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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4308-4316
Trivalent Antimonials Induce Degradation of the PML-RAR
Oncoprotein and Reorganization of the Promyelocytic Leukemia
Nuclear Bodies in Acute Promyelocytic Leukemia NB4 Cells
By
Stefan Müller,
Wilson H. Miller Jr, and
Anne Dejean
From the Unité de Recombinaison et Expression
Génétique, INSERM U 163, Institut Pasteur, Paris, France;
and the Lady Davis Institute for Medical Research, McGill University,
Montreal, Canada.
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ABSTRACT |
Acute promyelocytic leukemia (APL) is characterized by a specific
t(15;17) chromosomal translocation that fuses the genes encoding the
promyelocytic leukemia protein (PML) and the retinoic acid receptor 
(RAR). The resulting PML-RAR protein induces a block in the
differentiation of the myeloid progenitor cells, which can be released
by retinoic acid (RA) in vitro and in vivo. The RA-induced
differentiation of APL blasts is paralleled by the degradation of the
fusion protein and the relocation of wild-type PML from aberrant
nuclear structures to its normal localization in nuclear bodies.
Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce
complete remission in both RA-sensitive and -resistant APL patients.
Intriguingly, As2O3 was also shown to induce
degradation of the PML-RAR chimera and to reorganize PML nuclear
bodies. Here we show that trivalent antimonials also have striking
effects on RA-sensitive and RA-resistant APL cells. Treatment of the
APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony
trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the
antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of
antimony action is likely to be similar to that of
As2O3, notably both substances induce the
attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of
PML-RAR. From these data, we propose that, in analogy to
As2O3, antimonials might have a beneficial
therapeutic effect on APL patients, perhaps with less toxicity than
arsenic.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ACUTE PROMYELOCYTIC leukemia (APL)
represents about 10% of acute myeloid leukemia cases. APL is
characterized by a specific differentiation block of the myeloid
progenitor cells at the promyelocytic stage. At the molecular level, in
the vast majority of cases (>95%), APL blasts harbor the balanced
t(15;17) chromosomal translocation, which fuses the promyelocytic
leukemia protein (PML) gene located on chromosome 15 to
the retinoic acid receptor (RAR) gene on chromosome
17.1-5 The PML-RAR fusion protein retains most of the
functional domains of the parental PML and RAR proteins. The key
role of the chimera in the differentiation block has initially been
shown in in vitro models,6,7 and its oncogenic potential
has more recently been confirmed in transgenic mice.8-11 A
unique feature of APL blasts is their ability to undergo terminal
differentiation after retinoic acid (RA) treatment in vitro as well as
in vivo.12-14 Indeed, oral administration of
all-transRA, the natural ligand for RAR, induces complete
remission in t(15;17) APL patients, making APL the first example of
differentiation therapy in treatment of advanced cancer. Several
clinical studies have conclusively shown that the combination of RA
with chemotherapy has improved the survival of patients with
APL.15-17
The PML-RAR chimera may exert its dominant negative effect on
myelocyte differentiation by interfering with both RAR or PML
signaling pathways. Whereas the role of retinoids and their receptors
in cellular differentiation processes have at least been partially
elucidated, the biological function of PML is still poorly understood.
An important observation was the detection of its altered subcellular
localization in APL cells. In normal cells, PML has been shown to
localize to distinct subnuclear domains, the so-called PML nuclear
bodies (NBs), ND 10 or PODs,18-20 that are tightly
associated with the nuclear matrix. Whereas normal cells contain 10 to
30 NBs per nucleus, in APL cells, NBs are highly disorganized into
numerous and aberrant microstructures containing both PML and
PML-RAR. Strikingly, RA treatment induces a drastic reorganization
of the NBs back to their normal number and morphology, suggesting that
the delocalization of PML or other NB-associated proteins may play a
role in APL pathogenesis. More recent data have shown that RA-induced
restoration of PML bodies is paralleled by a selective degradation of
the PML-RAR fusion protein,21,22 suggesting that NB
restoration is a direct consequence of PML-RAR disappearance.
