Autonomous Rexinoid Death Signaling Is Suppressed by Converging Signaling Pathways in Immature Leukemia Cells

G. R. Benoit, M. Flexor, F. Besançon, L. Altucci, A. Rossin, J. Hillion, Z. Balajthy1, L. Legres2, E. Ségal-Bendirdjian, H. Gronemeyer and M. Lanotte

INSERM U-496 (G.R.B., M.F., J.H, Z.B., L.L., E.S.-B., M.L.) Centre G. Hayem Hôpital Saint-Louis 75010 Paris, France
INSERM U-365 (F.B.) Institut Curie 75248 Paris Cedex 05, France
Institut de Génétique et de Biologie Moléculaire et Cellulaire (L.A., A.R., F.H.G.) Centre Nationale de la Recherche Scientifique/INSERM/ULP BP 163, 67404 Illkirch Cedex C. U. de Strasbourg, France
Istituto di Patologia generale e Oncologia (L.A.) Seconda Università degli studi di Napoli Piazzetta S. Andrea delle Dame 2 80138, Napoli, Italy


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
On their own, retinoid X receptor (RXR)-selective ligands (rexinoids) are silent in retinoic acid receptor (RAR)-RXR heterodimers, and no selective rexinoid program has been described as yet in cellular systems. We report here on the rexinoid signaling capacity that triggers apoptosis of immature promyelocytic NB4 cells as a default pathway in the absence of survival factors. Rexinoid-induced apoptosis displays all features of bona fide programmed cell death and is inhibited by RXR, but not RAR antagonists. Several types of survival signals block rexinoid-induced apoptosis. RAR{alpha} agonists switch the cellular response toward differentiation and induce the expression of antiapoptosis factors. Activation of the protein kinase A pathway in the presence of rexinoid agonists induces maturation and blocks immature cell apoptosis. Addition of nonretinoid serum factors also blocks cell death but does not induce cell differentiation. Rexinoid-induced apoptosis is linked to neither the presence nor stability of the promyelocytic leukemia-RAR{alpha} fusion protein and operates also in non-acute promyelocytic leukemia cells. Together our results support a model according to which rexinoids activate in certain leukemia cells a default death pathway onto which several other signaling paradigms converge. This pathway is entirely distinct from that triggered by RAR agonists, which control cell maturation and postmaturation apoptosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoids regulate complex physiological events during development, control maintenance of homeostasis, and induce or inhibit cellular proliferation, differentiation, and death. Due to their strong differentiative and antiproliferative activity, retinoids are used as cancer therapeutic agents and may be able to exert cancer-preventive activities (1, 2, 3). The prototype of a cancer that can be successfully treated with retinoids is acute promyelocytic leukemia (APL), but also the treatment of squamous cell carcinoma of the cervix and the skin (4) and Kaposi sarcoma (5) have been reported. Moreover, retinoids can suppress oral premalignancy and prevent second primary head-and-neck tumors (6). Most, if not all, biological responses to retinoids originate from the transcriptional control of gene programs by the cognate nuclear receptors (7). Malfunction due to genetic defects associated with these receptors or their downstream mediators, which may alter or interrupt retinoid signaling, can cause major pathologies and may account for therapeutic failures. Consequently, the comprehension of retinoid signaling has been a major task in cell biology during this decade (reviewed in Ref. 7).

The highly pleiotropic effects of retinoids result from the combinatorial action of their six receptors [retinoic acid receptors (RAR{alpha}, ß, and {gamma}), and retinoid X receptors (RXR{alpha}, ß, and {gamma})], which can heterodimerize and act as ligand-inducible transcription-regulatory factors. In addition, RXRs can also form heterodimers with various other nuclear receptors [e.g. vitamin D receptor (VDR), thyroid hormone receptor (TR), peroxisome proliferator activated receptor (PPAR), and orphan receptors], thereby modulating multiple signaling pathways (reviewed in Refs. 8, 9). To assess the contributions of the individual heterodimeric partners and to investigate whether both RAR and RXR can autonomously induce cognate signaling pathways, retinoid panagonists/antagonists or receptor-selective agonists/antagonists have been developed (10, 11, 12, 13). Using such reagents, RXR, which was previously considered a nonautonomous signaling partner in the RXR-RAR heterodimer (14, 15), we have shown recently that rexinoids can signal autonomously in the context of an activated protein kinase A (PKA) pathway (16). This novel signaling paradigm operates independently of RARs and promyelocytic leukemia (PML)-RAR{alpha}, even in the presence of RAR antagonists, and triggers the maturation not only of promyelocytic NB4 (17) but also of retinoid-resistant NB4-R2 cells (18, 19), thus bypassing the genetic defects of the resistant cells (16).

Significant insight has been gathered in recent years in the genetic basis of APL. Due to a t(15;17) chromosomal translocation, a fusion protein between the retinoic acid receptor {alpha} (RAR{alpha}) and PML (20), the function of which is still poorly understood (21, 22, 23), is formed and causes a differentiation block at the promyelocytic stage. It is believed that this fusion protein acts as a dominant-negative mutant that impairs the action of residual RAR{alpha} expressed from the second allele, but pharmacological doses of retinoic acid lead to a destabilization of the fusion protein and/or relieve its dominant negative activity, with concomitant differentiation of the leukemic blasts (reviewed in Refs. 24, 25, 26). In APL cells, all-trans-retinoic acid (ATRA)-induced maturation is followed by late cell death process, which exhibits all the hallmarks of apoptosis (reviewed in Refs. 27, 28, 29, 30), but whether maturation and apoptotic cell death are triggered by the same or distinct signaling pathways has remained elusive (21). The formation of PML-RAR{alpha} may affect the kinetics/efficiency of postmaturation apoptosis in APL cells: in fact, the ectopic expression of PML induces apoptosis (31, 32, 33), and it is conceivable that PML-RAR{alpha} impairs this function of PML. Indeed, PML-RAR{alpha} has been shown to exert antiapoptotic effects (33, 34, 35) and PML-RAR{alpha} degradation induced by ATRA may facilitate the onset of apoptosis observed after terminal maturation of APL cells (36). Accordingly, maturation-resistant APL cells might be restrained in their ability to embark on the apoptosis program, even when the corresponding machinery is functional. Apoptosis and maturation are likely mechanistically coupled since numerous genes potentially involved in the apoptotic process are regulated during maturation (c-myc, p53, Bcl-2, Bcl-xL, PML-RAR{alpha}, PML). Although several reports indicate that retinoids induce apoptosis in cells defective for maturation of APL cells (37, 38) or non-APL cells (39), convincing evidence that retinoids induce cell death independently of cell maturation in retinoid-responsive cells is lacking.

