) to confirm equal loading of protein. Immunolabeling of blotted membranes was then carried out as previously described [8].
RESULTS
Bax and Bcl-2 Adopted Distinct Expression and Localization Patterns during RA-Induced Apoptosis We have demonstrated previously by histological analysis that Bcl-2 is not downregulated by 9-cis RA
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FIG. 1. Comparative analysis of the Bcl-2 expression and apoptotic responses to 9-cis RA in NB4 (A) and NB4-R1 cells (B). Cells were incubated with 9-cis RA (1 mM) for 48, 72, and 96 h. Apoptotic and viable cells (500 cells scored) were counted after May– Grunewald– Giemsa staining (values represent means { SD of five independent morphological scores). Western blot analysis of Bcl-2 was carried out comparatively on crude cytosolic NB4 and NB4-R1 cell extracts, and membranes were scanned for quantification. Values are expressed in arbitrary units.
in NB4-R1 cells undergoing apoptosis [8]; however, Bcl2 downregulation occurred normally in NB4 cells undergoing maturation. This point is illustrated using a quantitative Western blot analysis in NB4 and NB4R1 cells following treatment with 9-cis RA (Fig. 1). Apoptosis was assessed in these experiments by morphometric analysis after May –Grunewald staining. The morphological cell death patterns were confirmed with DNA fragmentation analysis by gel electrophoresis (not shown). These results suggest that NB4-R1 cells could be dying prematurely because of a functional defect of Bcl-2 since a high expression persisted and/ or because of an enhanced expression of Bax. Bcl-2 and Bax proteins were then analyzed simultaneously in NB4 and NB4-R1 cells by double immunofluorescence (Fig. 2). We found that Bcl-2 exhibited a limited and perinuclear distribution in NB4-R1 cells, whereas in NB4 cells Bcl-2 exhibited both perinuclear and punctuated cytosolic localization. In contrast, Bax was more uniformly distributed in the cytoplasm of NB4 and NB4-R1 cells. Bax was also found in the peripheral area of the cytoplasm of NB4-R1 cells where Bcl-2 was absent (Fig. 2F). Double-labeling experiments clearly demonstrated that Bcl-2 and Bax did not colocalize in NB4-R1 cells, whereas substantial colocalization was observed in the NB4 cell line (see in legend to Fig. 2). Since the pattern of Bcl-2 expression in NB4 and NB4-R1 cells differed with respect to the perinuclear area, we wanted to determine whether the peculiar
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localization of Bcl-2 in NB4-R1 cells was the result of a physical association to the nuclear membrane. Membranes, cytosol, and nuclei-containing fractions were prepared and analyzed comparatively. Quantitative analyses of the distribution of Bcl-2 (Fig. 3A) and Bax (Fig. 3B) in these subcellular compartments were carried out by Western blot after cell fractionation. It confirmed (see in legend to Fig. 3) an increased amount of Bcl-2 physically associated to nuclei in subcellular fractions. We speculated that a causal relation might exist between the Bcl-2/Bax localization and the cell susceptibility to programmed cell death. Bcl-2/Bax expression was assessed following cell death induction by 9-cis RA. In apoptotic cells, Bcl-2 expression remained high, but its localization was altered by the structural modification of the cell. Bcl-2 lost its preferential perinuclear localization and became more diffusely cytosolic. Simultaneously, Bax was translocated in a discreet area of the cytoplasm where its concentration became extremely high (Fig. 4). Despite the altered distribution of Bcl-2 and Bax proteins in apoptotic cells, their subcellular localization remained in general nonoverlaping. Bcl-2 Overexpression in Stably Transfected NB4-R1 Clones Enhanced Resistance to Apoptosis NB4-R1 cells were stably transfected with a fulllength Bcl-2 [23], and the expression of Bcl-2 protein
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FIG. 2. Confocal analysis of Bcl-2 and Bax expression in NB4 and NB4-R1 cells. Double-immunofluorescence labeling was carried out and localization analyzed in a single cell. Notice that red (bcl-2) (A) and green (bax) (B) colors overlapped to form yellow color (C) in NB4 cells, while these labels occupied distinct nonoverlapping areas in NB4-R1 cells (F). Notice also in (F) that in the perinuclear area where Bcl-2 and Bax are both expressed, Bcl-2 and Bax likely associated to distinct subcellular structures since the two colors (red and green) do not overlap (see for comparison in (C) that yellow color formed, indicating that BCL-2 and Bax colocalized). FIG. 4. Distinct topographical localization of Bcl-2 and Bax upon induction of apoptosis in NB4-R1 cells by retinoids. Confocal analysis were carried out under conditions similar to those reported in Fig. 1. Immunocytological analyses were carried out after 72 h of incubation with 9-cis RA (1 mM). (A, C) Controls; (B, D) 9-cis RA treatments; (A, B) anti-Bcl-2 antibody labeling; (C, D) anti-bax antibody labeling.