Despite the broad success of RA therapy in APL, a significant
percentage (20% to 30%) of patients relapse after initial remission and subsequently develop resistance to RA treatment. The clinical outcome of these patients is poor, as an effective substitute for RA is
not yet available. Recently, arsenic trioxide
(As2O3), a component of antileukemic drugs used
in Chinese traditional medicine, was proposed as an alternative therapy
in the treatment of APL.23-25 In vitro studies performed in
freshly isolated APL blasts and in the APL-derived NB4 cell line showed
that As2O3 exerts a dose-dependent dual effect
on APL cells. At concentrations of 1 µmol/L and above, it induced
preferentially apoptotic cell death, whereas at lower concentrations
partial differentiation of APL cells was observed.23,24
However, when given in vitro in combination with RA,
As2O3 inhibited differentiation, and RA inhibited As2O3-induced apoptosis in APL cell
lines and patient samples.26 Most intriguingly, similarly
to RA, As2O3 drastically changed the metabolic
stability of the fusion protein, triggering PML-RAR degradation
within 6 to 8 hours, and provoked the restoration of enlarged PML
NBs.24,26-28 Recently, we and others have shown that both
PML and PML-RAR are posttranslationally modified by covalent linkage
with the ubiquitin-like SUMO-1 protein,28-30 formerly named
PIC-1.31 The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO-1 modified forms are compartmentalized exclusively in the PML NBs, indicating that covalent
modification of PML with SUMO-1 is implicated in its targeting to these
structures.28 Strikingly, As2O3
treatment alters the profile of SUMO-1 modification by inducing the
attachment of multiple SUMO-1 molecules to both PML and PML-RAR.
Whereas the poly-SUMO-1-modified forms of wild-type PML are
metabolically stable and concentrate in NBs, poly-SUMO-1 modification
of PML-RAR is accompanied by a decrease in the metabolic stability
of the fusion protein by a yet unknown mechanism.
It has been shown that trivalent antimonials can interfere in a number
of biological processes in a similar manner to trivalent arsenics, a
phenomenon that has been explained by the chemical similarity of both
substances.32,33 Arsenic and antimony are elements from
group V of the periodic table, classified as metalloids. The potential
of their trivalent salts to interact with biochemical reactions has
been ascribed in particular to their ability to target vicinal thiol
groups in proteins with quite a high specificity.34 In this
study we report that trivalent antimonials, such as antimony trioxide
(Sb2O3) and potassium antimonyl tartrat (PAT),
can perfectly mimic the effects of As2O3 on APL
cells. Like arsenic, the antimonials are effective in triggering
PML-RAR degradation, NB reorganization, and conjugation with the
SUMO-1 modifier. In addition, they preferentially provoke apoptosis of
APL cells. Based on these observations we suggest that, in analogy to
As2O3, antimonials might have a beneficial effect in the treatment of APL patients.
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MATERIALS AND METHODS |
Cell culture and treatment of cells.
NB4 cells,35 NB4R4,36 and U937 cells were
cultured in RPMI medium (GIBCO-BRL, Gaithersburg, MD)
supplemented with antibiotics, glutamine, and 10% fetal calf serum.
The HeLa cell line stably overexpressing PML(F) was as previously
described.28 The cells were grown at 37°C in 5%
CO2 in Dulbecco's modified minimal essential medium
(GIBCO-BRL), supplemented with antibiotics, glutamine, 10% fetal calf
serum and with G418 (Geneticin, GIBCO-BRL; 750 µg/mL).
All-trans RA (Sigma Chemical Company, St Louis, MO) was prepared as
a 10 mmol/L stock solution in ethanol; As2O3
(Sigma) as a 1 mmol/L stock solution in PBS; PAT (Aldrich), and
meglumine antimonate (Glucantime; Rhône-Poulenc, Paris, France)
as 10 mmol/L stock solutions in H2O.
Sb2O3 (Fluka, Buchs, Switzerland)
and Bi2O3 (Sigma) were dissolved in
a minimal volume of HCl and a 100 µmol/L stock solution prepared in
100 mmol/L HEPES, pH 7.5.
Antibodies.
The polyclonal anti-PML antibody and the monoclonal anti-SUMO-1
antibody (21C7) were as described previously.19,37 The anti-human RAR rabbit polyclonal antibody (RPF-115)38
and the anti-F antibody Ab(F3)39 ascites fluid and
hybridoma culture supernatant were provided by M.-P. Gaub,
D. Metzger, and P. Chambon (IGBMC, Illkirch, France).
Preparation of cell extracts and Western blotting.