Here we provide the first evidence for an autonomous rexinoid-induced default apoptosis program that is operative in immature NB4 APL cells (17) and is entirely distinct from RAR agonist-controlled cell maturation and subsequent postmaturation apoptosis. Moreover, we demonstrate that rexinoid signaling is integrated in, and controlled by, contextual signaling paradigms that affect NB4 cell growth and differentiation. Altogether our results strongly support the idea that an autonomous rexinoid pathway for apoptosis exists in APL cells that operates independently of the RAR agonist-dependent pathway for cell maturation and postmaturation apoptosis. Rexinoid-induced apoptosis is not an isolated feature of NB4 cells, which were derived from an APL patient classified FAB M3 (17), as we observed it also in PLB985 cells that have been established from a patient with myelomonocytic leukemia (FAB M4) (40). Together, our results suggest the existence of a novel RXR-selective cell biological activity that could correspond to a basal death-by-default program of APL, and possibly other cell types. Apparently, cell life and proliferation in the presence of rexinoid agonists require survival signals of very different characteristics, three of which we have identified. It thus appears that RXR may be a valuable pharmacological target for anticancer therapy.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rexinoids Induce Apoptosis of Immature NB4 Cells in Low Serum Cell Culture Conditions
That rexinoids can cross-talk in an RAR-independent manner with other signaling pathways to induce cell differentiation (16) could imply the existence of an autonomous RXR signaling pathway, the activity of which is regulated positively or negatively by other signals. Thus, the apparent absence of any biological effects of rexinoids in cell culture systems could be due to the masking by other signaling pathways. To limit the impact of such pathways and to exclude at the same time possible interference from serum-borne retinoic acids, NB4 cells were adapted and permanently grown in low serum media (0.5% instead of 10% FCS) supplemented with retinoid-free essential growth regulators (see Materials and Methods).

Notably, while rexinoids have no apoptogenic effect on NB4 cells grown in high serum, they induce rapid and massive cell death with all typical features of apoptosis when serum factor(s) are limiting. Cell death induced by the rexinoid agonist SR11237 (0.125 µM) occurred between 60 and 72 h of treatment with a sequence of events typical for apoptosis: cell shrinkage, nuclear fragmentation, altered cytoskeleton architecture, and sudden cell disruption (Fig. 1Go and data not shown). DNA fragmentation was confirmed by classical agarose gel electrophoresis (Fig. 1BGo) and flow cytometry analysis (TUNEL, Fig. 1CGo). Cell morphology changes and DNA cleavage were observed concomitantly with caspase 3 activation and poly-ADP-ribose polymerase (PARP) cleavage (not shown; see also Fig. 4DGo). Rexinoid-induced apoptosis required a transcriptionally active RXR, as the rexinoid antagonist BMS287 rescued NB4 cells from SR11237-induced apoptosis in a dose-dependent manner (Fig. 1DGo), also demonstrating that the rexinoid is not toxic per se to these cells; we also did not notice any toxicity with other cell types (data not shown). No sign of significant cell maturation, as assessed by nitroblue tetrazolium (NBT) staining or CD11c cell surface marker positivity, accompanied rexinoid (SR11237)-induced cell death (Fig. 1EGo, lanes 5). Degradation of PML-RAR{alpha}, a hallmark of retinoid-induced maturation of APL cells, was not observed upon rexinoid treatment of NB4 cells in low serum (Fig. 1FGo; compare the PML-RAR{alpha} and {Delta}PML-RAR{alpha} bands in ATRA, rexinoid-treated cells, and controls). In keeping with these data we did not find any alteration in the micropunctate staining of PML nuclear bodies during rexinoid exposure (data not shown). We conclude that rexinoid-induced NB4 cell apoptosis (in low serum conditions) occurs without prior differentiation and results from (bona fide) RXR-mediated gene programming in the absence of transcriptionally active RARs.



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Figure 1. RAR{alpha} and RXR Agonists Induce Distinct Biological Responses in NB4 Promyelocytic Leukemia Cells Cultured under Limiting Serum Conditions

A, Dose-response (2 nM to 500 nM) to BMS753 (RAR{alpha} agonist; green curve) and SR11237 (RXR agonist; red curve) of NB4 cell growth measured after 72 h. Cell viability was measured by the WST-1 colorimetric assay. Data (mean values of triplicates) were expressed in percent of the untreated control. At concentrations ranging from 2 to 20 nM, SR11237 has no significant effect on cell proliferation and viability, while at 100 nM, SR11237 induces complete cell death. BMS753 causes growth arrest and cell maturation at concentrations greater than 100 nM. No cell death was observed for the highest concentration used (500 nM). The cell morphologies (May-Grünwald Giemsa staining) corresponding to the indicated treatments are shown in insets. Apoptotic cells exhibited massive nuclear fragmentation and chromatin spreading followed by cell disintegration. B, Electrophoretic analysis of DNA fragmentation during rexinoid-induced NB4 cell death on agarose gels. Apoptotic chromatin cleavage was monitored by the formation of DNA ladders to compare the apoptogenic activities of BMS753 (500 nM) and SR11237 (125 nM). No DNA fragmentation was detected after a treatment for 60 h, while SR11237-induced DNA fragmentation became apparent as early as 48 h and was massive after 60 h ligand exposure. C, Flow cytometry analysis of DNA fragmentation by the TUNEL method. Cells were treated as indicated in Fig. 1BGo and analyzed at 72 h. At this time 28% of the SR11237 (125 nM)-treated cells displayed DNA labeling indicative of apoptosis (untreated control, 2%). However, due to massive apoptosis and cell disintegration the flow cytometry underestimates apoptosis, as disrupted cells and debris are lost during the cell washes. No DNA fragmentation was detected in BMS753 (500 nM)-treated cells. D, RXR-dependent induction of apoptosis by the RXR agonist SR11237. NB4 cells were treated with increasing concentrations of the RXR{alpha} agonist SR11237 and the RXR antagonist BMS287, as indicated. Cell viability was analyzed at 72 h as described in Fig. 1AGo, using O.D. units in the WST-1 colorimetric assay for representation. Note that at 500 nM the RXR antagonist significantly neutralizes the activity of 250 nM SR11237 (30%). About 60% of the SR11237 activity is abolished by BMS287 (500 nM) when the agonist concentration is lowered to 125 nM. This 4:1 ratio is in keeping with the differences in binding affinity for RXR of the two compounds used in competition. E, Rexinoid apoptosis occurs without cell maturation. Cell differentiation was not observed by morphological criteria (not shown), surface membrane markers (CD11c expression), or by functional assay (NBT reduction). Analyses were performed at 48 h (first signs of apoptosis in the culture) and 72 h. Values correspond to the percentage of positive cells. Lane 1, Untreated control; lane 2, 9-cis RA (200 nM); lane 3, ATRA (200 nM); lane 4, BMS753 (500 nM); lane 5, SR11237 (125 nM). In lane 5 (72 h) the low counts further indicate massive apoptosis. F, Rexinoid apoptosis in low serum condition does not involve PML-RAR{alpha} proteolysis. NB4 cells were incubated for 36 h in either low (0. 5%; L) or high serum (10%; H) culture media with ATRA (1 µM), cAMP (200 µM), ATRA (1 µM) + cAMP (200 µM); SR11237 (0.2 µM) and SR11237 (0.2 µM) + cAMP (200 µM). Cell responses were determined by morphological examination of stained microscope slides. (M, maturation; A, apoptosis; "—", neither maturation nor apoptosis). Cell extracts were analyzed by SDS-PAGE and membranes were probed with a specific antiserum raised against human RAR{alpha} [RP{alpha} (F)]. The {Delta}-PML-RAR{alpha} specific degradation (97-kDa band) is only detected after ATRA or ATRA + cAMP treatment inducing cell maturation in both low and high serum conditions; no PML-RAR{alpha} degradation was observed during rexinoid-induced maturation in either condition.