expression in transfected and control NB4-R1 cells was compared on Western blot. Quantitative scanning of the immunostained membranes indicated an 8-fold overexpression of a 26-kDa Bcl-2 protein in the transfected cells (Fig. 5). Bcl-2-transfected cells were then tested for induction of apoptosis. NB4-R1/Bcl-2 showed
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a significant increase in resistance to RA-induced apoptosis. No cell death was detectable with micromolar concentrations of retinoids after 72 h of incubation, while up to 65% of cells were irreversibly committed to death in the control cultures (Fig. 6). Transfected cells could even resist, and proliferate with, a 20-fold in-
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FIG. 3. Intracellular compartimentalisation of Bcl-2 and Bax in NB4 and NB4-R1 cells. Western blot analysis of Bcl-2 expression associated to the nuclei (lanes 1 and 4) and microsomal (lanes 2 and 5) and cytosolic (lanes 3 and 6) fractions of NB4 and NB4-R1 cells. By scanning the gel membranes loaded with the same subcellular fractions of NB4 and NB4-R1 cells, the relative contents of Bcl2 and Bax in nuclear, cytosolic, and microsomal fractions has been evaluated as 6%/1%/93% in NB4 cells and 21%/1%/78% in NB4-R1 cells for Bcl2, while it was 26%/1%/73% in NB4 cells and 1%/0%/99% in NB4-R1 cells for Bax.
crease in RA concentration, without showing any of the morphological signs of apoptosis. Induction of apoptosis and its suppression in Bcl-2 expressing cells was confirmed by flow cytometry and DNA electrophoresis (not shown).
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FIG. 6. Comparative analyses of the sensitivity of NB4-R1 and NB4-R1 transfected cells to apoptosis induced by retinoids. Cell viabilities in treated and control cultures were evaluated comparatively. Values (triplicates; 500 cells scored for apoptotic features on May– Grunewald – Giemsa-stained slides) were expressed as percentages of the control.
lyzed comparatively, followed by the identification of Bax in the same cell. In transfected cells, Bcl-2 showed a uniform distribution in the cytoplasm, a feature which closely resembles the case of NB4 cells. Strikingly Bcl-2 protein was no longer limited to the perinuclear area (Fig. 7). Importantly, double labeling in sin-
The Blockade of RA-Induced Apoptosis Is Correlated with Bcl-2 and Bax Colocalization in Bcl-2Transfected Cells The patterns of expression of Bcl-2 in transfected cells and untransfected control cells were then ana-
FIG. 5. Quantitative analysis of Bcl-2 overexpression by Western blot in NB4-R1-transfected cells. Analyses were carried out on crude cytoplasmic extracts.
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FIG. 7. Confocal analyses of Bcl-2 and Bax expression patterns in NB4, NB4-R1, and NB4-R1/Bcl-2 cells.
