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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4300-4310
Combined Arsenic and Retinoic Acid Treatment Enhances
Differentiation and Apoptosis in Arsenic-Resistant NB4 Cells
By
Maurizio Giannì,
Marcel H.M. Koken,
Mounira K. Chelbi-Alix,
Gérard Benoit,
Michel Lanotte,
Zhu Chen, and
Hugues de Thé
From the Centre National de la Recherche Scientifique Unité
Propre de Recherche 9051, Laboratoire associé au Comité de
Paris de la Ligue Contre le Cancer, UIH, Université Paris VII,
Service de Biochimie B, Hôpital St Louis, Paris; INSERM
Unité 496, Hôpital St Louis, Paris, France; Shanghai
Institute of Hematology, Rui-Jin Hospital, Shanghai, China.
 |
ABSTRACT |
In the acute promyelocytic leukemia (APL) cell line NB4, as well as
in APL patients' cells, arsenic trioxide
(As2O3) leads to incomplete cell maturation,
induction of apoptosis, as well as to the degradation of the oncogenic
PML/RAR fusion protein. We have isolated an arsenic-resistant NB4
subline (NB4-AsR), which fails to undergo apoptosis, but
maintains the partial differentiation response to this drug. When grown
in the presence of As2O3, NB4-AsR
cells degrade PML/RAR, slightly differentiate, and become more sensitive to serum deprivation-induced apoptosis. Similarly, in RA-resistant NB4-R1 cells, RA induced a significant PML/RAR
degradation and yet failed to induce cell maturation. Thus,
As2O3- or retinoic acid (RA)-induced PML/RAR
degradation may be a prerequisite, but is not sufficient for the full
differentiative/apoptotic response to these drugs. Strikingly,
RA-triggered differentiation and apoptosis were greatly accelerated in
As2O3-treated NB4-AsR cells. The
synergism between these two agents in this setting could provide an
experimental basis for combined or sequential RA/As2O3 therapies.
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INTRODUCTION |
ACUTE PROMYELOCYTIC leukemia (APL) is a
rare disease representing 10% to 15% of all the acute myelogeneous
leukemias of adults. APL is characterized by developmental arrest of
granulopoiesis at the promyelocytic stage and is generally associated
with a specific t(15;17) translocation, which fuses the PML and
retinoic acid receptor (RAR) genes to yield a PML/RAR fusion
protein.1 RAR belongs to the steroid receptor
superfamily and activates transcription in response to all-trans
retinoic acid (RA).2 PML is a zinc finger protein
containing a coiled-coil domain, which has growth and transformation
suppressor activity.3-5 Immunofluorescence analysis
indicates that PML is localized on discrete subnuclear structures
called PML nuclear bodies (NBs) whose function remains unknown.6-9 In APL cells, both the PML/RAR fusion
protein and the normal RAR and PML proteins are
expressed.10 Recent results, obtained in vivo with
PML/RAR transgenic mice, show that this chimeric protein is
sufficient to impair neutrophilic differentiation and initiate
development of leukemia.11-13 PML/RAR bears most of the
functional domains of both PML and RAR and consequently may
interfere with the normal function of these proteins. However, it
remains to be elucidated how leukemogenesis relates to altered RAR
and/or PML function(s). PML/RAR (as well as a dominant
negative RAR mutant) interferes with nuclear receptor function and
myeloid differentiation.14,15 In addition, PML/RAR
displaces PML and several other NB antigens from NBs to other
ill-defined nuclear sites, presumably through the formation of
PML:PML/RAR heterodimers.6-9 Such loss of specific PML
localization could antagonize its growth suppressive properties and
contribute to deregulated cell growth. Finally, the PML/RAR fusion
may exhibit new functional properties, as suggested by the observation
that PML/RAR homodimers recognize new DNA target
sites.16,17
Paradoxically, the APL cells are exquisitely sensitive to the
differentiating action of pharmacologic concentrations
(10 6 mol/L) of RA, leading to high rates of
temporary clinical remissions.18,19 In fact, in APL cells
or in the APL cell line, NB4, RA administration causes the degradation
of the fusion protein20 associated with the reaggregation
of PML and other NB-antigens to yield their normal pattern of nuclear
speckled localization.8 Arsenic trioxide (As2O3) has also proven to be an effective drug
in the treatment of APL patients, inducing remission even in
RA-resistant APL cases.21-23 As2O3
triggers apoptosis at micromolar concentrations, and this is associated
with a strong downregulation of the bcl-2 protein,21 while
at lower concentrations, some differentiation can be
observed.23 In addition, we have shown that, in NB4 cells,
As2O3 targets PML and PML/RAR onto NBs and
induces the degradation of both proteins.24 Thus,
As2O3 and RA target the PML and RAR moities of
the fusion protein, respectively, and both induce its degradation,
providing the first example of oncogene-targeted therapy.