 



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Figure 4. Rexinoid-Dependent Signaling for Cell Death Is Suppressed by RAR Agonists

A, Rescue of rexinoid-induced NB4 cell death by RAR{alpha} ligands occurs concomitantly with the induction of cell maturation. Cell viability was estimated by WST-1 assay and expressed in percent of the untreated control (as described above). Cell morphology was assayed by MGG staining. Cells were cultured for 72 h and analyzed. Increasing concentrations of the RAR{alpha} agonist BMS753 (2–500 nM) were combined with a constant concentration of the RXR agonist (SR11237; 250 nM). B, Retinoids and rexinoids induce the expression of distinct sets of proliferation and (anti)apoptosis mediators. Modulation of the mRNA levels of a number of key factors (denoted at the right) known to be involved in the regulation of proliferation (p19) and apoptosis (TRAFs, IAPs, Bfl1) as assessed by multiplex RNAse protection assays. NB4 cells grown in low serum conditions were exposed to the agents displayed at the top for 0, 12, 24, 36, and 48 h (subsequent lanes for each treatment). Only sections of the corresponding gels are shown; the bottom panel gives a representative example of the expressions of L32 and GAPDH used as the invariant internal controls for calibration. Note that the invariant controls were equivalent to the one shown in all cases displayed here. C, Retinoid-dependent rescue from rexinoid-induced apoptosis correlates with the induction of antiapoptogenic gene programs. Multiplex RNAse protection assays with bcl2 family members. Expression of the antiapoptotic bfl-1 gene is induced when an excess of the RAR{alpha} agonist BMS753 is added to NB4 cells exposed to the rexinoid BMS749 (lanes 6–9). Exposure times were 0 (lane 1) and 12 h, 24 h, 36 h, and 48 h (lanes 2–5, 6–9, and 10–13). "Probe" corresponds to the nondigested multiplex probe; lines point to the smaller gene expression-indicative fragments after hybridization and RNAse treatment. D, Action of the ligands of various RXR partners on rexinoid-induced apoptosis. Open circles, Untreated control; black filled squares, dose-response to ligands for the RXR partner; red squares and red curves, dose-response to the RXR agonist (SR11237); the arrowed red square indicates the response to 200 nM SR11237; this concentration (200 nM SR11237) is used together with increasing concentration of the various ligands for the RXR partners (open blue squares and blue curve). The concentrations of the various ligands for the RXR partners [RAR{alpha}, BMS753 (panel 1); RARß, BMS641 (panel 2); RAR{gamma}, BMS961 (panel 3); VDR, vitamin D3, (panel 4); TR, T3 (panel 5); PPAR pan agonists, BMS 990 (panel 6), BMS530 (panel 7), BMS972 (panel 8)] ranged from 2 nM to 500 nM. Cell viability was evaluated after 72 h of treatment using the WST-1 assay (O.D. arbitrary unit, means of triplicates, values in % of the untreated control).

 
In contrast to rexinoid signaling, retinoid action was not affected by low serum concentrations. Natural retinoids (ATRA, preferentially binding to RARs, and 9-cis RA, binding to both RARs and RXRs), as well as RAR{alpha}-specific agonists (BMS753), induced NB4 cell maturation similarly as in 10% serum (Fig. 1Go, A and E; see Ref. 16 for high serum data). Importantly, no apoptosis could be observed after 60 h or 72 h treatment with the retinoid BMS753, whereas under identical conditions massive apoptosis occurred in rexinoid-treated cells (Fig. 1Go, B and C). Note, however, that postmaturation apoptosis becomes apparent after prolonged exposure to retinoid agonists (data not shown). The above data show that the change in the concentration of serum factors affects RXR/rexinoid but not RAR/retinoid-mediated signaling. Correspondingly, several events associated with postmaturation apoptosis of NB4 cells, such as Bcl-2 down-regulation, were not observed during rexinoid apoptosis, while they could be seen after retinoid treatment of these cells also in low serum (data not shown).

RAR Antagonists Turn pan-RAR/RXR Agonists into Apoptotic Inducers
Given that distinct biological activities are induced by RAR (maturation) and RXR (apoptosis) agonists in conditions of limiting serum factors, we tested whether blocking the RAR activity of the pan-RAR/RXR agonist 9-cis RA would switch between the two responses. Indeed, while 9-cis-RA enhanced proliferation at low concentrations (<100 nM) and induced growth arrest and cell maturation at high concentrations as early as 48 h (Fig. 2AGo) without any sign of cell death (Fig. 2BGo), the cotreatment with 9-cis RA and the RAR{alpha} antagonist BMS614 induced rapid cell death (Fig. 2Go, A and B) in the absence of any cell maturation. Neither morphological changes, nor up-regulation of CD11c, nor NBT reduction was observed (data not shown). Note that on its own BMS614 was neither inhibiting cell growth nor inducing apoptosis (Fig. 2Go, A and B).