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gle cells clearly indicated that Bcl-2 and Bax colocalized in the Bcl-2-transfected cells (Fig. 7). Treatments of NB4-R1 and NB4-R1/Bcl-2 cells with RA carried out comparatively showed that the overexpression of Bcl2 in transfected cells prevented the translocation and concentration of Bax in an area of the cytoplasm free of Bcl-2, as we observed in NB4-R1 apoptotic cells. DISCUSSION
We observed that the intracellular distribution of Bcl-2 was associated with the susceptibility of NB4-R1 cells to undergoing apoptosis induction by retinoids. In NB4-R1 cells Bcl-2 was limited to the perinuclear area and despite the fact that Bax also was detected in this area, Bcl-2 did not significantly overlap with the localization of Bax. We speculate that the greater sensitivity of NB4-R1 cells to apoptosis induction compared to NB4 cells is related to the intracellular distribution of Bcl-2 and its ability to colocalize or physically interact with Bax [37, 38]. Bcl-2 has been shown to block cell death following a variety of stimuli [21, 27–30] and the suppression of apoptosis is due to its interaction with ‘‘killer’’ partners, like Bax [21]. Bcl-2 is an integral membrane protein [23, 31] with organelle targeting and anchoring determined by a single 20-amino-acid COOH terminus domain [32, 33]. Bcl-2 localizes to membranes of mitochondria, endoplasmic reticulum, and nuclear envelope [23, 31, 34, 35], but it is, as yet, unclear whether distinct specific mechanisms are determined by differential targeting. It might well result from specialized import systems which can saturate hierarchically. It is conceivable, therefore, that a structural modification in Bcl-2 itself, affecting either its cytoplasmic localization or its ability to associate with membranes [33, 36], may result in a reduction in Bcl-2-mediated cell death suppression. Enforced expression of Bcl-2 in NB4-R1 cells conferred resistance to apoptotic cell death induction which was associated with a more widely distributed pattern of Bcl-2 protein within the cell. The distribution of Bcl-2 within the transfected NB4-R1 cells resembled that of Bcl-2 in the NB4 cells and showed significant colocalization with Bax. These findings indicate that the subcellular distribution of Bcl-2 may be an important determinant of Bcl-2-mediated cell death suppression. These results are in accordance with data reported by others [22] using mutated forms of Bcl-2 showing altered function and subcellular localization. Therefore, the molecular interaction of positive and negative regulators of apoptosis, such as Bcl-2 and Bax, is conditioned by their subcellular colocalization. This could provide a general basis for the regulation of programmed cell death and is reminiscent of CED-9 and
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CED-4 localization and function in Caenorhabditis elegans [39, 40]. Our findings indicate that despite high levels of expression of endogenous Bcl-2 in NB4-R1 cells, they were still sensitive to retinoid-induced cell death. This work underlines the uncertainty of using the level of expression of proteins which belong to the Bcl-2 family in the prognosis of tumor cell fate during chemotherapeutic treatments inducing programmed cell death. It shows the importance of further investigation on the impact of subcellular localization of Bcl-2 and Bax proteins to better understand how the cell execution program is ultimately controlled by these regulators. Basically, it remains to elucidate what structural modification can facilitate or compromise the localization and function of these molecules. These events could provide efficient targets for manipulating dysregulated apoptosis in cell pathology. Supported in part by grants from the Fondation contre la Leuce´mie (Fondation de France), Fondation Saint-Louis, Association pour la Recherche contre le Cancer (A.R.C.), Ligue Nationale contre le Cancer, INSERM, CNRS (SDI 16954.I), and EU (CT93-1530, Biomed I). This work also benefited from ‘‘Euroconferences on Apoptosis’’ (EU, Human Capital and Mobility Program and Training and Mobility of Researcher Program).
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Received December 18, 1996 Revised version received March 2, 1997
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Altered Sensitivity to Retinoid-Induced Apoptosis Associated with Changes in the Subcellular Distribution of Bcl-2 A. Bruel,* E. Karsenty,* M. Schmid,† T. J. McDonnell,‡ and M. Lanotte*,1 *INSERM U-301 and †INSERM U-462, Centre G. Hayem, Hoˆpital Saint-Louis, 75010 Paris, France; and ‡M. D. Anderson Cancer Research Center, Houston, Texas 77225
In the acute promyelocytic leukemia cell line NB4, Bcl-2 downregulation occurred as a late event of retinoid-induced differentiation. In the maturation-resistant NB4-R1 subclone, retinoids failed to downregulate Bcl-2 even in the situation of apoptosis massively induced by pan-agonists and RXR-selective agonists. We observed that NB4 and NB4-R1 cells differed with respect to the intracellular localization of Bcl-2 which showed a perinuclear localization in NB4-R1 cells, while Bax was broadly expressed in the cytoplasm and to only a minor extent in the perinuclear area. Therefore, the distinct intracellular localization of Bcl-2 and Bax was in general nonoverlaping. Bcl-2 remained massively expressed until cell disruption. Bax was not significantly upregulated in cells committed to death. However, Bax localization changed from a diffuse pattern to concentrate in few specific cytoplasmic area at a stage preceding the formation of apoptotic bodies. A human Bcl-2 transgene was transiently overexpressed in NB4-R1 cells which showed increased resistance to apoptosis induced by retinoids. Stably transfected clones of NB4-R1 cells showed an increased expression of Bcl-2 and a marked resistance to apoptosis. Interestingly, the overexpression of Bcl-2 restored a pattern of uniform Bcl-2 labeling in the cytoplasm and, remarkably, the colocalization of Bcl-2 with Bax. This work demonstrates that the ability of retinoid-induced cells to undergo apoptosis depends on the level of expression and the functional interaction between Bcl-2 and Bax. q 1997 Academic Press
INTRODUCTION
Retinoids have recently been shown potent therapeutic drugs for t(15;17) acute promyelocytic leukemia [1, 2] and other tumor cells (reviewed in [3 –5]. By terminal granulocytic maturation, retinoid acid (RA), partic1
To whom correspondence and reprint requests should be addressed at INSERM-U301, Centre G. Hayem, 1, rue Claude Vellefaux, Hoˆpital Saint-Louis, F-75475-Paris Cedex, France. Fax: 33 1 42 40 95 57. E-mail: [email protected].