For Western blots, approximately 5 × 106 cells were
washed twice in phosphate-buffered saline (PBS), lysed in 250 µL
sodium dodecyl sulfate (SDS)-sample buffer, and boiled for 10 minutes. Proteins were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-C extra (Amersham, Arlington Heights, IL) membranes. Membranes were blocked in
5% nonfat dry milk in phosphate-buffered saline-0.05%
Tween (PBST) and incubated for 2 hours with the various antibodies
diluted in PBST. The anti-PML antibody was used at a dilution of
1:2,500, the anti-F Ab(F3) hybridoma cell culture supernatant at 1:200, and anti-RAR (RPF-115) at 1:500. After primary antibody
incubation, blots were extensively washed in PBST and incubated for 1 hour with the appropriate peroxidase coupled secondary antibodies
(Amersham). Enhanced chemiluminescence reagents (ECL; Amersham) were
used for detection.
Indirect immunofluorescence and confocal laser microscopy.
NB4 or NB4R4 cells were washed twice in PBS, 1 × 106
cells were resuspended in 100 µL PBS, and attached on poly-Lysine
(Sigma) treated coverslips. Cells were fixed in 3.7% paraformaldehyde (PFA) in PBS for 10 minutes at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 20 minutes at room temperature. After fixation and permeabilization, cells were rinsed twice in PBS and
once in PBST, incubated with primary antibodies for 1 hour, washed in
PBS and PBST, and further incubated with the appropriate secondary
antibodies conjugated with either fluorescein (Sigma) or Texas red
(Amersham). Primary antibodies were used at a dilution of 1:200 for
anti-PML and 1:500 for anti-SUMO-1, and secondary antibodies were used
at a dilution of 1:200. After three washes in PBS, the samples were
mounted in VectaShield (Vector Laboratories, Burlington, CA). Confocal
laser scanning microscopy was performed with an LEICA SM microscope
(Heidelberg, Germany) using excitation wavelengths of 488 nm (for
fluorescein) and 543 nm (for Texas red). The two channels were recorded
independently and pseudocolour images were generated and superimposed.
The acquired digital images were processed using Adobe Photoshop v.3.1
software (San Jose, CA).
Apoptosis assays.
Five-milliliter cultures of NB4, NB4R4, or U937 cells at a density of 2 × 105 cells/mL were incubated with 1 µmol/L
As2O3, 1 µmol/L
Sb2O3, or 1 µmol/L PAT. Aliquots of the cells
were removed after 24 hours and 48 hours, and the cells were bound to
poly-Lysine (Sigma) treated coverslips. Cells were fixed with 3.7% PFA
in PBS for 10 minutes at room temperature and then stained for 5 minutes with PBS containing 1 µg/mL Hoechst 33258 (Sigma). After
rinsing with PBS, morphological assessment of cells was made by
epifluorescence microscopy.
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RESULTS |
Trivalent antimonials trigger the degradation of PML-RAR in
RA-sensitive and -resistant APL cells.
The potential to induce degradation of the PML-RAR fusion protein
seems to be critical for the antileukemogenic activity of both RA and
As2O3. As antimonials share several chemical
properties with arsenics, we were tempted to determine the effect of
antimony trioxide (Sb2O3) on the
PML-RAR level in the APL-derived NB4 cells. To this aim, cellular
extracts were prepared from untreated NB4 cells and from cells treated
with either 1 µmol/L As2O3 or 1 µmol/L
Sb2O3 for 12 hours. For direct comparison, an
extract from NB4 cells treated for 36 hours with 1 µmol/L RA was
included in the experiment. An extract from cells treated with 1 µmol/L bismuth trioxide (Bi2O3), another
trivalent metalloid from group V of the periodic table, served as a
control to determine whether group V metals in general can act on
PML-RAR. Proteins from the extracts were separated by SDS-PAGE and
the PML-RAR protein levels in the samples were measured by
immunoblotting with an anti-RAR antibody (Fig
1, lanes 1 through 5). The wild-type RAR
receptor, migrating as a doublet-band at approximately 50 kD, was
detected in equal amounts in all extracts. In extracts from untreated
cells (lane 1), a major PML-RAR band migrating at 120 kD together
with two higher anti-RAR reactive bands at 140 kD and 160 kD, were visible. We have recently shown that these higher molecular weight species correspond to PML-RAR forms where one or two molecules of
the ubiquitin-like SUMO-1 protein are covalently linked to the PML
moiety of the fusion protein.28 As expected, incubation of
the cells with either RA or As2O3 leads to a
complete disappearance of all of these PML-RAR bands (lanes 2 and
3). The novel anti-RAR reactive band migrating at 80 kD observed
after RA treatment (lane 2) represents a PML-RAR cleavage product
described previously.19,21,27 Interestingly, treatment of
cells with 1 µmol/L Sb2O3 also induces an
almost complete degradation of PML-RAR (lane 4). In contrast, Bi2O3 treatment had no effect on the level of
the PML-RAR protein (lane 5), underlining the specificity of
As2O3 and Sb2O3. In
addition to Sb2O3, we tested two other antimony
compounds, the PAT and the pentavalent antimonial meglumine antimonate,
for their potential to induce PML-RAR downregulation in NB4 cells.