In the present work, the isolation and the characterization of an
As2O3-resistant NB4-derived population,
NB4-AsR, is reported. These cells, when grown in the
presence of As2O3, are more differentiated than
parental NB4 cells, constantly degrade PML/RAR, and display a normal
speckled NBs pattern. NB4-AsR cells treated with the
combination of As2O3 and RA show accelerated differentiation and/or a dramatic induction of apoptosis. By
showing that RA and As2O3 synergize for
induction of differentiation and apoptosis and that cells resistant to
one agent are sensitive to the other, our results provide a rational
for combined therapies.
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MATERIALS AND METHODS |
Cell culture conditions and reagents.
The NB4 APL cell line and the NB4-derived lines (NB4-R1 and NB4-R2 )
were kindly provided by M. Lanotte.25-27 All
cell lines were grown in RPMI-1640 supplemented with 10% fetal calf
serum (FCS) (GIBCO-BRL, Gaithersburg, MD) under the same conditions as
previously reported for the isolation and maintenance of the NB4 cell
line.25 All-trans RA and As2O3 were
purchased from Sigma-Aldrich (St Louis, MO).The 102
mol/L RA stock solution was prepared by dissolving the compound in
dimethyl sulfoxide (DMSO). A 100 mmol/L stock solution of
As2O3 was obtained by dissolving
As2O3 in 1 N NaOH and dilution in
H2O.
Selection of NB4-AsR cells.
An As2O3-resistant NB4 subline was derived from
the NB4 cell line by growth in medium supplemented with
107 mol/L As2O3. While most
of the cell population died, rare
As2O3-resistant cells became evident in about 4 months of selection in 107 mol/L
As2O3. The few living cells were recovered
following the method described by Andersen and Junker.28 To
restore the required minimal cell density, the surviving cells
(1/105 cells) were replated at 30 cells/well in a 96-well
microtiter plate and selection continued. After 2 months in the
presence of 106 mol/L As2O3,
a pool of resistant cells was isolated, which was used in the
subsequent experiments.
Characterization of cell differentiation.
Morphology and granulocytic differentiation was evaluated by light
microscopy of May-Grünwald-Giemsa-stained cytospin preparation. Differentiation of NB4 and NB4-AsR cells was also assessed
by the ability of the cells to produce superoxide. This was measured by
the degree of reduction of nitroblue-tetrazolium (NBT, Sigma-Aldrich)
achieved by the cells over a 30-minute period at 37°C in the
presence of phorbol myristate acetate (PMA,
Sigma-Aldrich).29
Measurement of leukocyte alkaline phosphatase (LAP) activity.
A total of 106 cells were harvested, washed once with
phosphate-buffered saline (PBS), and pelleted. The cell pellet was
resuspended in homogenization buffer30 and disrupted by
vigorous pipetting. The homogenate was used for the LAP assay, which
was performed using p-nitrophenol phosphate (Sigma-Aldrich) as a
substrate. LAP activity was normalized for the protein content in the
sample. Protein concentration was measured according to the Bradford
method using bovine serum albumin (BSA) fraction V (Sigma) as a
standard and a commercially available kit (BioRad, Hercules CA). One
unit of LAP activity was defined as the amount of enzyme able to
transform 1 nmol of substrate in 1 minute at 37°C. Enzyme assays
were performed in conditions of linearity relative to the substrate and
to the concentration of proteins.
Measurement of plastic adherence.
After incubation with medium alone or with medium supplemented with the
different compounds, NB4 and NB4-AsR cells still growing in
suspension were collected and counted, the plastic wells were then
washed twice with PBS, and adhering cells were detached with
trypsin-EDTA and counted. Adherence was estimated as the percentage of
cells in each well adhering to the plastic (ie, adhering cell
number/total (adhering + nonadhering) cell number × 100%).
Analysis of cytodifferentiation markers and interferon
(IFN) titration.
The myeloid surface markers CD33, CD11b, CD11c, and CD14 were
determined by flow cytometry analysis using appropriate antibodies purchased from DAKO, Glostrup, Denmark (CD11b, CD11c) or Coulter Corp,
Miami, FL (CD33 and CD14). The percentage of positive cells (%) and
the mean associated fluorescence were quantitated using a FACScan
analyzer (LYSIS II software, Becton Dickinson, Mountain View, CA). IFN
was titrated on Madin Darby bovine kidney (MDBK) cells as
described.31
Apoptosis and cell cycle distribution.
DNA degradation was estimated by labelling DNA strand breaks with
terminal deoxynucleotidyl transferase (TdT). The tailing reaction named
TUNEL was performed according to the manufacturer's instructions
(Boehringer, Mannheim GmbH, Germany) except that the paraformaldehyde
fixation was replaced by a 4% formaldehyde fixation. Cells were
analyzed by fluorescence microscopy and by flow cytometry using a
FACScan. All data were collected, stored, and analyzed by LYSIS II
software (Becton Dickinson). Cells were also analyzed for their DNA
content and cell cycle distribution (CELLFIT cell cycle analysis,
Becton Dickinson) by flow cytometry analysis of propidium iodine
(PI)-stained nuclei.32
Immunofluorescence and Western blot analysis.