ularly all-trans RA, induced a clonal extinction of leukemia cells after terminal maturation. However, it has been shown in vivo that maturation resistance, whose causes are not yet clarified, occurred after prolonged treatments with retinoids [6]. This has justified the use of combined therapies based on both maturation by retinoids and apoptosis induced by chemotherapeutic drugs [7]. Recent reports suggest that therapies based on retinoid-induced apoptosis could be developed [8, 9]. Apoptosis is a physiological process of cell death that is controlled by effector genes, either suppressors or activators (see ref. 10 and 11). Importantly, apoptosis suppression or a decreased susceptibility of clonogenic cells to undergo apoptosis induction might constitute an early event in leukemogenesis. This is suggested by the fact that several translocations or mutations involve genes known to regulate apoptosis (myc, Bcl-2, abl, p53). It has been proposed that the chimeric protein PML-RARa resulting from the t(15;17) translocation [12 –14] was responsible for an increased resistance to apoptosis [15]. Therefore in leukemia cells, increased resistance to apoptosis is quite often a feature acquired by means of genetic alterations. Alternatively, transient resistance to apoptosis might result from induction of maturation. Treatment of leukemia cells with retinoids committed cells to a maturation program in which they become temporarily less susceptible to apoptosis induction [16]. When this maturation program fails in its completion, then apoptosis is delayed. It has been shown in non-APL (acute promyelocytic leukemia) HL60 cells [17] and in NB4 cells [18] that retinoid-induced maturation was accompanied by a marked downregulation of Bcl-2 which did not result in apoptosis [8, 9]. Finally, the retinoid-Xreceptor (RXR)-specific retinoids were highly efficient inducers of apoptosis in cells independent of cell maturation and, in the case of NB4-R1 cells [19, 20], this occurred despite high levels of Bcl-2 protein [8]. Bcl-2 belongs to an ever-expanding family of genes implicated in the control of apoptosis [10, 11]. These proteins show diverse cytoplasmic localization, membranous, mitochondrial, or nuclear membranes, or adopt a diffuse cytoplasmic pattern. Functionally these
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0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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proteins belong to two distinct categories:suppressors of apoptosis (Bcl-2, Bcl-xL) and inducers of apoptosis (Bax, Bad, Bik, BclxS). Bcl-2 family members are able to physically interact, resulting in homo- or heterodimer formation [21]. Interestingly, the function of these proteins might be modulated by their subcellular localization and the finding of dimerization partners in these particular cell compartments [22]. Therefore, apoptosis is exquisitely tuned by the balanced expression of these regulators in a single cell. The biological response can be altered by (1) imbalanced expression, (2) ectopic expression which modifies dimerization, (3) posttranslational modification of the proteins, or (4) alterations in subcellular localization. In NB4-R1 cells, in addition to the unusually high cell sensitivity to retinoid-induced apoptosis previously reported [8], we show here a specific localization of Bcl2 in the perinuclear area. We have analyzed the distinct patterns of expression of endogenous Bcl-2 and Bax, in relation to the susceptibility of cells to undergoing apoptosis. The effects of enforced expression of Bcl2 after gene transfer are reported. MATERIALS AND METHODS Reagents. Anti-human Bcl-2 mouse monoclonal antibody and goat anti-mouse polyclonal antibody conjugated to peroxidase were furnished by DAKO A/S (Glostrup, Denmark). An anti-human Bax mouse monoclonal antibody was previously described [21]. A 7.2-kb expression vector was constructed in the splenic focus-forming virus (SFFV) using a human Bcl-2 cDNA expression plasmid, as previously reported [23, 24]. All-trans retinoic acid was purchased from Sigma. The 9-cis retinoic acid was a generous gift from Dr. R. Heyman (Ligand Pharmaceutical Inc., CA). Stock solutions (1002 M) were made by dissolving the compounds in either ethanol or DMSO, stored in the dark at 0307C, and routinely used at 1 mM. Cell lines and cell cultures. The human promyelocytic