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
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ABSTRACT
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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
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
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.
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INTRODUCTION
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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
, ß, and
), and retinoid X receptors (RXR
, ß, and
)], 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
, 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
(RAR
) 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
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
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
impairs this function
of PML. Indeed, PML-RAR
has been shown to exert antiapoptotic
effects (33, 34, 35) and PML-RAR
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
, 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.
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RESULTS
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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. 1
and data not shown). DNA fragmentation
was confirmed by classical agarose gel electrophoresis (Fig. 1B
) and
flow cytometry analysis (TUNEL, Fig. 1C
). 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. 4D
). 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. 1D
), 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. 1E
, lanes 5). Degradation of PML-RAR
, a hallmark of
retinoid-induced maturation of APL cells, was not observed upon
rexinoid treatment of NB4 cells in low serum (Fig. 1F
; compare the
PML-RAR
and
PML-RAR
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 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 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. 1B 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 agonist SR11237
and the RXR antagonist BMS287, as indicated. Cell viability was
analyzed at 72 h as described in Fig. 1A , 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 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 [RP (F)]. The -PML-RAR 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
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 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 agonist BMS753 (2500 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 agonist BMS753 is added to NB4
cells exposed to the rexinoid BMS749 (lanes 69). Exposure times were
0 (lane 1) and 12 h, 24 h, 36 h, and 48 h (lanes
25, 69, and 1013). "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 , BMS753 (panel 1);
RARß, BMS641 (panel 2); RAR , 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).
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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
-specific agonists (BMS753),
induced NB4 cell maturation similarly as in 10% serum (Fig. 1
, 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. 1
, 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. 2A
) without any sign of
cell death (Fig. 2B
), the cotreatment with 9-cis RA and the
RAR
antagonist BMS614 induced rapid cell death (Fig. 2
, 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. 2
, A and B).

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Figure 2. The RAR 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 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 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 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. 2A . Cultures were analyzed after
48 h of treatment. Cell viability was measured by the WST-1 assay
(lanes 14). 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. 1 ). 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. 1B .
(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. 1B . The corresponding morphological features of cells are shown in
panels at the right.
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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. 2C
). 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. 2D
) and TUNEL
analysis (compare BMS749 in Fig. 2E
, third panel from top,
with SR11237 in Fig. 1C
, 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. 2C
).
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
fusion protein,
we adapted myelomonocytic PLB985 cells (40) to low serum condition and
exposed the cells to the rexinoid BMS749 or the RAR
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. 3A
) and annexin positivity
(Fig. 3B
; see Fig. 3C
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. 3
, AC; 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
fusion protein.

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Figure 3. Rexinoid-Induced Apoptosis Is Operative in
Myelomonocytic PLB985 Cells That Are Devoid of PML-RAR
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.
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The RAR
-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
-selective agonist, BMS753, were mixed
together (Fig. 4
).
SR11237 was used at 250 nM, a concentration not
allowing any survival at 72 h (see Figs. 1A
and 4A
, lane 2).
BMS753, used at 500 nM, induced NB4 cell
maturation but no cell death could be detected at 72 h (Fig. 1C
, bottom panel; Fig. 2E
, second panel; Fig. 4A
inset 9). Note that the decrease in viable cell counts at
this time (Fig. 1A
; Fig. 4A
, lane 9) reflects the growth inhibition
associated with granulocytic maturation (compare the cell morphologies
depicted in insets 1 and 9 of Fig. 4A
). Notably,
increasing the concentration of BMS753 efficiently inhibited SR11237
rexinoid-induced apoptosis in a dose-dependent manner (Fig. 4A
, lanes
28) with equimolar concentrations of SR11237 and BMS753 resulting in
cell maturation (lane 7). We conclude that residual RAR
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
agonists (Fig. 4B
), suggesting that these factors may contribute
to the antagonistic effect of BMS753 on rexinoid-induced apoptosis.
Indeed, bfl-1 (Fig. 4C
) 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
agonist BMS753. No up-regulation
of these genes was seen with pure rexinoids (Fig. 3
, 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
(BMS961), nor for the VDR, TR, or
PPAR
, -ß, and -
receptors had any antiapoptotic effect
(Fig. 4D
). 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
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. 5A
) and PARP cleavage
(Fig. 5B
). Importantly, in the absence of a functional PML-RAR
, the
pan-RAR/RXR agonist 9-cis RA was also able to induce
apoptosis (Fig. 5C
, middle panel). This is in keeping with
our results demonstrating that RAR
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. 1C .
Note that 9-cis-RA induces apoptosis in NB4-R2 cells. As
mentioned in legend to Fig. 1C , 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. 6
).
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. 4A ).
|
|
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. 7
). This rescue occurred also in the
presence of RAR antagonists, thus confirming that it corresponded to a
signaling phenomenon different from that triggering RAR
-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-
B (NF-
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. 4
, B and C)
and only tumor necrosis factor-
(TNF
) 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-
B (NF-
B)-mediated signaling, the activation of NF-
B by
retinoids, rexinoids, and P 75 A agonists was tested in low and high
serum conditions by electrophoretic mobility shift assay (EMSA) (Fig. 8
). Clearly, NF-
B activation
correlated with cell maturation induced by either RAR
agonists or
rexinoid/PKA cross-talk, but not with rexinoid-induced cell death.
Importantly, serum factors did not change this pattern of NF-
B
nuclear activation. Moreover, the observations that serum rescue from
rexinoid apoptosis does not involve the induction of antiapoptotic
genes and NF-
B, as is the case for RAR
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- 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 (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
agonists and serum factors
involves distinct survival programs.
 |
DISCUSSION
|
---|
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
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
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
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. 9
),
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
(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
agonists suffice to induce maturation and postmaturation apoptosis
(Fig. 9
). 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
fusion protein.
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
|
---|
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. 15596018). The RNAse protection assay
was performed according to the suppliers instructions
(PharMingen, San Diego, CA). Briefly, the corresponding
template sets (PharMingen) were labeled with
[
-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-
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
[
-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. 
2 Present address: Service dAnatomie-Pathologie, Centre G. Hayem,
Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75010 Paris,
France. 
Received for publication November 10, 2000.
Revision received February 14, 2001.
Accepted for publication March 12, 2001.
 |
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