Immunofluorescence was performed as previously described.6
PML/RAR and PML were revealed by rabbit polyclonal
antibodies6 followed by a fluorescein isothiocyanate
(FITC)-labeled antirabbit polyclonal antibody (Biosys, Compiegne,
France). RAR was revealed by monoclonal antibodies
(kindly provided by B. Allegretto, Ligand Co, La Jolla, CA) followed by
a Texas Red antimouse polyclonal antibody (Amersham, Little Chalfont,
Buckinghadshire, UK). For Western blot analysis, cells
were washed and resuspended in PBS, lysed in 1 X hot Läemmli
sample buffer, and boiled for 5 minutes. About 30 µg of protein was
analyzed on a 12% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gel, and transferred to nitrocellulose by
semidry blotting. Membranes were blocked with 10% skimmed milk in Tris
buffered saline (TBS)-0.1% Tween for 1 hour and incubated overnight with the following specific rabbit polyclonal antibodies; anti-RAR (RPF') (kindly provided by P. Chambon, Strasbourg, France), anti-STAT1,
anti-P21WAF1/CIP1 (Santa Cruz Biotechnology, Santa Cruz,
CA), as well as with antihuman Bcl-2 mouse monoclonal antibody (DAKO
Corp, Carpinteria, CA). These initial incubations were followed by
incubation with antirabbit IgG or antimouse IgG horse radish
peroxidase-conjugated antibodies (Biosys). Antibody
complexes were detected by chemiluminescence using the ECL kit
(Amersham Aylesbury). To estimate the apparent molecular mass of
polypeptides, kaleidoscope prestained standards from BioRad were used
(BioRad Laboratories, Richmond, CA).
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RESULTS |
Arsenic triggers differentiation, but not apoptosis in the
NB4-AsR subline.
Arsenic-resistant cells were selected from NB4 cells treated with
106 mol/L As2O3 over a
4-month period (see Materials and Methods). In normal medium or in low
doses (107 mol/L) of arsenic, NB4, and
NB4-AsR cells have similar growth rates
(Fig 1A and see Fig 7A)
. At higher doses
(106 mol/L) of As2O3, a
sharp difference in growth was observed between parental and resistant
cells: the later grow, although at a reduced rate, while parental cells
die (Fig 1A). Indeed, a sharp reduction in
As2O3-induced apoptosis was demonstrated by
TUNEL in resistant compared with parental cells (Fig 1B). Resistant
cells withdrawn from arsenic for over a month still achieve a
significant resistance to apoptosis on de novo arsenic exposure (not
shown). Thus, NB4-AsR cells remain sensitive to growth
retardation of arsenic (a property also seen in HL60 or U937 cells),
but fail to apoptose at high concentrations (Fig 1B). Consistent with
this result, two proteins, p21 and Bcl-2, which are modulated during
As2O3 triggered NB4 apoptosis, failed to
respond to As2O3 in resistant cells (see Fig
8). As2O3 content of NB4 and
NB4-AsR cells was very similar (respectively 0.025 ± 0.003 µmol/L/107 cells v 0.021 ± 0.004 µmol/L/107 cells when grown for 24 hours in
107 mol/L As2O3
and 0.105 ± 0.008 µmol/L/107 cells v
0.097 ± 0.007µmol/L/107 cells when
grown for 24 hours in 106 mol/L
As2O3). This suggests that the intracellular
As2O3 content of resistant cells was
unaffected, in contrast to, for instance, As2O3-resistant bacteria, which actively
exclude arsenicals.33
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| Fig 1.
Effect of As2O3 on NB4 and
NB4-AsR cell growth. (A) Exponentially growing cells were
seeded at 105 cells/mL and incubated for 4 days without
As2O3 (NB4 ( ), NB4-AsR cells
( ), or with 106 mol/L As2O3
(NB4-AsR ( ), NB4 ( ). Each value represents the mean ± standard deviation (SD) of three independent
determinations. (B) Apoptotic effect of As2O3
on NB4 or NB4-AsR cells. NB4 () and NB4-AsR
() were treated for 5 days with the concentrations indicated in the
plot. The percentage of apoptosis (%) was determined by a TUNEL
assay.
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| Fig 7.