leukemia cell line NB4 [18] and the resistant promyelocytic leukemia cell line NB4-R1 [8, 19, 20] were cultured as previously described. Briefly, cultures were initiated by seeding 2r105 cells/ml of fresh RMPI 1640 medium (Gibco-BRL) supplemented with L-glutamine (2 mM), antibiotics, and 10% (v/v) of selected batches of fetal calf serum (Boehringer). Cultures were maintained at 3 to 5 1 105 cell/ml by a daily adjustment of the cell concentration, adding fresh culture medium with supplements when necessary. Cells were incubated at 377C in an air/5% CO2 atmosphere. Differentiation was evaluated by morphology with May– Grunewald –Giemsa and by nitroblue tetrazolium (NBT, Sigma) reduction assay. Apoptosis was assessed by morphological criteria, DNA fragmentation, in situ TdT assay, and flow cytometry, as previously described [8]. Cell proliferation and/or viability were evaluated by the WST-1 colorimetric assay (Boehringer), using a Pasteur Diagnostic (Paris, France) double-wavelength automated spectrophotometer. All assays were carried out in triplicate. Cell transfection and selection of stable transfectants. NB4-R1 cells were transfected by electroporation as described by Mu et al. [25]. Briefly, NB4-R1 cells growing exponentially were washed twice in phosphate-buffered saline (PBS). Cells at a concentration of 5 1 106 cells in 500 ml of ice-cold PBS were mixed with 25 mg of expression plasmid and 250 mg of denatured salmon DNA was kept on ice for 5 min. Cells were then electroporated twice with a GenePulser (BioRad Laboratories, Richmond, CA) at a capacitor setting of 500 mF and 200 mV, with a pulse length of 15 to 20 ms. Cells were immediately
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transferred in 10 ml RPMI 1640 containing 20% fetal calf serum (Gibco-BRL) and incubated for 24 h at 377C in an air/CO2 incubator; then the cultures were replenished with fresh medium supplemented with serum. The efficacy of transfection was controlled before antibiotic selection by evaluating transient expression by Western blots in cellular extracts from aliquots of cultures. Two days after electroporation was performed, G418 (500 mg/ml) was added in cultures. Selection was carried out for 3 weeks, until cultures grew rapidly. Pools of transfected cells were frozen and stored at 0807C. Clones selected by limiting dilution in medium supplemented with G418 and expanded for 2 weeks in antibiotic-free medium. Plasmid insertion was controlled by Southern blot and stable expression confirmed using a neo probe on Northern blot. Immunohistological labeling and confocal analyses. Histological slides were prepared by cytocentrifugation, as previously described [8], and fixed. They were then incubated with normal goat serum (2% in PBS) for 15 min to reduce nonspecific antibody binding. Each incubation, performed in a humidified chamber, was followed by 5min washes in PBS. The slides were then successively incubated with the monoclonal mouse anti-human Bcl-2 immunoglobulin (1 mg/ ml; DAKO A/S) for 24 h at 47C and then with the bridging goat antibody anti-mouse immunoglobulin (10 mg/ml) and with alkaline phosphatase-coupled goat anti-mouse (0.5 mg/ml) for 30 min each at room temperature. After a final rinsing the Bax and Bcl-2 proteins were revealed with FITC-conjugated (Dako-France) or anti-mouse IgG CY3-labeled (Sigma) fluorescent second anti-rabbit antibodies, respectively. Fluorescent preparations were examined by confocal laser scanning microscopy using a Bio-Rad MRC-600 confocal imaging system (Bio-Rad Microscience Ltd., Hertfordshire, UK) and an inverted Diaphot 300 Nikon microscope. Images were collected using an oil immersion lens (60X, NA I.4 plan Apochromat). For fluorescein and CY3 excitation a krypton/argon ion laser (Ion Laser Technology Inc., Salt Lake City, UT) operating with the 488-nm line was used. For DAPI excitation, an Entreprise ion laser (Coherent Laser Group, Santa Clara, CA) operating with a 353 nonline was used. Images of FITC, CY3, and DAPI were merged and pseudo-colored green for FITC, red for CY3, and blue for DAPI. Each image represents a single section for which the confocal system was adjusted to allow a field depth of about 0.8 mm. Preparation of subcellular fractions. Cells were harvested by centrifugation at 800g for 10 min at 47C, washed with cold PBS, and resuspended at 2r107 cell/ml. The