Whereas PAT was as effective as Sb2O3 in
triggering degradation of the fusion protein, the pentavalent antimony
salt had no effect on PML-RAR levels (data not shown).
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| Fig 1.
Trivalent antimonials trigger the degradation of
PML-RAR in RA-sensitive and -resistant APL cells. Cellular extracts
from NB4 cells (lanes 1 through 5) and from the RA-resistant NB4R4
cells (lanes 6 through 10) were prepared in SDS sample buffer. Cells
were untreated (lanes 1 and 6), treated with either 1 µmol/L RA for
36 hours (lanes 2 and 7) or 1 µmol/L As2O3
(lane 3 and 8), or 1 µmol/L Sb2O3 (lanes 4 and 9) or 1 µmol/L Bi2O3 (lanes 5 and 10) for
12 hours. Proteins were separated on a 7.5% SDS-PAGE gel, transferred
to a nitrocellulose membrane, and probed with an anti-RAR polyclonal
antibody. The 120-kD PML-RAR and the 50-kD RAR proteins are
indicated by arrowheads. The SUMO-1-PML-RAR conjugates are
indicated by open triangles and the 80-kD PML-RAR cleavage product
observed after RA treatment is indicated by an asteriks.
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To see whether Sb2O3 can similarly induce
PML-RAR degradation in RA-resistant APL cells, we used the
RA-resistant NB4R4 cell line as a model system. In this cell line, the
PML-RAR chimera harbors a point mutation in the ligand-binding
domain of the RAR moiety, preventing ligand binding and RA-induced
differentiation.36,40 NB4R4 cells were cultured as
described above in the presence of the different chemicals, and their
respective effect on PML-RAR levels was determined by Western
blotting (Fig 1, lanes 6 through 10). In untreated cells, PML-RAR is
seen as the typical triplet of bands corresponding to both unmodified
and SUMO-1-modified chimera species (lane 6). As reported
before,21 RA is not able to trigger PML-RAR degradation
in these cells (lane 7). In contrast, both
As2O3 (lane 8) and
Sb2O3 (lane 9) treatment, but not incubation with Bi2O3 (lane 10), drastically reduce the
amount of PML-RAR to hardly detectable levels.
Taken together these data show that similarly to RA and
As2O3, trivalent antimonials induce a targeted
degradation of the PML-RAR oncoprotein. Like arsenic, the potential
of antimonials to degrade PML-RAR is not abrogated in an
RA-resistant APL cell line, indicating that both compounds act by a
mechanism that is distinct from that of retinoids.
Trivalent antimonials induce poly-SUMO-1 modification of wild-type
PML.
We have recently shown that As2O3 targets the
PML moiety of PML-RAR by inducing the attachment of multiple SUMO-1
molecules to PML.28 This posttranslational modification is
most easily detected on Western blots from PML-overexpressing cells, as
the As2O3-induced formation of
poly-SUMO-1-PML conjugates leads to a dramatic change in the
electrophoretic mobility of these PML forms in SDS-PAGE. To examine
whether antimonials have any effect on SUMO-1 modification of PML, we
incubated cells from a HeLa cell line overexpressing PML, tagged with
the F region of the human estrogen receptor, with either 1 µmol/L
Sb2O3, PAT, or meglumine antimonate for 6 hours. Extracts from untreated cells and from cells treated with RA or
Bi2O3 were used as controls. The PML and the
PML-SUMO-1 conjugates were detected by immunoblotting with a
monoclonal antibody directed against the F tag (Fig
2). In untreated cells (lane 1) as well as
in RA-treated cells (lane 2), the antibody detects a major PML(F) form
showing an apparent molecular mass of 100 kD. In addition, four higher
molecular weight PML species are seen migrating between 20 and 60 kD
above this major form. These bands correspond to SUMO-1-PML
conjugates, where one or more SUMO-1 molecules are covalently attached
to PML. As reported previously, As2O3 induces
the conversion of these oligo-SUMO-1-modified PML forms toward
poly-SUMO-1-modified species, migrating from 160 kD toward the top of
the gel (lane 3). Strikingly, the same phenomenon was observed in
cellular extracts from cells that had been treated with either
Sb2O3 (lane 4) or PAT (lane 5). In contrast, incubation of cells with either the pentavalent meglumine antimonate (lane 6) or Bi2O3 (lane 7) did not alter the
profile of SUMO-1-modified PML forms.