(A) Effect of As2O3
and/or RA on NB4 and NB4-AsR cell growth. NB4 and
NB4-AsR were seeded at 105 cells/mL and
incubated with 107 mol/L As2O3
( ), with 106 mol/L RA (), with the combination of
106 mol/L RA and 107 mol/L
As2O3 ( ) or without
As2O3 (). A representative experiment among
three separate assays is shown. Each value represents the mean ± SD
of three determinations. (B) Effect of As2O3
and/or RA on NB4 or NB4-AsR cell apoptosis. NB4 and
NB4-AsR were treated for 3 days as indicated in the
pictures. The percentage of apoptosis (%) was determined by a TUNEL
assay (abscissa) and PI staining (ordinate), and analyzed by flow
cytometry.
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| Fig 2.
Morphologic features of NB4 and NB4-AsR cell
lines on RA, As2O3 or the combined treatment.
NB4 and NB4-AsR cells were treated for 5 days,
respectively, without (A), with 106 mol/L RA (B),
106 mol/L As2O3 (C) or with the
combination of 106 mol/L RA plus 106
mol/L As2O3 (D).
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| Fig 8.
Western blot analysis of Bcl-2, STAT1, p21
expressions, and IFN production in NB4 and NB4-AsR cells
(A) NB4-AsR, maintained in 107 mol/L
As2O3, and NB4 cells were treated with medium
alone or 106 mol/L RA. The time-course of synthesis of
STAT1 protein is depicted. (B) Time-course of IFN secretion. NB4
cells were treated with medium alone () or 106 mol/L
RA (). NB4-AsR cells were treated with
107 mol/L As2O3 (completely
coinciding with ) or 107 mol/L
As2O3 plus 106 mol/L RA ().
(C and D) NB4 or NB4-AsR cells were grown for 36 hours with
the different treatments indicated in the figure.
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Morphologic analysis of May-Grünwald-Giemsa stained cells showed
that untreated NB4-AsR cells were identical to their
parental cells. However, when grown for 3 days in
106 mol/L As2O3-supplemented
medium, the resistant cells became significantly larger than untreated
NB4 cells. Moreover, a higher fraction of cells presented an excentric
nucleus. The nucleus/cytoplasm ratio, azurophic granules, and basophily
were reduced, and in the cytoplasm, neutrophilic granules and vacuoles
were increased (Fig 2). Nevertheless, despite these changes, the resistant cells maintained immature features, as previously noted in NB4 cells treated with low doses of
As2O3 (107
mol/L).23 An increased rate of spontaneous adherence to
plastic was also noted (data not shown). To gain further insight into the phenotypic changes of the NB4-AsR cell line, surface
markers specific for granulocytic or monocytic differentiation were
analyzed (Table 1). NB4 cells have a high expression of CD33, absence of CD14 (a marker of the monocytic lineage), and a low expression of CD11b (a marker of the granulocytic lineage, like CD11c). When NB4 cells were grown 3 days in
108 mol/L or 107 mol/L
As2O3, CD33 expression was weakly
downregulated, while CD11b, CD11c, and CD14 expression
increased.23 NB4-AsR cells grown without
As2O3 have a more differentiated
immunophenotype than the parental cell lines. These cells grown in
108 mol/L or 107 mol/L
As2O3 showed a sharp increase in CD11b and
CD11c expression, while CD33 and CD14 expression remained almost
unchanged (Table 1). Because maturation along both the granulocytic and
monocytic lineages is associated with the ability of myeloid cells to
produce superoxide, NB4-AsR cells were examined for this
ability with a NBT reduction assay. An increase of superoxide
production was found in the As2O3-treated resistant or parental cells, which was much weaker than the one induced
by RA (Fig 3). LAP (a marker of terminal
granulocytic differentiation) was absent from NB4 and
NB4-AsR grown in the absence of
As2O3 and only modestly induced by
RA.34 However, a sharp induction was caused by arsenic
treatment at 3 days in both cell lines (Fig 3). Importantly,
NB4-AsR cells respond to RA like the parental clone in
their surface antigen expression, NBT reduction, LAP activity, and
morphologic differentiation, showing that resistance to
As2O3-triggered apoptosis did not impair RA
response. In conclusion, NB4-AsR are apoptosis-resistant
cells that remain sentitive to As2O3- or
RA-triggered differentiation.
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| Fig 3.
(A) Effects of As2O3, RA, or
their combination on the NBT-reducing activity of NB4 and
NB4-AsR cells. Aliquots of 5 × 105 cells were
treated for 3 days as indicated under the plot. (B) Effects of
As2O3, RA, or the combination of the two
compounds on LAP enzymatic activity. Aliquots of 106 cells
were treated with RA, As2O3, or the combination
of 106 mol/L RA plus As2O3 for 3 days. Each value represents the mean ± SD of three independent
measurements.
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| Fig 4.
Western blot analysis of PML/RAR and RAR expression
in NB4 and NB4-AsR cells. (A) NB4 cells were treated for 1 day with medium alone or 106 mol/L RA.