following steps were performed at 47C, as previously described [26]. Briefly, cells were disrupted with a type B Dounce homogenizer in a 10 mM KCl, 1.5 mM MgCl2, 1 mM PMSF, 10 mM Tris– HCl buffer at pH 7.2. The nuclei were separated by centrifugation at 1000g for 30 min at 47C and then immediately stored frozen at 0807C. The supernatant was centrifuged for 30 min at 105,000g at 47C (TL100, Beckman, Palo Alto, CA). The pellet constituted the membrane fraction and the supernatant was the cytosol. All fractions were stored frozen at 0807C. For SDS–PAGE electrophoresis, the protein content in subcellular fractions was determined by the Sigma ‘‘Protein Assay’’ (Sigma-Chimie, France). Western blot analysis. Nuclear and cytoplasmic protein extracts (100 mg) were boiled in the presence of 5% b-mercaptoethanol and loaded (25 ml/slot) on 12% sodium dodecyl sulfate (SDS)– polyacrylamide gels, electrophoresed, and blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After transfer to nitrocellulose membranes, proteins were visualized with Ponceau S (Sigma) to confirm equal loading of protein. Immunolabeling of blotted membranes was then carried out as previously described [8].
RESULTS
Bax and Bcl-2 Adopted Distinct Expression and Localization Patterns during RA-Induced Apoptosis We have demonstrated previously by histological analysis that Bcl-2 is not downregulated by 9-cis RA
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FIG. 1. Comparative analysis of the Bcl-2 expression and apoptotic responses to 9-cis RA in NB4 (A) and NB4-R1 cells (B). Cells were incubated with 9-cis RA (1 mM) for 48, 72, and 96 h. Apoptotic and viable cells (500 cells scored) were counted after May– Grunewald– Giemsa staining (values represent means { SD of five independent morphological scores). Western blot analysis of Bcl-2 was carried out comparatively on crude cytosolic NB4 and NB4-R1 cell extracts, and membranes were scanned for quantification. Values are expressed in arbitrary units.
in NB4-R1 cells undergoing apoptosis [8]; however, Bcl2 downregulation occurred normally in NB4 cells undergoing maturation. This point is illustrated using a quantitative Western blot analysis in NB4 and NB4R1 cells following treatment with 9-cis RA (Fig. 1). Apoptosis was assessed in these experiments by morphometric analysis after May –Grunewald staining. The morphological cell death patterns were confirmed with DNA fragmentation analysis by gel electrophoresis (not shown). These results suggest that NB4-R1 cells could be dying prematurely because of a functional defect of Bcl-2 since a high expression persisted and/ or because of an enhanced expression of Bax. Bcl-2 and Bax proteins were then analyzed simultaneously in NB4 and NB4-R1 cells by double immunofluorescence (Fig. 2). We found that Bcl-2 exhibited a limited and perinuclear distribution in NB4-R1 cells, whereas in NB4 cells Bcl-2 exhibited both perinuclear and punctuated cytosolic localization. In contrast, Bax was more uniformly distributed in the cytoplasm of NB4 and NB4-R1 cells. Bax was also found in the peripheral area of the cytoplasm of NB4-R1 cells where Bcl-2 was absent (Fig. 2F). Double-labeling experiments clearly demonstrated that Bcl-2 and Bax did not colocalize in NB4-R1 cells, whereas substantial colocalization was observed in the NB4 cell line (see in legend to Fig. 2). Since the pattern of Bcl-2 expression in NB4 and NB4-R1 cells differed with respect to the perinuclear area, we wanted to determine whether the peculiar
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localization of Bcl-2 in NB4-R1 cells was the result of a physical association to the nuclear membrane. Membranes, cytosol, and nuclei-containing fractions were prepared and analyzed comparatively. Quantitative analyses of the distribution of Bcl-2 (Fig. 3A) and Bax (Fig. 3B) in these subcellular compartments were carried out by Western blot after cell fractionation. It confirmed (see in legend to Fig. 3) an increased amount of Bcl-2 physically associated to nuclei in subcellular fractions. We speculated that a causal relation might exist between the Bcl-2/Bax localization and the cell susceptibility to programmed cell death. Bcl-2/Bax expression was assessed following cell death induction by 9-cis RA. In apoptotic cells, Bcl-2 expression remained high, but its localization was altered by the structural modification of the cell. Bcl-2 lost its preferential perinuclear localization and became more diffusely cytosolic. Simultaneously, Bax was translocated in a discreet area of the cytoplasm where its concentration became extremely high (Fig. 4). Despite the altered distribution of Bcl-2 and Bax proteins in apoptotic cells, their subcellular localization remained in general nonoverlaping. Bcl-2 Overexpression in Stably Transfected NB4-R1 Clones Enhanced Resistance to Apoptosis NB4-R1 cells were stably transfected with a fulllength Bcl-2 [23], and the expression of Bcl-2 protein