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| Fig 2.
Trivalent antimonials induce poly-SUMO-1 modification of
wild-type PML. Cellular extracts from HeLa cells stably overexpressing
PML(F) were prepared in SDS sample buffer. Cells were either untreated
(lane 1) or treated with 1 µmol/L RA (lane 2), 1 µmol/L
As2O3 (lane 3), 1 µmol/L
Sb2O3 (lane 4), 1 µmol/L PAT (lane 5), 1 µmol/L meglumine antimonate (lane 6), or 1 µmol/L
Bi2O3 (lane 7). Proteins were run on a 7.5%
SDS-PAGE gel, transferred to a nitrocellulose membrane, and the blot
was immunostained with a monoclonal antibody directed against the F
tag. The unmodified 100-kD PML form is indicated by an open triangle.
The oligo-SUMO-1-modified PML forms migrating between 120 and 160 kD
are indicated by arrowheads and the high molecular
poly-SUMO-1-modified PML species forming a smear toward the top of
the gel by a square bracket. The ~68-kD band represents a protein
cross-reacting with the anti-F antibody that has been described
previously.39
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In summary, these result indicate that, like
As2O3, trivalent antimonials act specifically
on the PML moiety of the PML-RAR fusion protein by inducing
the formation of poly-SUMO-1- modified PML conjugates.
Trivalent antimonials trigger the reorganization of the PML NBs in
APL cells.
The observation that both RA and As2O3 are able
to redirect wild-type PML from its aberrant subnuclear distribution in
APL cells to its normal localization led us to examine PML localization after treatment with antimony. With regard to the apparent role of the
modification by SUMO-1 in targeting PML to the NBs, the intracellular
distribution of SUMO-1 was determined in parallel. Indirect
immunofluorescence microscopy was done on untreated NB4 cells, as well
as on cells treated for 36 hours with RA or for 10 hours with either
As2O3, Sb2O3,
Bi2O3, PAT, or meglumine antimonate. Double
labeling was performed with a polyclonal anti-PML antibody and an
anti-SUMO-1 monoclonal antibody, and the localization of both proteins
was analyzed by confocal laser microscopy
(Fig 3 and data not shown).
In untreated NB4 cells, PML shows the microspeckled appearance typical
for APL cells (Fig 3A). SUMO-1 staining reveals an intense nuclear
diffuse signal and, in addition, some concentration in nuclear punctate
structures (Fig 3B). Superposition of PML and SUMO-1 stainings shows
the colocalization of both proteins in the APL-specific microspeckles,
which can be seen more easily in the largest of these structures (Fig
3C). After RA treatment, PML is predominantly found in the restored PML
NBs, although a nonnegligible fraction remains diffusely distributed in
the nucleoplasm (Fig 3D). SUMO-1 undergoes a similar relocalization
process and, in addition to its nuclear diffuse localization, the
protein can now be clearly detected in distinct subnuclear aggregates
(Fig 3E), where it colocalizes with PML (Fig 3F). Incubation of NB4 cells with As2O3 induces a characteristic
redistribution of PML (Fig 3G) together with SUMO-1 (Fig 3H) from the
microspeckled structures to intact NBs, where both proteins colocalize
(Fig 3I). However, in contrast to RA, As2O3
also induces the concentration of the nuclear diffuse PML and SUMO-1
fractions into the NBs, which, in consequence, are significantly larger
than the bodies reformed in RA-treated cells. Treatment of NB4 cells
with Sb2O3 similarly leads to a drastic
reorganization of the immunofluorescence patterns. Both PML and SUMO-1
are corecruited onto dramatically enlarged NBs, whereas the nuclear
diffuse SUMO-1 staining is greatly diminished (Fig 3, J through L).