NB4-AsR cells were grown continuously in 107
mol/L As2O3, then washed with medium and
treated for 1 day with 107 mol/L
As2O3, 106 mol/L RA plus
107 mol/L As2O3 or grown for 2 weeks without As2O3 and treated for 1 day with
medium alone or 106 mol/L RA. PML/RAR indicates a
cleavage product of PML/RAR. (B) NB4 cells were treated for 1 day
with medium alone or 106 mol/L
As2O3. NB4-AsR cells were treated
for 1 week with medium alone, As2O3 at
106 mol/L, 107 mol/L, 108
mol/L. CBB waived gels are shown below to ensure equal protein loading.
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NB4-AsR-resistant cells degrade PML/RAR on
As2O3 exposure.
Western blot analysis with anti-RAR antibodies was performed on NB4
cells treated for 1 day with As2O3
(Fig 4B) or RA (Fig 4A).
As2O3 caused a near total degradation of
PML/RAR,24 while 106 mol/L RA led to
the degradation of PML/RAR and the generation of a cleavage
intermediate,  PML/RAR (Fig 4A). Essentially identical results
were found for NB4-AsR cells, showing that
As2O3-triggered degradation of the fusion protein is not sufficient to trigger apoptosis (Fig 4A). RA also induced the degradation of RAR proteins after an overnight treatment in both parental and arsenic-resistant cells (Fig 4A). Interestingly, NB4-AsR cells exposed for a week to different
concentrations of As2O3 presented a
dose-dependent RAR degradation (Fig 4B). Arsenic-triggered apoptosis
precluded analysis of long-term effects of
As2O3 on RAR protein expression in parental
NB4 cells. In both cell lines, PML/RAR and RAR transcripts
(assessed by Northern blot analysis) were almost unchanged on 1 day
treatment with As2O3 or RA, showing that the
decrease in the corresponding proteins was due to protein catabolism
(data not shown).
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| Fig 5.
Confocal laser microscopy analysis of NB4 and
NB4-AsR cells double-labeled with anti-PML antibodies
(revealed with a Texas Red-labeled conjugate) and anti-RAR
antibodies (revealed by a FITC-labeled conjugate). (A) NB4; (B)
NB4-AsR; (C) NB4-AsR treated for 1 day with
106 mol/L RA; or (D) NB4-AsR treated for 1 day with 106 mol/L As2O3.
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NB4-AsR cells grown 24 hours or more in
As2O3, lacked the microspeckled RAR
staining, but presented a strong PML staining on nuclear speckles (NBs)
(Fig 5D). Contrary to RA-treated NB4 cells, in which approximatively 10 NBs were found in each nucleus, NB4-AsR cells maintained in As2O3,
had a lower number of NBs that appeared smaller than RA-induced NBs in
NB4 (data not shown) or in NB4-AsR cells (Fig 5C). This
suggests that there was a further degradation of PML and NBs
aggregation in As2O3-treated
NB4-AsR cells, consistent with our previous
work.24 NB4-AsR cells withdrawn from
As2O3 for a month recovered a normal synthesis of PML/RAR and RAR proteins (Fig 4A and B) and hence a
microspeckled PML and RAR staining like in NB4 cells (Fig 5A and B),
but rechallenge with arsenic led again to PML/RAR degradation and
relocalization of NBs. These observations suggest that
As2O3 resistance was not due to a genetic
defect affecting PML/RAR, PML, or RAR expression and could rather
be ascribed to other As2O3-induced events.
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| Fig 6.
Effects of FCS depletion on NB4 and NB4-AsR
apoptosis. NB4 (left graph) and NB4-AsR cells (right graph)
were grown in the absence () or in the presence () of
107 mol/L As2O3 for 3 days.
Medium was supplied with the concentrations of FCS indicated in the
plot. Apoptosis (%) was determined, after a TUNEL assay, by flow
cytometry. Each value represents the mean ± SD of three
independent measurements.
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Arsenic facilitates induction of apoptosis in NB4-AsR
cells.
The property of NB4-AsR cells to express or degrade
PML/RAR protein in the presence or in the absence of
As2O3 and the proposed links between PML/RAR
expression and apoptosis14,35 led us to study the role of
the fusion protein on NB4 cell survival. We submitted NB4 and
NB4-AsR cells to serum deprivation in the
presence or absence of 107 mol/L
As2O3 (which led to the complete degradation of
PML/RAR). Arsenic sensitized NB4 and NB4-AsR
cells to both cell death (measured by Trypan blue exclusion) and
apoptosis (assessed by TUNEL) triggered by serum deprivation (Fig 6). Although we cannot rule out a
direct action of arsenic on apoptotic pathways, these results are
consistent with the idea that PML/RAR degradation favors apoptosis
triggered by serum deprivation.
RA and arsenic synergize to induce differentiation and apoptosis.