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FIG. 2. Confocal analysis of Bcl-2 and Bax expression in NB4 and NB4-R1 cells. Double-immunofluorescence labeling was carried out and localization analyzed in a single cell. Notice that red (bcl-2) (A) and green (bax) (B) colors overlapped to form yellow color (C) in NB4 cells, while these labels occupied distinct nonoverlapping areas in NB4-R1 cells (F). Notice also in (F) that in the perinuclear area where Bcl-2 and Bax are both expressed, Bcl-2 and Bax likely associated to distinct subcellular structures since the two colors (red and green) do not overlap (see for comparison in (C) that yellow color formed, indicating that BCL-2 and Bax colocalized). FIG. 4. Distinct topographical localization of Bcl-2 and Bax upon induction of apoptosis in NB4-R1 cells by retinoids. Confocal analysis were carried out under conditions similar to those reported in Fig. 1. Immunocytological analyses were carried out after 72 h of incubation with 9-cis RA (1 mM). (A, C) Controls; (B, D) 9-cis RA treatments; (A, B) anti-Bcl-2 antibody labeling; (C, D) anti-bax antibody labeling.
expression in transfected and control NB4-R1 cells was compared on Western blot. Quantitative scanning of the immunostained membranes indicated an 8-fold overexpression of a 26-kDa Bcl-2 protein in the transfected cells (Fig. 5). Bcl-2-transfected cells were then tested for induction of apoptosis. NB4-R1/Bcl-2 showed
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a significant increase in resistance to RA-induced apoptosis. No cell death was detectable with micromolar concentrations of retinoids after 72 h of incubation, while up to 65% of cells were irreversibly committed to death in the control cultures (Fig. 6). Transfected cells could even resist, and proliferate with, a 20-fold in-
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FIG. 3. Intracellular compartimentalisation of Bcl-2 and Bax in NB4 and NB4-R1 cells. Western blot analysis of Bcl-2 expression associated to the nuclei (lanes 1 and 4) and microsomal (lanes 2 and 5) and cytosolic (lanes 3 and 6) fractions of NB4 and NB4-R1 cells. By scanning the gel membranes loaded with the same subcellular fractions of NB4 and NB4-R1 cells, the relative contents of Bcl2 and Bax in nuclear, cytosolic, and microsomal fractions has been evaluated as 6%/1%/93% in NB4 cells and 21%/1%/78% in NB4-R1 cells for Bcl2, while it was 26%/1%/73% in NB4 cells and 1%/0%/99% in NB4-R1 cells for Bax.
crease in RA concentration, without showing any of the morphological signs of apoptosis. Induction of apoptosis and its suppression in Bcl-2 expressing cells was confirmed by flow cytometry and DNA electrophoresis (not shown).
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FIG. 6. Comparative analyses of the sensitivity of NB4-R1 and NB4-R1 transfected cells to apoptosis induced by retinoids. Cell viabilities in treated and control cultures were evaluated comparatively. Values (triplicates; 500 cells scored for apoptotic features on May– Grunewald – Giemsa-stained slides) were expressed as percentages of the control.
lyzed comparatively, followed by the identification of Bax in the same cell. In transfected cells, Bcl-2 showed a uniform distribution in the cytoplasm, a feature which closely resembles the case of NB4 cells. Strikingly Bcl-2 protein was no longer limited to the perinuclear area (Fig. 7). Importantly, double labeling in sin-
The Blockade of RA-Induced Apoptosis Is Correlated with Bcl-2 and Bax Colocalization in Bcl-2Transfected Cells The patterns of expression of Bcl-2 in transfected cells and untransfected control cells were then ana-
FIG. 5. Quantitative analysis of Bcl-2 overexpression by Western blot in NB4-R1-transfected cells. Analyses were carried out on crude cytoplasmic extracts.