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Figure 2. The RAR{alpha} Antagonist BMS614 Converts the panRAR,RXR Agonist into a Death Inducer

A, Apoptosis dose-response of 9-cis RA in the presence of the RAR{alpha} antagonist BMS614 (2 µM). Control cultures comprised untreated cells (black square, 100%); BMS614 (2 µM) treated cultures (white square); dose-response to 9-cis-RA (from 2 nM to 500 nM) (blue curve). NB4 cells were cultured for 48 h in presence of BMS614 (2 µM) plus increasing concentrations of 9-cis-RA (from 2 nM to 500 nM) (red curve). The estimated number of viable cells (O.D., arbitrary unit, means of triplicates) is given as percent of the untreated control (gray square; 100%). Under these conditions the values for 9-cis-RA were significantly above the control from 6 nM to 50 nM indicating growth stimulation; growth inhibition associated with cell maturation was observed for concentrations above 100 nM (blue curve); no apoptosis was detected. The cotreatment with 9-cis-RA (increasing concentrations) and a constant concentration of RAR{alpha} antagonist (BMS614; 2 µM) shows a steep decrease in cell viability above 20 nM, associated with massive apoptosis (also apparent from cell morphology or DNA fragmentation, not shown). B, RAR{alpha} antagonist converts a maturation-inducing retinoid into an apoptotic inducer. BMS614 and 9-cis-RA were used alone or combined as indicated in the figure. The colors of histograms correspond to the color labels in Fig. 2AGo. Cultures were analyzed after 48 h of treatment. Cell viability was measured by the WST-1 assay (lanes 1–4). Cell morphology was analyzed after Giemsa staining (insets 1 to 4). BM614 (2 µM) affects neither cell proliferation nor cell viability. After 48 h (lane 4, inset 4) the combination of the two drugs induces massive cell death, making this combination more efficient that SR11237 (see Fig. 1Go). Note that 9-cis RA (0.1 µM) induces no growth arrest at 48 h when the first sign of morphological maturation is already visible (inset 2). C, Comparative analysis of the apoptogenic potential of RXR-specific ligands and bifunctional rexinoids in NB4 cells. Dose response to bifunctional rexinoids (BMS749, blue squares; BMS772, red squares) compared with the RXR-specific agonist SR11237 (green triangles). Cell viability was evaluated as described above. D, Comparative analysis of the apoptotic potential of RXR specific ligands and bifunctional rexinoids in NB4 cells. Electrophoretic analysis of DNA fragmentation during rexinoid-induced NB4 cell death on agarose gels. The experimental conditions are reported in the legend to Fig. 1BGo. (SR11237, 125 nM; BMS749, 50 nM). E, Flow cytometry analysis of DNA fragmentation in NB4 cells by the TUNEL method. Cells were treated (BMS753, 200 nM; BMS749, 200 nM) and analyzed at 48 h as indicated in Fig. 1BGo. The corresponding morphological features of cells are shown in panels at the right.

 
That RAR antagonists liberate the apoptotic activity associated with pan-RAR/RXR agonists suggests that certain bifunctional ligands, i.e. RXR agonists that display intrinsic RAR antagonistic activity, may be superagonists for rexinoid-induced apoptosis. Such a ligand has been described previously (BMS749; Ref. 16). Indeed, compared with SR11237, BMS749 displays a left shift of two logs (EC50 400 nM and 8 nM, respectively, at 48 h) for its apoptotic activity (Fig. 2CGo). After 72 h no surviving cells were found in cultures treated with BMS749 at 8 nM. Moreover, apoptosis occurred earlier after treatment with BMS749 than with SR11237 (48 h vs. 72 h). The different apoptogenic potencies of BMS749 and SR11237 were also clear from the different kinetics of fragmentation of chromosomal DNA (Fig. 2DGo) and TUNEL analysis (compare BMS749 in Fig. 2EGo, third panel from top, with SR11237 in Fig. 1CGo, middle panel). Note that a second bifunctional compound displaying the same activity as BMS749, BMS772, acted also as a super death agonist in this system (Fig. 2CGo).

Rexinoids Induce Apoptosis in Myelomonocytic PLB985 Cells
To assess whether rexinoid-induced apoptosis is an isolated feature of APL cells or of the NB4 cell model, and whether it can operate independently of the presence of the PML-RAR{alpha} fusion protein, we adapted myelomonocytic PLB985 cells (40) to low serum condition and exposed the cells to the rexinoid BMS749 or the RAR{alpha} agonist BMS753. BMS753 retarded moderately the proliferation of PLB985 cells and induced differentiation but exerted no apoptogenic effect, as is obvious from the absence of sub-G1 apoptotic bodies (Fig. 3AGo) and annexin positivity (Fig. 3BGo; see Fig. 3CGo for the effect on proliferation). In contrast, whereas the BMS749 rexinoid had no effect on PLB985 cells in 10% serum, an exposure of cells adapted to 1% serum resulted in a G1 block and more than 50% apoptosis after 3 days (Fig. 3Go, A–C; compare the cells grown in 1% serum in the absence and presence of BMS749). Thus, rexinoids have apoptogenic potential also for non-APL cells, and apoptosis occurs independently of the PML-RAR{alpha} fusion protein.



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Figure 3. Rexinoid-Induced Apoptosis Is Operative in Myelomonocytic PLB985 Cells That Are Devoid of PML-RAR{alpha}

A, Flow cytometry analysis (propidium-iodide staining) of PLB985 cells grown in 10%, or adapted to 1%, serum. Cells were treated for 72 h with 1 µM BMS749 or BMS753, as indicated. The percentage of sub-G1 particles representing apoptotic bodies are given. B, Percentage of annexin V-positive cells in high and low serum after 96 h exposure to the indicated ligands. C, Proliferation curve of PLB985 cells treated as indicated in 1% serum-containing medium.

 
The RAR{alpha}-Induced Terminal Maturation Program Acts Dominantly over the Rexinoid-Induced Program That Triggers Apoptosis of Immature Blasts
The observation that exposure of NB4 cells to the pan-RAR/RXR agonist 9-cis RA results in cell maturation suggests that the RAR activity (maturation) associated with this ligand can override the RXR activity (apoptosis). If true, this is an important aspect relevant to the design and use of rexinoids because most of the rexinoids available to date possess (traces of) retinoid activity that could potentially limit rexinoid action. To address this issue directly, we carried out experiments in which the pure RXR-specific agonist, SR11237, and the RAR{alpha}-selective agonist, BMS753, were mixed together (Fig. 4Go). SR11237 was used at 250 nM, a concentration not allowing any survival at 72 h (see Figs. 1AGo and 4AGo, lane 2). BMS753, used at 500 nM, induced NB4 cell maturation but no cell death could be detected at 72 h (Fig. 1CGo, bottom panel; Fig. 2EGo, second panel; Fig. 4AGo inset 9). Note that the decrease in viable cell counts at this time (Fig. 1AGo; Fig. 4AGo, lane 9) reflects the growth inhibition associated with granulocytic maturation (compare the cell morphologies depicted in insets 1 and 9 of Fig. 4AGo). Notably, increasing the concentration of BMS753 efficiently inhibited SR11237 rexinoid-induced apoptosis in a dose-dependent manner (Fig. 4AGo, lanes 2–8) with equimolar concentrations of SR11237 and BMS753 resulting in cell maturation (lane 7). We conclude that residual RAR{alpha} activity masks or even blunts rexinoid signaling.