The effects of As2O3, RA, or both agents on the
differentiation and apoptosis of NB4 cells were examined. When parental
or arsenic-resistant NB4 cells were treated with 107
mol/L As2O3 and 106 mol/L RA
in combination, a sharp decrease in cell growth was observed (Fig 7A)
and these drugs synergized for induction of apoptosis (assessed by
TUNEL and PI labeling) (Figs 2 and 7B). Such massive induction of
apoptosis makes morphologic assessment of differentiation difficult
(Fig 2); nevertheless, immunophenotypic analysis showed a weak synergy
between As2O3 and RA for CD33 and CD14
expressions (Table 1). Under similar conditions, at both 3 (not shown)
and 5 days (Fig 2), dual treated NB4-AsR cells presented
highly differentiated features (an increased number of cells presented
lobed or multiple nuclei) and more neutrophilic granules and vacuoles
than RA-treated cells, as well as a smaller cell size and a less
basophilic cytoplasm (Fig 2). Increased plastic adhesion was also noted
(data not shown). The NBT reduction assay, LAP activity, and surface
marker analysis support the idea of enhanced differentiation with the
combination of RA and As2O3. Therefore, these
agents appear to synergize for the induction of apoptosis
and/or differentiation, primarily in arsenic-resistant, but
also in NB4 cells.
The expression of several key genes involved in differentiative
and/or apoptotic pathways and known to be directly or
indirectly responsive to RA, was then studied. Bcl-2 protein was
expressed at the same levels in parental or resistant NB4 cells. In NB4 cells, the protein was downregulated by 106 mol/L
As2O3 or 106 mol/L RA
(see Fig 9).21,36 Arsenic did
not modulate this protein in NB4-AsR cells, while RA
treatment did (data not shown). However, increasing the
As2O3 concentration in the presence of RA did
lead to a further decrease in bcl-2 protein
(Fig 8C), which was not due to cell death,
as STAT1 expression sharply increased (see below). RA induces the
STAT1 protein and also stimulates IFN secretion.31,37 In NB4 or in NB4-AsR, 107 mol/L
As2O3 did not modify STAT1 protein levels
(data not shown), while 106 mol/L RA induced
expression of this protein (Fig 8A and not shown). RA and
As2O3 combination sharply shortened the
response time in NB4-AsR cells (2 days) compared with NB4
cells (4 days) for both STAT1 synthesis (Fig 8A) and IFN
secretion (Fig 8B). p21WAF1/CIP1 is a primary target gene
of both RA38 and STAT1.39 p21 was strongly
upregulated by either As2O3 or RA in NB4 cells
(Fig 8D). NB4-AsR cells have an elevated baseline of p21
expression and 106 mol/L
As2O3 induced a moderate increase of p21, while
RA alone or with As2O3 induced high levels of
p21 expression. Taken together, these findings show that RA and
As2O3 strongly cooperate in their activation of
effectors of differentiation, growth control, and apoptosis.
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| Fig 9.
(A) Effect of As2O3
and/or RA on NB4, NB4-R1, and NB4-R2 cell apoptosis. NB4,
NB4-R1, and NB4-R2 were treated for 3 days with 106
mol/L As2O3 or 106 mol/L RA, as
indicated. Apoptosis was determined, after a TUNEL assay and
PI-staining, by flow cytometry, and the % of apoptotic cells is
reported in the plots. The abscissa of the plot represents TUNEL
(apoptosis) and the ordinate represents PI (DNA quantity). (B) Western
blot analysis of Bcl-2 in NB4 and NB4-R1 cells. Cells were grown for 48 hours in the presence of the indicated treatments. (C) Effect of
As2O3 and/or RA on PML/RAR and
RAR expression in NB4-R1 cells. The cells were treated for 2 days
with either medium, 106 mol/L RA, 107
mol/L As2O3, or the combination of
106 mol/L RA plus 107 mol/L
As2O3. The two right lanes represent sequential
treatments: 106 mol/L RA for 1 day followed by
107 mol/L As2O3 for the next day
or 107 mol/L As2O3 for 1 day
followed by 106 mol/L RA for the next day
(far right lane).
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RA-resistant cell lines are sensitive to
As2O3 apoptotic effects.
NB4-R1 and NB4-R2 are two stable RA-resistant cell lines.27
The As2O3-induced apoptosis in NB4-R1 and
NB4-R2 cell lines (with or without RA) appeared faster compared with
parental cells. The whole cell population died after a 4-day incubation
with As2O3, while at the same time, only 50%
of the parental NB4 cells were apoptotic (Fig 9A). This observation is
consistent with the clinical finding that RA-resistant patients are
As2O3-sensitive and that another RA-resistant
subclone is As2O3 sensitive.23,40
The NB4-R1 and NB4-R2 apoptosis pattern was reproducibly different from
parental NB4 cells and may present two apoptotic populations. Surprisingly, RA and/or As2O3-treated
NB4-R1 cells presented high levels of Bcl-2 protein (Fig 9B and not
shown), again suggesting that Bcl-2 protein does not play a key role in
NB4 apoptosis.36 Western blot analysis confirmed that
untreated NB4-R1 cells expressed PML/RAR and RAR proteins (Fig
9C), while NB4-R2 no longer expressed the PML/RAR
protein.41 In NB4-R1, as in NB4 cells, a 2-day RA treatment
caused the degradation of PML/RAR, a partial degradation of RAR
proteins, and the stabilization of PML/RAR (Fig 9C). Moreover,
sequential treatment with As2O3 and RA showed
that the PML/RAR cleavage product, once generated by RA exposure,
was resistant to the degradative action of
As2O3 both in NB4 (not shown) and NB4-R1 cells
(Fig 9C). This suggests that an initial proteolytic step triggered by
RA renders PML/RAR insensitive to As2O3.