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FIG. 7. Confocal analyses of Bcl-2 and Bax expression patterns in NB4, NB4-R1, and NB4-R1/Bcl-2 cells.
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gle cells clearly indicated that Bcl-2 and Bax colocalized in the Bcl-2-transfected cells (Fig. 7). Treatments of NB4-R1 and NB4-R1/Bcl-2 cells with RA carried out comparatively showed that the overexpression of Bcl2 in transfected cells prevented the translocation and concentration of Bax in an area of the cytoplasm free of Bcl-2, as we observed in NB4-R1 apoptotic cells. DISCUSSION
We observed that the intracellular distribution of Bcl-2 was associated with the susceptibility of NB4-R1 cells to undergoing apoptosis induction by retinoids. In NB4-R1 cells Bcl-2 was limited to the perinuclear area and despite the fact that Bax also was detected in this area, Bcl-2 did not significantly overlap with the localization of Bax. We speculate that the greater sensitivity of NB4-R1 cells to apoptosis induction compared to NB4 cells is related to the intracellular distribution of Bcl-2 and its ability to colocalize or physically interact with Bax [37, 38]. Bcl-2 has been shown to block cell death following a variety of stimuli [21, 27–30] and the suppression of apoptosis is due to its interaction with ‘‘killer’’ partners, like Bax [21]. Bcl-2 is an integral membrane protein [23, 31] with organelle targeting and anchoring determined by a single 20-amino-acid COOH terminus domain [32, 33]. Bcl-2 localizes to membranes of mitochondria, endoplasmic reticulum, and nuclear envelope [23, 31, 34, 35], but it is, as yet, unclear whether distinct specific mechanisms are determined by differential targeting. It might well result from specialized import systems which can saturate hierarchically. It is conceivable, therefore, that a structural modification in Bcl-2 itself, affecting either its cytoplasmic localization or its ability to associate with membranes [33, 36], may result in a reduction in Bcl-2-mediated cell death suppression. Enforced expression of Bcl-2 in NB4-R1 cells conferred resistance to apoptotic cell death induction which was associated with a more widely distributed pattern of Bcl-2 protein within the cell. The distribution of Bcl-2 within the transfected NB4-R1 cells resembled that of Bcl-2 in the NB4 cells and showed significant colocalization with Bax. These findings indicate that the subcellular distribution of Bcl-2 may be an important determinant of Bcl-2-mediated cell death suppression. These results are in accordance with data reported by others [22] using mutated forms of Bcl-2 showing altered function and subcellular localization. Therefore, the molecular interaction of positive and negative regulators of apoptosis, such as Bcl-2 and Bax, is conditioned by their subcellular colocalization. This could provide a general basis for the regulation of programmed cell death and is reminiscent of CED-9 and
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CED-4 localization and function in Caenorhabditis elegans [39, 40]. Our findings indicate that despite high levels of expression of endogenous Bcl-2 in NB4-R1 cells, they were still sensitive to retinoid-induced cell death. This work underlines the uncertainty of using the level of expression of proteins which belong to the Bcl-2 family in the prognosis of tumor cell fate during chemotherapeutic treatments inducing programmed cell death. It shows the importance of further investigation on the impact of subcellular localization of Bcl-2 and Bax proteins to better understand how the cell execution program is ultimately controlled by these regulators. Basically, it remains to elucidate what structural modification can facilitate or compromise the localization and function of these molecules. These events could provide efficient targets for manipulating dysregulated apoptosis in cell pathology. Supported in part by grants from the Fondation contre la Leuce´mie (Fondation de France), Fondation Saint-Louis, Association pour la Recherche contre le Cancer (A.R.C.), Ligue Nationale contre le Cancer, INSERM, CNRS (SDI 16954.I), and EU (CT93-1530, Biomed I). This work also benefited from ‘‘Euroconferences on Apoptosis’’ (EU, Human Capital and Mobility Program and Training and Mobility of Researcher Program).
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Received December 18, 1996 Revised version received March 2, 1997
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