An initial screening of the activity of key factors involved in the regulation of apoptosis revealed an increased expression of several antiapoptosis genes, such as bfl1, c-IAP1, c-IAP2, NAIP, as well as the tumor suppressor p19ARF, in the presence of RAR{alpha} agonists (Fig. 4BGo), suggesting that these factors may contribute to the antagonistic effect of BMS753 on rexinoid-induced apoptosis. Indeed, bfl-1 (Fig. 4CGo) and the other above mentioned antiapoptotic genes were induced when the cells were exposed to both the BMS749 rexinoid and an excess of the RAR{alpha} agonist BMS753. No up-regulation of these genes was seen with pure rexinoids (Fig. 3Go, B and C). Thus, in the presence of retinoids, the induction of an antiapoptotic gene program apparently counteracts the rexinoid-induced apoptosis. In view of these results, it is possible that serum-borne retinoic acids have "disguised" rexinoid signaling in cell culture systems, thus explaining why this pathway had not been detected earlier.

Given that RXR is a promiscuous heterodimerization partner for a great number of nuclear receptors, we wondered whether a ligand for any partner of RXR in a heterodimeric receptor complex could antagonize rexinoid-induced apoptosis similarly as retinoids, even though (with the exception of VDR) these receptors are not involved in mediating NB4 cell maturation. However, neither ligands specific for other RAR isotypes [RARß (BMS641), RAR{gamma} (BMS961), nor for the VDR, TR, or PPAR{alpha}, -ß, and -{gamma} receptors had any antiapoptotic effect (Fig. 4DGo). These results suggest that retinoids may simultaneously induce expression of the mature phenotype and inhibit a default apoptosis pathway triggered by RXR agonists.

Rexinoid-Induced Apoptosis Is Operative in Retinoid-Resistant NB4-R2 Cells
Rexinoid signaling can still function in retinoid-resistant APL cells, such as the NB4-R2 cell line in which resistance is due to a point mutation that truncates the ligand-binding domain of the PML-RAR{alpha} fusion protein (16, 19). We therefore investigated whether rexinoid signaling would operate in low serum conditions to induce apoptosis of NB4-R2 cells. Both SR11237 (data not shown) and the bifunctional RXR agonist RAR-antagonist BMS749 induced cell death under these conditions in both NB4 and NB4-R2 cells involving caspase 3 activation (Fig. 5AGo) and PARP cleavage (Fig. 5BGo). Importantly, in the absence of a functional PML-RAR{alpha}, the pan-RAR/RXR agonist 9-cis RA was also able to induce apoptosis (Fig. 5CGo, middle panel). This is in keeping with our results demonstrating that RAR{alpha} signaling overrides the rexinoid apoptosis signaling.



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Figure 5. Rexinoid-Induced Apoptosis in the Retinoid-Resistant NB4-R2 Cells

A, Caspase 3 activity as measured by cleavage of the colorimetric substrate DEVD-pNA (see Materials and Methods). B, Western blot analysis of caspase-3 and PARP cleavages in response to rexinoid treatment in NB4 and NB4-R2 cells. C, Flow cytometry analysis of DNA fragmentation in NB4-R2 cells during retinoid and rexinoid treatments. The experimental conditions are those decribed in Fig. 1CGo. Note that 9-cis-RA induces apoptosis in NB4-R2 cells. As mentioned in legend to Fig. 1CGo, apoptosis in BMS749- treated cultures is underestimated due to cell disruption. In contrast to the situation in wt cells, BMS753 did not abrogate BMS749-induced apoptosis in NB4-R2 cells.

 
PKA Agonists Switch the Rexinoid Response from Apoptosis to Differentiation
The above data suggest that rexinoid-induced apoptosis of immature APL cells and retinoid-induced maturation of these cells are mutually exclusive phenomena. In view of our recent demonstration of the existence of a novel NB4 cell maturation pathway that involves a cross-talk between rexinoids and PKA agonists (16), we investigated whether also this alternative differentiation pathway would be incompatible with rexinoid apoptosis under low serum conditions. Indeed, addition of PKA agonists, such as 8-chloro-phenyl-thio-cAMP (8CPT-cAMP), blunted BMS749-rexinoid-induced apoptosis and triggered maturation not only in NB4 but, notably, also in the retinoid-resistant NB4-R2 cells (Fig. 6Go). These results indicate the existence of several independent types of "check points" or "controlling systems" that allow the cell to switch on or off the rexinoid-dependent cell death or maturation pathways.



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Figure 6. Antiapoptotic Action of cAMP Analog in Cultures

NB4 and NB4-R2 cells were cultured in similar conditions, with a constant concentration of the RXR agonist BMS749 (500 nM) and with increasing concentration of 8-CPT-cAMP in culture medium [RPMI1640 supplemented with essential factors in HY supplement (1% vol/vol), FCS (0.5% vol/vol)]. Cell viability was evaluated using WST-1 assay (as described above). Data (O.D., arbitrary unit, means of triplicates) are expressed in percent of the untreated control in basal media. In the absence of 8-CPT-cAMP, NB4 and NB4-R2 cell cultures showed no viable cells (gray square). The curves (NB4, black squares; NB4-R2, white squares) show the death rescue by increasing concentration of cAMP. Note that viable cell counts also reflect growth arrest associated with maturation, as also observed for the action of BMS753 (see in Fig. 4AGo).

 
Rexinoid Apoptosis Is Rescued by Serum Factors
Our initial rationale for using low serum conditions in the experiments described above was to, 1) exclude the possible "contamination" with serum-borne retinoids to study "pure" rexinoid action, and 2) to exclude or limit a possible cross-talk between rexinoids and signaling pathways induced by serum factors. To reveal the possible role of serum factors in rexinoid-induced apoptosis in the absence of any cell differentiation, we studied the effect of increasing serum concentrations (0.5% to 10% FCS) on BMS749 (500 nM)-induced NB4 cell death. Serum efficiently rescued the cells from the apoptopic action of the BMS749 rexinoid (Fig. 7Go). This rescue occurred also in the presence of RAR antagonists, thus confirming that it corresponded to a signaling phenomenon different from that triggering RAR{alpha}-dependent maturation. Also serum depleted of hormones by charcoal treatment showed similar capacity to inhibit apoptosis (not shown), indicating that the serum component(s) that gives rise to the "rescue" effect is not a small molecule that can be readily absorbed to active surfaces. Importantly, rexinoid apoptosis was inhibited by serum in the absence of any sign of NB4 cell maturation (data not shown). We conclude that a nonretinoid activity in serum is able to suppress rexinoid apoptosis.