Overnight As2O3 treatment again caused the
complete degradation of PML/RAR, but not RAR, in NB4-R1 cells
(Fig 9C). By showing that As2O3 and RA induce
PML/RAR degradation in NB4 sublines resistant to their respective
actions, our data demonstrate that PML/RAR degradation may be
required, but is not sufficient for the induction of differentiation or
apoptosis.
| |
DISCUSSION |
In this report, an arsenic-resistant NB4 population,
NB4-AsR, was characterized and used to show that arsenic
and RA cooperate to induce differentiation and/or apoptosis.
Resistance of NB4-AsR cells to apoptosis was not due to a
decreased As2O3 uptake in presence of the drug
or to altered PML/RAR As2O3-induced
degradation. Rather, resistance to the apoptotic effects of
As2O3 is linked to alterations in some unknown
effectors. Evidence for this process comes from the failure of
As2O3 to regulate expression of bcl-2 or p21
proteins in As2O3-resistant NB4 cells. However,
NB4-AsR cells remain sensitive to some of the effects of
As2O3, such as induction of partial
differentiation or growth retardation and thus provide a model system
in which the two effects of As2O3 (differentiation and apoptosis) are dissociated. The
NB4-AsR cell line shows an As2O3
concentration-dependent degradation of PML/RAR and thus provides a
powerful means by which to study the effects of PML/RAR (such as NB
antigens dispersion8 and/or control of
apoptosis14,35) in the context of a myeloid cell. In NB4
cells, the degradation of PML/RAR would be expected to restore
myeloid maturation by relieving the differentiation block. We
previously argued that during As2O3 treatment,
differentiation may not be apparent because of the rapid onset of
apoptosis.24 Indeed, in NB4 cells grown in low
As2O3 concentrations, as well as in
NB4-AsR treated with high As2O3
concentrations, some differentiation is observed. Although we cannot
exclude that As2O3 might activate specific
genes involved in myeloid differentiation (such as LAP), it could be
suggested that PML/RAR degradation is responsible for the partial
myeloid differentiation. Consistent with this hypothesis, in NB4-R1
cells, RA-induced PML/RAR decrease is also associated with changes
in surface markers but not terminal maturation.27 Nevertheless, our data both in As2O3- and in
RA-resistant cells clearly show that induction of PML/RAR
degradation may be a first step, but is not sufficient for the full
response to these two agents.
Besides PML/RAR degradation, long-term, but not short-term,
As2O3 exposure induced a pronounced and
dose-dependent downregulation of RAR protein. We have previously
shown that As2O3 leads to modifications in
RAR phosphorylation, which could induce modifications in the
half-life of the RAR protein.24 In addition, a major, lactacystin (a proteasome-inhibitor)-sensitive RA-induced RAR degradation was consistently observed in all cell lines examined to
date (data not shown). Together with the recent observation that RA
also induces the catabolism of the PML/RAR20,42 and
PLZF/RAR43 fusion proteins, this observation strongly
suggests that RA induces the degradation of RAR-containing proteins.
The less pronounced downregulation of RAR proteins compared with
PML/RAR may be due to the concomitant RA induction of RAR
genes.2 Future studies should delineate the mechanisms
responsible for the catabolism of RAR induced by RA, as well as the
links between RAR degradation and the RA target gene activation.
When NB4-AsR cells are primed with
As2O3, RA induces apoptosis. This apoptosis
could reflect the terminal differentiation of the myeloid cells, as
strongly suggested from the sharp synergy between
As2O3 and RA for morphologic differentiation.
Alternatively, as some retinoids have been shown to induce apoptosis
independently from differentiation, RA may directly trigger apoptosis
in As2O3-primed cells.44-46
Reciprocally, arsenicals may induce apoptosis, and RA-mediated
PML/RAR catabolism (which sensitize the cells to cell death), could
favor As2O3-triggered apoptosis and thus
account for the RA/As2O3 synergy in NB4 cells.