Treatment of cells with Bi2O3 had no
effect on PML or SUMO-1 localization, when compared with untreated controls (Fig 3, compare M through O with A through C). Among the two other antimonials tested, the trivalent PAT was as
effective as Sb2O3 in triggering NB
reorganization, whereas meglumine antimonate could neither alter PML
nor SUMO-1 localization (data not shown). In the RA-resistant NB4
subclone NB4R4, arsenic and the trivalent antimonials, but not RA,
provoked the reorganization of the NBs accompanied by recruitment of
SUMO-1 onto these structures (data not shown). After exposure of both
NB4 and NB4R4 cells for 24 to 36 hours to arsenic or 48 hours to
antimonials, the immunofluorescence staining of PML and SUMO-1 became
more diffuse and the NBs were hardly detectable. In cells that show
morphological signs of apoptosis such as micronucleation and chromosome
condensation (see below), a complete loss of PML and SUMO-1 staining
was seen (not shown).
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| Fig 3.
Trivalent antimonials trigger the
reorganization of PML NBs in APL Cells. NB4 cells were subjected to
double-immunofluorescence staining with a polyclonal anti-PML antibody
and a monoclonal anti-SUMO-1 antibody. Labeling was performed on
untreated cells (A through C), cells treated for 36 hours with 1 µmol/L RA (D through F), or cells treated for 10 hours with either 1 µmol/L As2O3 (G through I), 1 µmol/L
Sb2O3 (J through L), or 1 µmol/L
Bi2O3 (M through O). The staining pattern was
analyzed by confocal laser microscopy. The red signal (PML) is obtained
with an anti-rabbit Ig Texas red-conjugated secondary antibody, the
green signal (SUMO-1) with an anti-mouse Ig fluorescein-conjugated
secondary antibody. Superimposing of the two colors (merge) results in
a yellow signal, where both proteins colocalize.
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The data from immunofluorescence show that trivalent antimonials can
efficiently trigger PML NB reorganization. In contrast to the NB
restoration provoked by RA, the arsenic/antimony-induced reorganization
of NBs is also accompanied by an important swelling of the structures,
followed by a loss of PML staining.
Trivalent antimonials induce apoptosis of retinoid-sensitive and
-resistant APL cells.
It has been suggested that the therapeutic effect of
As2O3 might, at least partially, be due to its
potential to induce apoptosis in APL cells.23-26 To
determine whether antimonials have an apoptotic effect on APL cells,
NB4 and NB4R4 cells were cultured in the presence of 1 µmol/L
As2O3, 1 µmol/L
Sb2O3, or 1 µmol/L PAT. After 24 hours and 48 hours, cells were stained with the DNA colorant Hoechst 33258 to detect
apoptotic nuclei. Hoechst-stained NB4 cells after incubation for 24 hours with the different substances are shown in
Fig 4. In cultures of untreated NB4 cells,
apoptotic cells are rarely detected (Fig 4A). In contrast, as shown
previously,23,24,26 the number of cells showing apoptotic
features, such as chromatin condensation and formation of micronuclei,
has strongly increased after arsenic treatment (Fig 4B). Similarly,
after incubation of the cells with Sb2O3 (Fig
4C) and PAT (Fig 4D) the percentage of cells with morpohological signs
of apoptosis is significantly augmented. Similar results were obtained
in the RA-resistant NB4R4 cells (data not shown). A detailed,
quantitative comparison of the apoptotic potential of
As2O3 and antimonials in the NB4 and NB4R4
cells is shown in Fig 5. In the absence of
any treatment, a low percentage of cells (1% to 3%) show
morphological signs of apoptosis. After incubation with
As2O3, the percentage of apoptotic cells in cultures of NB4 and NB4R4 cells is about 18% to 20% after 24 hours and 31% to 35% after 48 hours. As can be seen, the apoptotic potential of Sb2O3 and PAT is very similar,
although slightly weaker than that of As2O3.
After incubation of NB4 or NB4R4 cells for 24 hours with these two
compounds, 11% to 14% of the cells can be considered as apoptotic
and, 48 hours posttreatment, the percentage of apoptotic cells is
increased up to 19% to 25%. In contrast to NB4 cells, U937 cells, a
non-APL myeloid leukemia cell line, showed only slightly elevated
levels of apoptotic cells following treatment with either
As2O3 or antimonials.
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| Fig 4.
Trivalent antimonials induce apoptosis of APL cells.
Untreated NB4 cells (A) or cells treated for 24 hours with either 1 µmol/L As2O3 (B), 1 µmol/L
Sb2O3 (C), or 1 µmol/L PAT (D) were bound to
poly-Lysine treated coverslips, fixed and stained with Hoechst 33258. Apoptotic cells show chromatin condensation and micronuclei.