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Figure 7. Antiapoptotic Action of Serum Factors from Serum in Cultures

NB4 cells were cultured with increasing concentrations (%) of serum supplement in culture medium [RPMI 1640 supplement with essential factors in HY supplement (1% vol/vol), defined as basal media]; opened squares (curve A). In a second series of cultures, in conditions similar to panel A, a fixed concentration of the RXR agonist BMS749 (500 nM) was added (curve B). Cell viability was evaluated using WST-1 assay (as described above). Data (O.D., arbitrary unit, means of triplicates) were expressed as the percentage of the untreated control in basal media. The inset shows the value of the ration B/A at identical serum concentration in cultures.

 
Nuclear Factor-{kappa}B (NF-{kappa}B) Is Activated during Retinoid-Induced Maturation but Serum Factors Do Not Use This Survival Pathway to Suppress Rexinoid-Dependent Death Signaling
That rexinoid-induced death of immature NB4 cells is entirely different from that subsequent to retinoid- induced differentiation is strongly supported by the analysis of the expression patterns of several apoptosis-regulatory key genes. In particular, the expression of a number of antiapoptosis genes that are induced by retinoids is not affected during rexinoid death signaling (Fig. 4Go, B and C) and only tumor necrosis factor-{alpha} (TNF{alpha}) expression was augmented in the panel of genes tested when NB4 cells were exposed to rexinoids under low serum conditions (data not shown). To investigate whether serum factors would determine the cell fate via TNF-elicited nuclear factor-{kappa}B (NF-{kappa}B)-mediated signaling, the activation of NF-{kappa}B by retinoids, rexinoids, and P 75 A agonists was tested in low and high serum conditions by electrophoretic mobility shift assay (EMSA) (Fig. 8Go). Clearly, NF-{kappa}B activation correlated with cell maturation induced by either RAR{alpha} agonists or rexinoid/PKA cross-talk, but not with rexinoid-induced cell death. Importantly, serum factors did not change this pattern of NF-{kappa}B nuclear activation. Moreover, the observations that serum rescue from rexinoid apoptosis does not involve the induction of antiapoptotic genes and NF-{kappa}B, as is the case for RAR{alpha} agonists, indicates that serum factors use a distinct survival pathway to suppress rexinoid-dependent death signaling.



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Figure 8. EMSA Measurement of NF-{kappa}B Activation by Serum Retinoids, Rexinoids, and cAMP in NB4 Cells

NB4 cells were treated with the indicated agents (serum, 10% vol/vol; SR11237, 500 nM; 8-CPT-cAMP, 200 µM; BMS753, 500 nM) for 48 h with the exception of TNF{alpha} (5 h). EMSA was carried out as described in Materials and Methods. Cell maturation and/or apoptosis was evaluated in parallel on the same cultures. Biological responses are indicated as an inset on the figure (no biological response detected (-); apoptosis (A); maturation (M). The migration shift of the probe bound to NF-kB was shown by autoradiography. Autoradiograms were scanned and analyzed with Image Quant computer program. Values (%) were expressed as increase of binding compared with the untreated cell control (lane 1). Lane 10 shows background control (no nuclear extract).

 
Together these results demonstrate that 1) retinoid and rexinoid signaling activate distinct biological programs in NB4 cells and 2) rescue from rexinoid apoptosis by RAR{alpha} agonists and serum factors involves distinct survival programs.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rexinoids Induce an Autonomous Death Pathway in Promyelocytic Leukemia Cells
Several lines of evidence indicate that we have identified a novel rexinoid-dependent apoptogenic signaling pathway that is operative in immature NB4 cells and is distinct from previously investigated postmaturation apoptosis. These conclusions are supported by the following observations: 1) rexinoid apoptosis requires a transcriptionally active RXR independently of prior cell differentiation, 2) differentiation blocks rexinoid apoptosis of immature cells, 3) rexinoid apoptosis is operative in retinoid-resistant cells and is even enhanced in the presence of RAR{alpha} antagonists that inhibit cell differentiation, 4) serum factors block rexinoid apoptosis but not retinoid-induced cell differentiation and postmaturation apoptosis, 5) rexinoid apoptosis is fully functional in retinoid-resistant cells that do not differentiate or undergo postmaturation apoptosis, and 6) the differential expression of known key genes regulating cell life and death indicates that rexinoid apoptosis and postmaturation death are two completely distinct gene programs. This latter conclusion is further supported by the observation that retinoids rescue cells from rexinoid apoptosis whereas they synergize with RXR ligands for maturation and postmaturation death.

Note that rexinoid apoptosis is a signaling pathway that is entirely distinct from the apoptosis observed by so-called "pseudo-retinoids" such as CD437 or 4-HPR (41, 42, 43). This conclusion is based on the observations that 1) CD437 and 4-HPR have been reported to induce apoptosis in high serum (41, 42, 43); 2) CD437-induced NB4 cell apoptosis in low serum occurs with the same potency as in high serum media; 3) neither PKA nor RAR{alpha} agonists could diminish the CD437-dependent apoptosis of NB4 cells (G. Benoit and M. Lanotte, unpublished); and 4) both CD437 and 4-HPR are devoid of any measurable rexinoid activity in reporter cell assays (C. Gaudon and H. Gronemeyer, unpublished).

RXR within the RAR-RXR heterodimer is believed to be silenced by apo-RAR (a phenomenon also termed "RXR subordination") but may synergize with holo-RAR although the mechanistic basis of this phenomenon is still a matter of controversy (12, 14, 44, 45, 46, 47). In addition to its signaling through RAR-RXR heterodimers, RXR homodimers and a great number of alternative RXR heterodimers can signal in target cells (9). What could be the RXR signaling entity that triggers immature APL cell apoptosis? Our study does not support an implication of RAR-RXR heterodimers that are believed to mediate differentiation and postdifferentiation apoptosis of NB4 and F9 cells (12, 48, 49), mainly because bifunctional ligands, such as BMS749, do apparently not generate a transcriptionally active RAR-RXR heterodimer. The observation that a transcriptionally active RXR is required for rexinoid apoptosis suggests an implication in this phenomenon of either RXR homodimers, for which so far neither a separate signaling pathway nor cognate target genes (or so-called "permissive" heterodimers) have been identified. Clearly further genetic studies, involving, for example, mutants that are deficient in RXR homo- or heterodimerization, are required to provide evidence for the existence of a potentially existing RXR homodimer death signaling pathway.