Bcl-2 protein is downregulated during RA-induced NB4 differentiation,
while in NB4-R1 cells, RXR-specific agonists induce apoptosis despite
high levels of bcl-2 protein.36 Moreover, the protein
levels of bcl-2 are unchanged during
As2O3-induced apoptosis of NB4-R1 cells (Fig
9B) and an NB4-R1 cell-line that overexpresses bcl-2
protein47 presented the same kinetics of arsenic-induced
apoptosis as parental cells (data not shown). Altogether, these results
imply that Bcl-2 modulation per se is not significant for apoptosis of
NB4 cells. STAT1 signaling pathway could induce apoptosis through
the induction of interleukin converting enzyme (ICE)
genes48 or other unknown ways. However, although STAT1
induction precedes RA-triggered apoptosis, it is not upregulated during
arsenic-induced apoptosis, questioning its role in apoptosis. A
candidate gene, which could participate in both
As2O3 and RA-induced apoptosis, is p21, which
blocks the cell cycle at both the G1/S and G2/M transitions during the
apoptosis process after DNA damage49 and whose upregulation
by either As2O3 or RA always precedes induction
of apoptosis.
Several nonexclusive hypotheses can be put forward to account for the
arsenic/RA effects on differentiation. Either, arsenic alone leads to a
significant differentiation not dependent on RAR, which would lead
to additive effects between As2O3 and RA. This
can be a property of arsenic per se due, for example, to modifications in phosphorylation of transcription factors such as
AP150 or to DNA hypomethylation, which in turn, facilitates
the expression of some genes.51 In this respect, it is
worth noting that As2O3 sharply induced LAP
activity (Fig 3B). Arsenic could also control differentiation through
PML/RAR degradation and hence modulation of putative
PML/RAR-regulated genes involved in myeloid differentiation, which
are not controlled by RA.16 Alternatively,
As2O3 could affect the RA response, for
instance through changes in phosphorylation of RAR or other proteins
involved in RA transduction pathways, leading to cooperative effects.
Although arsenic did not alter the RA response in transient
transfection assays, our demonstration that long-term
As2O3 exposure leads to RAR degradation
could relate to its activation.24 Finally,
As2O3-induced PML/RAR degradation might be
expected to facilitate the RA response through the normal receptor by
liberating DNA binding sites and/or RAR cofactors. However,
that the STAT1 gene induction by RA was identical in NB4 cells
pretreated or not with 107 mol/L
As2O3 (not shown) does not favor this
hypothesis. Our results are compatible with all three possibilities:
there are additive effects on differentiation, facilitated responses
for STAT1, NBT reduction, and synergistic effects on LAP activity
and apoptosis. That As2O3- or RA-resistant
cells not only show no cross-resistance but, indeed, an enhanced
sensitivity to the other agent, has important implications for APL,
supporting the idea that the combination of RA and
As2O3 treatments will be of clinical benefit.
| |
FOOTNOTES |
Submitted October 3, 1997;
accepted January 20, 1998.
Supported by grants from the "Association pour la Recherche
sur le Cancer," "Ligue contre le Cancer" (Nationale,
Comité de Paris and Hauts-de-Seine), and "Fondation St
Louis," Paris, France. M.G. was supported by La "Ligue Contre le
Cancer" and partially supported by the "FIRC" (Fondazione
Italiana per la Ricerca sul Cancro, Milano, Italy).
Address reprint requests to Hugues de Thé, MD, PhD, CNRS, UPR
9051, Hôpital St Louis, Paris, 1 Avenue Vellefaux, 75475 Paris Cedex 10, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Prof. P. Chambon for the generous gifts of anti-RAR
antibodies. We thank E. Garattini and all members of the de Thé's laboratory for critical reading of the manuscript. We also thank M.T. Daniel for critical comments on "Giemsa stained"
NB4-AsR cells and M. Schmid for his help in the confocal
microscopy.
| |
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J. Hu, Y.-F. Liu, C.-F. Wu, F. Xu, Z.-X. Shen, Y.-M. Zhu, J.-M. Li, W. Tang, W.-L. Zhao, W. Wu, et al.
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T. Sternsdorf, E. Puccetti, K. Jensen, D. Hoelzer, H. Will, O. G. Ottmann, and M. Ruthardt
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Deconstructing a Disease: RAR{alpha}, Its Fusion Partners, and Their Roles in the Pathogenesis of Acute Promyelocytic Leukemia
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L. Mologni, I. Ponzanelli, F. Bresciani, G. Sardiello, D. Bergamaschi, M. Gianni, U. Reichert, A. Rambaldi, M. Terao, and E. Garattini
The Novel Synthetic Retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene Carboxylic Acid (CD437) Causes Apoptosis in Acute Promyelocytic Leukemia Cells Through Rapid Activation of Caspases
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R. E. Gallagher
Arsenic -- New Life for an Old Potion
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Retinoic acid (RA) and As2O3 treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature of the leukemogenic process induced by the PML-RARalpha and PLZF-RARalpha oncoproteins
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Abstract
Introduction
Abstract