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| Fig 5.
Comparison of the apoptotic potential of
As2O3 and antimonials on RA-sensitive and
-resistant APL cells and U937 cells. NB4, NB4R4, or U937 cells were
incubated for 24 or 48 hours with 1 µmol/L
As2O3, 1 µmol/L
Sb2O3, or 1 µmol/L PAT, and the percentage of
apoptotic cells was determined by Hoechst 33258 staining. Untreated
cells were used as control.
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Taken together these data show that trivalent antimonials at a
concentration of 1 µmol/L can efficiently and specifically induce
apoptotic cell death in RA-sensitive and RA-resistant APL cells.
Comparison with the apoptotic potential of arsenic shows that
antimonials are only slightly less effective than arsenic in inducing
apoptotic cell death of NB4 and NB4R4 cells.
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DISCUSSION |
In this report we show that, in vitro, trivalent antimonials have
profound effects on APL cells, which are similar to the effects of the
antileukemic agents RA and As2O3. The most
striking observation is that all three substances can selectively
induce degradation of the PML-RAR oncogene and trigger
reorganization of the PML NBs. Our results indicate that the mechanisms
of both antimony and arsenic action may be identical, as both target
specifically the PML moiety of the PML-RAR fusion protein by
altering its posttranslational modification profile by SUMO-1. The
potential of antimonials to degrade PML-RAR and to reorganize PML
NBs is retained in RA-resistant NB4 cells indicating that, as for
As2O3, there is no cross-resistance between RA
and antimony. As has been reported for As2O3,
trivalent antimonials induce apoptotic cell death in RA-sensitive and
RA-resistant NB4 cells. Thus, in analogy to
As2O3, trivalent antimonials might provide a
therapeutic alternative to circumvent RA resistance in APL patients.
A critical feature of the antileukemic activity of both RA and
As2O3 seems to be their potential to degrade
the PML-RAR oncogene. The most current hypothesis to explain the
mechanism of RA-induced degradation is that the conformational change
upon ligand binding to the RAR moiety results in the formation of a
specific cleavage product. Our recent data28 and the data
reported here show that As2O3 and antimony act
on the PML moiety of PML-RAR by inducing the attachment of multiple
SUMO-1 molecules to PML. Although in the case of the native PML
protein, SUMO-1 modification is most likely involved in subcellular
partitioning rather than in protein degradation processes, it is
possible that in the context of the fusion protein, PML-RAR
undergoes a conformational change that allows its recognition by the
cellular degradation machinery. The exact molecular mechanism for the
induction of poly-SUMO-1 modification of PML by arsenic and antimony
is not known. Although the process of SUMO-1 modification resembles
ubiquitination, there is increasing evidence that the enzymes
implicated in SUMO-1 modification of target proteins are distinct from
the enzymes used in classical ubiquitination.41,42 We
suggest that As2O3 and antimonials can
specifically interact with an enzyme involved in the SUMO-1 modification or demodification process of PML and PML-RAR. The potential to trigger poly-SUMO-1 modification is restricted to trivalent arsenicals and antimonials indicating that binding to vicinal
thiol groups is implicated in arsenic/antimony action. Consistent with
this hypothesis, pentavalent antimonials and trivalent bismuth salts,
which both cannot target vicinal thiols, are not able to induce SUMO-1
attachment to PML. Identification and characterization of the enzymes
involved in posttranslational modification by SUMO-1 should help to
understand the exact molecular mechanism underlying arsenic/antimony
action.
The RA-induced degradation of PML-RAR is paralleled by the
restoration of PML NBs, suggesting that NB reorganization may just only
be a simple consequence of PML-RAR dissappearance. After arsenic and
antimony treatment of NB4 cells, NBs are not only restored but are also
dramatically enlarged compared with the normal NBs in non-APL cells.
This reorganization may result from two distinct phenomena: (1) the
loss of PML-RAR and (2) the poly-SUMO-1 modification of the
remaining wild-type PML followed by the recruitment of these
poly-SUMO-1-PML conjugates to the NBs.