Rexinoid Death Signaling: A Default Pathway in Disguise?
The observation that the knockout of RXR{alpha} generates a lethal phenotype indicates that RXR is more than simply a silent heterodimerization partner (50). Our results strongly support the implication of RXR (ligands) in the regulatory mechanisms controlling the cell life and death balance (Fig. 9Go), which had probably gone unnoticed because of the suppressive action of serum factors that are present in virtually all experiments done with cultured cells or because its action was masked by retinoids and/or other signaling pathways. In this respect, it will be of interest to assess the rexinoid responsivity of hematopoietic and nonhematopoietic cells other than NB4 under conditions of limiting serum factors: notably, that RXR is required for apoptosis in cells of nonhematopoietic origin, which is based on the observation that retinoid-induced apoptosis is blunted in mouse F9 embryo carcinoma cells lacking RXR{alpha} (49, 51). In this case, however, it has remained unclear whether RXR apoptogenic signaling is autonomous. Also in non-APL HL60 cells, RXR ligands were required for postmaturation apoptosis but only after prior exposure of the cells to retinoids and subsequent cell maturation (52). Again, it will be interesting to assess whether rexinoids have the capacity to signal autonomously in these cells. It is worth noting that retinoid-rexinoid signaling in HL60 is apparently distinct from that in APL cells where RAR{alpha} agonists suffice to induce maturation and postmaturation apoptosis (Fig. 9Go). The underlying mechanism(s) accounting for distinct action of retinoids and rexinoids in these two cell lines are not yet elucidated but may be linked to the differential expression of the PML-RAR{alpha} fusion protein.



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Figure 9. Two Default Signaling Pathways Triggered by Retinoids and Rexinoids Determine Life and Death of NB4 Promyelocytic Leukemia Cells

Simplified schematic illustration of retinoid and rexinoid signaling pathways that affect NB4 cell maturation, survival, and apoptosis. The rexinoid pathway (top), activated by rexinoid (i.e. RXR selective) agonists and mediated by either so-called "permissive" (see text for details and references) RXR heterodimers (RXR-"X") with an unknown nuclear receptor heterodimerization partner or RXR homodimers [(RXR)2], leads by default to immediate apoptosis of immature NB4 cells. This pathway is characterized by its insensitivity toward RAR{alpha} antagonists. Several alternative signaling options can rescue NB4 cells from rexinoid-induced apoptosis, including RAR{alpha} and PKA agonists, both of which activate pathways that lead to cell maturation, and presently uncharacterized serum factors that induce survival. The second default signaling pathway (bottom) is dependent on RAR{alpha} agonists, abrogated by RAR{alpha} antagonists, and leads to cell maturation followed by postmaturation apoptosis. The receptor species involved in this signaling have not been unequivocally determined and may involve RXR-RAR{alpha} or RXR-PML-RAR{alpha} heterodimers or oligomers (57 ) of PML-RAR{alpha} [(PML-RAR{alpha})x]. Note that coordinate activation of RAR{alpha} and RXR leads to synergistic activation of cell maturation.

 
It is tempting to speculate that (endogenous) rexinoids may even correspond to death inducers, depending on the signaling context (e.g. hormones, cytokines, extracellular matrix; see Ref. 16 for PKA action) of a cell at a certain time in development or position within the cell lineage. Indeed, evidence for the possible existence of endogenous rexinoids has been obtained with transgenic "reporter" mice (53, 54). Thus, RXRs may correspond to attractive targets for drug design, possibly in combination with compounds that alter the inhibitory activity of retinoid agonists (preferably in the form of a bifunctional retinoid, such as BMS749) or endogenous signals that correspond to the unknown serum factors observed in this study. Further investigation to determine the cascade of events downstream from the RXR-dependent transcriptional regulation and of the nature of the interfering signals in vivo should provide new insights on how natural retinoids control cell fate in cells and developing organisms.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents and Drugs
ATRA, 9-cis retinoic acid (9-cis RA), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). The BMS753, BMS649 (SR11237), BMS614, BMS493, BMS009, BMS287, BMS772, and BMS749, provided by Bristol-Myers Squibb (Princeton, NJ), are synthetic retinoids with receptor selectivity, the features of which have been reported previously (16).

Cell Lines, Cultures, and Analysis of Cell Maturation and Cell Viability
NB4, NB4-R2, and PLB985 cells (17, 18, 40) were adapted to culture conditions with minimal serum addition in the synthetic media and allowing optimal cell proliferation and/or differentiation and long-term survival. To this purpose, cells were maintained in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 1% (vol/vol) HY (Life Technologies, Inc.), 0.5% (NB4 and NB4R2) and 1% (PLB985) (vol/vol) FCS (Bayer Corp., Elkhart, IN), glutamine (2 mM), and antibiotics in a humidified incubator at 37 C with 5% CO2. Morphological studies were performed on smears stained with May-Grünwald-Giemsa (MGG; Sigma). Cell maturation was measured by NBT reaction. Results are expressed as percentage of NBT-positive cells after a count on 300 cells. Cell viability was measured by the WST-1 colorimetric assay (Roche Molecular Biochemicals, Indianapolis, IN). Data (mean values of triplicates) were expressed in percent of the untreated control.

Analyses of Apoptotic Features
Apoptosis was assessed by the TUNEL method, propidium iodide staining, or annexin V immunostaining using the annexin V detection kit (Roche Molecular Biochemicals); samples were analyzed by as recommended by the supplier. Briefly, cells were incubated in buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) containing annexin- V-fluorescein isothiocyanate (1 µg/ml) for 10 min in the dark. After resuspension in 1 ml labeled buffer, samples were analyzed using the FACScan flow cytometer. For the TUNEL assays the Fluorescent In Situ Cell Death Detection kit (Roche Molecular Biochemicals) was used according to the manufacturer protocol except that cells were fixed in PBS-4% formaldehyde. Labeled cells were analyzed using the FACSCALIBUR. Internucleosomal DNA cleavage was visualized after agarose gel electrophoresis. DNA was isolated from 2 x 106 cells according to the procedure described by Miller et al. (55), modified as we previously described (56). Caspase activities of total cell extracts were measured using a colorimetric procedure. Briefly, 2 x 106 cells were harvested and lysed in buffer A [50 mM Tris-HCl, pH 7.5, 0.03% NP40, 1 mM dithiothreitol (DTT)] after washing in PBS, pH 7.2. Unsoluble material was removed by centrifugation at 14,000 rpm for 15 min at 4 C. Protein concentration of the supernatant was measured using the BCA assay reagent (Pierce Chemical Co., Rockford, IL). The reaction was set up in 96-well plates by adding 0.2 mM of specific colorimetric substrate DEVD-pNa for Caspase-3 to 0.01 ml of lysate in caspase reaction buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1% 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, 10 mM DTT). The reaction was incubated at 37 C, and release of pNa was measured by absorbance reading at 405 nm once per hour during 5 h. Enzyme activity was measured as initial velocity of the enzymatic kinetic.