Initial in vitro studies with As2O3 on APL
cells indicate that its therapeutic effect is not related to cell
differentiation but is likely due to its ability to provoke apoptotic
cell death of APL blasts. In line with this data, we found that
trivalent antimonials at micromolar concentrations induce apotosis of
RA-sensitive and -resistant APL cells. Recent clinical data from
As2O3-treated APL patients showed that, in
vivo, As2O3 may have a significant cyto-differentiating effect, as partially differentiated granulocytes are detected in the bone marrow of APL patients after
As2O3 treatment.25 Consistent with
this observation, it has now been shown that, at subapoptotic
concentrations, As2O3 induces a partial
differentiation of APL cells in vitro.24 In analogy, we
detected an increased expression of myeloid and monocytic cellular
differentiation markers (CD11b and CD11c) in a small percentage of
cells when NB4 or NB4R4 cells were exposed to sub-apoptotic antimony
concentrations (<1 µmol/L) for 3 to 4 days (S.M. and A.D.,
unpublished results, February 1998). However, as with
arsenic, the percentage of cells expressing differentiation markers
remains low and terminal differentiation of cells is never achieved
with arsenic or antimonials. These data indicate that, like arsenic,
antimonials have a dual effect on APL cells, provoking preferentially
cell death at relatively high concentration and partial differentiation
at lower concentrations. This is in contrast to the effect of RA on APL
cells, where a complete terminal differentiation is achieved both in
vitro and in vivo. It is not known whether RA and antimony will
antagonize each other's effects when given in combination, as has been
reported for RA and arsenic.26
Taken together our data show that the biological effects of trivalent
antimonials on NB4 and NB4R4 cells are almost identical to the effects
of As2O3. Based on these observations, we
propose that trivalent antimonials might also be effective in the
treatment of APL patients. This may be of notable clinical relevance,
as an effective therapy for RA-resistant APL cases has not yet been established. Recent case reports from China show that
As2O3 treatment induces clinical remissions in
RA-sensitive and RA-resistant APL patients, and clinical trials in
Western countries to evaluate the pharmacology and toxicology of
As2O3, as well as its clinical activity, have
recently started. In contrast to As2O3, the
pharmacological characteristics of trivalent antimonials, especially
PAT, are already relatively well studied, as they have been used as
antiparasitics in the treatment of Leishmaniosis and Schistosomiasis
since the beginning of our century. In the treatment of Leishmaniosis,
the trivalent antimony salts have been replaced quite early by the less
toxic pentavalent antimonials, but in antischistosomal therapy PAT and
other trivalent antimonials were used until the late 1960s. The major
side effect observed in antischistosomal therapy with trivalent
antimonials was their cardiotoxicity, which, in particular at elevated
dose, led to severe complications.43 Based on our in vitro
data, we estimate that, for efficient in vivo treatment of APL, serum
antimony concentrations in the low micromolar range (~1 µmol/L)
should be sufficient. At that dose PAT is relatively well tolerated,
permitting treatment of schistosomiasis patients at moderate
toxicity.44,45 Thus, effective treatment of APL patients
with antimonials might be accomplished without provoking the side
effects known from antischistosomal therapy or from treatment with
arsenic.
In conclusion, we have shown that trivalent antimonials have biological
effects on APL cells that closely resemble the effects of
As2O3. It may thus be estimated that, in
analogy to As2O3, trivalent antimonials might
have an antileukemic potential in vivo. This could be rapidly tested in
the recently described PML-RAR transgenic mice,8-10
which provide a useful model for human APL. As trivalent antimonials
have widely been used in the past in the treatment of parasitic
diseases, their pharmacological characteristics are relatively well
studied. It is thus conceivable that antimonials might be included in
clinical trials in the near future to compare their therapeutic
potential and toxicity to that of arsenic.
| |
ACKNOWLEDGMENT |
We are indebted to Marie-Pierre Gaub, Daniel Metzger, Pierre Chambon,
Michael J. Matunis, and Günter Blobel for the generous gift of
antibodies used in these experiments. We are grateful to Emmanuelle
Perret for excellent help with confocal microscopy and Marie-Christine
Wagner for kind help with flow cytometry. We thank Pierre Tiollais for
support and all members of our group for stimulating discussions and
for providing reagents.
| |
FOOTNOTES |
Submitted April 3, 1998;
accepted July 31, 1998.
Supported by grants from the European Economic Community (EEC), the
Association pour la Recherche contre le Cancer, la Fondation pour la
Recherche Médicale et le Ministère de la Recherche et de la
Technologie. S.M. was supported by a TMR fellowship from the EEC.
W.H.M. is a Scholar of the Medical Research Council of Canada.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Anne Dejean, PhD, Unité
de Recombinaison et Expression Génétique, INSERM U 163, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France;
e-mail: adejean{at}pasteur.fr.
| |
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Abstract
Introduction