Immunofluorescence Analysis and Flow Cytometry Analysis of Cell Surface Antigen
Immunofluorescence analysis of Bcl-2 expression was performed as described previously. Briefly, after treatment by the indicated compound, cells were smeared on histological glass slides using a cytocentrifuge (Cytospin, Shandon). After overnight drying, cell smears were fixed in acetone at 4 C for 10 min and allowed to air dry for 20 min. The slides were then sequentially incubated with PBS for 15 min, and monoclonal mouse antibody was raised against human Bcl-2 protein (DAKO Corp., Carpenteria, CA) at a dilution of 1:400 in PBS for 1 h. After three washes in PBS, the slides were incubated with fluorescein-coupled antimouse antibody (Sigma) at a dilution of 1:200 in PBS for 30 min. After three washes in PBS, the slides were mounted with 5 µl of fluorescent mounting medium (DAKO Corp.) 0.2% DAPI. All incubations were at room temperature. Preparations were examined by Fluorescent Microscopy. Images were collected and digitalized using a CCD color camera and QWIN software (Leica Corp., Deerfield, IL). The expression of the membranous adhesion molecule CD11c integrin was analyzed by direct immunofluorescence. After incubation with the indicated compounds, cells were washed in PBS and labeled with antihuman CD11c PE mouse monoclonal antibodies (Becton Dickinson and Co., Franklin Lakes, NJ). Cells were then washed twice in PBS and fixed in 1% paraformaldehyde/PBS solution. Cells were analyzed using a FACSCALIBUR (Becton Dickinson and Co.) flow-cytometer.

Ribonuclease (RNAse) Protection Assays
Total RNA was extracted with the Trizol reagent (Life Technologies, Inc., cat. 15596–018). The RNAse protection assay was performed according to the supplier’s instructions (PharMingen, San Diego, CA). Briefly, the corresponding template sets (PharMingen) were labeled with [{alpha}-32P] uridine triphosphate. RNA (4 µg) and 6 to 8 x 105 cpm of labeled probes were used for hybridization. After RNAse treatments, the protected probes were resolved on a 5% urea-polyacrylamide-bis-acrylamide gel.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay
Cells were lysed in buffer A (10 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl, 1 mM DTT, 0.05% NP40, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin, antipain, and leupeptin) for 5 min on ice, and the cell lysate was centrifuged at 2,000 x g for 5 min. The nuclear pellet was then washed in buffer A without NP 40, resuspended in buffer B (20 mM Tris HCl, pH 8, 1.5 mM MgCl2, 600 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin, antipain, and leupeptin), frozen at -80 C, and centrifuged at 10,000 x g for 30 min to remove debris. Protein concentration of nuclear extracts was determined by the Bradford assay. For EMSA, the double-stranded consensus NF-{kappa}B probe, 5'-AGT TGA GGG GAC TTT CCC AGG C-3'; 3'-TCA ACT CCC CTG AAA GGG TCC G-5', was end-labeled using [{gamma}-32P] ATP and T4 polynucleotide kinase. Binding reactions were carried out in a 20 µl binding reaction mixture [10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 10% glycerol, 0, 2% NP40, and 4 µg of poly(dI-dC)(dI-dC)] containing 5 mg of nuclear proteins and 0.5 ng of the radiolabeled probe. Samples were incubated for 45 min on ice and fractionated by electrophoresis on a 6% nondenaturing polyacrylamide gel in TAE buffer (7 mM Tris, pH 7.5, 3 mM sodium acetate, 1 mM EDTA). Gels were run at 180 V for 2.5 h at 4 C, dried, and autoradiographed.

Protein Extraction and Western-Blot Analysis
Total protein extracts were prepared. Briefly, cultured cells were washed in PBS and pelleted by centrifugation at 400 x g for 5 min. Pellets of 2 x 106 cells were immediately lysed by adding 100 µl of a boiling Laemmli solution containing ß-mercaptoethanol and disrupted with a pestle. Samples were then boiled for 5 min and insoluble material was removed by centrifugation at 13,000 rpm for 5 min. Protein amount was quantified by a Coomassie Blue staining. Protein extracts (10 µg) were loaded on SDS-polyacrylamide gels, electrophoresed, and blotted onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA). After transfer, proteins were visualized with Ponceau S (Sigma) to confirm equal loading of protein. Membranes were blocked with 5% non-fat dry milk in PBS, pH 7.6, 0.1% Tween 20 (PBS-T), and then incubated with a specific antiserum raised against the indicated protein in PBS-T 0.5% milk for 18 h at 4 C. Membranes were incubated with horseradish peroxidase-coupled antibody (The Jackson Laboratory, Bar Harbor, ME) for 30 min at 25 C. Each of these steps was followed by three washes for 10 min in PBS-T 0.5% milk. Labeling was performed as described in the ECL detection kit (Amersham Pharmacia Biotech, Arlington Heights, IL).


    ACKNOWLEDGMENTS
 
We thank the Bristol-Myers-Squibb chemists for providing the synthetic retinoids. PLB985 cells were generously provided by Dr. Y. E. Cayre (Paris). L.A., A.R., and H.G. thank Michele Lieb for technical help and Emmanuelle Wilhelm for technical support and expert advice.


    FOOTNOTES
 
Address requests for reprints to: M. Lanotte, INSERM U-496, Centre G. Hayem, Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75010 Paris, France. E-mail: mlanotte{at}jupiter.chu-stlouis.fr or H.

This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, Bristol-Myers-Squibb, and grants to M.L. from the Ligue Nationale contre le Cancer and Association pour la Recherche contre le Cancer (ARC).

1 Present address: Department of Biochemistry, University Medical School, Debrecen, Nagyerdei krt. 98. H-4012, Hungary. Back

2 Present address: Service d’Anatomie-Pathologie, Centre G. Hayem, Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75010 Paris, France. Back

Received for publication November 10, 2000. Revision received February 14, 2001. Accepted for publication March 12, 2001.


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