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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2244-2251
Caspases Mediate Retinoic Acid-Induced Degradation of the Acute
Promyelocytic Leukemia PML/RAR Fusion Protein
By
Clara Nervi,
Fabiana F. Ferrara,
Mirco Fanelli,
Maria Rita Rippo,
Barbara Tomassini,
Pier Francesco Ferrucci,
Martin Ruthardt,
Vania Gelmetti,
Carlo Gambacorti-Passerini,
Daniela Diverio,
Francesco Grignani,
Pier Giuseppe Pelicci, and
Roberto Testi
From the Dipartimento di Istologia ed Embriologia Medica and
Dipartimento di Biotecnologie Cellulari e Ematologia, University of
Rome "La Sapienza," Rome; the European Institute of Oncology,
Department of Experimental Oncology, Milan; the Dipartimento di
Medicina Sperimentale e Scienze Biochimiche, University of Rome "Tor
Vergata," Rome; Istituto di Medicina Interna e Scienze Oncologiche,
Perugia University, Perugia; the Division of Experimental Oncology D,
Istituto Nazionale Tumori, Milan, Italy; and the Department of
Hematology, J.-W. Goethe University, Frankfurt, Germany.
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ABSTRACT |
All-trans-retinoic acid (RA) treatment induces morphological
remission in acute promyelocytic leukemia (APL) patients carrying the
t(15;17) and expressing the PML/RAR product by inducing terminal differentiation of the leukemic clone. RA treatment induces
downregulation of PML/RAR and reorganization of the PML-nuclear
bodies. These events have been proposed to be essential for the
induction of APL cell differentiation by RA. Here, we show that in the
APL-derived NB4 cell line as well as in myeloid precursor U937 cells
expressing the PML/RAR (U937/PR9) and in blasts from APL patients,
the PML/RAR fusion protein is cleaved by a caspase 3-like activity
induced by RA treatment. In fact, a caspase 3-like activity is
detectable in PML/RAR expressing cells after RA treatment, and
selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RAR deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the -helix
region of the PML component of the fusion protein. The extent of
PML/RAR cleavage directly correlates with the ability of RA to
restore the normal PML nuclear bodies (NBs) pattern. However,
RA-induced differentiation is not prevented by the persistence of the
fusion product and occurs in the absence of normally structured PML
NBs. These results indicate that PML/RAR is directly involved in
conferring RA sensitivity of APL cells and that the RA-induced
reassembly of PML NBs is the consequence of the disappearance of
PML/RAR.
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INTRODUCTION |
THE ACUTE PROMYELOCYTIC leukemia (APL)
phenotype depends on the expression of the PML/RAR fusion product,
which results from the recombination between the promyelocytic leukemia
(PML) gene on chromosome 15 and the retinoic acid (RA) receptor gene (RAR) on chromosome 17 (t15;17).1,2 PML, a member
of the RING finger family, is ubiquitously expressed in tissues within
large multiprotein nuclear structures, termed nuclear bodies (NBs), and
acts as a growth suppressor in vitro.3-6 RAR is one of
the nuclear retinoid receptors that mediates RA action on myeloid
differentiation.7-10 PML/RAR retains large portions of
the parental proteins, including the RING, B1-B2, and coiled-coil
regions of PML and the DNA and ligand binding domains of
RAR.1,2
The mechanism of the PML/RAR leukemogenic activity is poorly
understood. PML/RAR expression into hematopoietic precursor cell
lines induces differentiation block and promotes
survival.11 Both phenotypes depend on the integrity and
fusion of PML and RAR sequences.12 It has been proposed
that PML/RAR exerts a dominant negative action on wild-type PML and
RXR, an RAR cofactor, because expression of PML/RAR provokes PML
and RXR delocalization.3-5 Indeed, in APL cells and in bone
marrow cells from leukemic PML/RAR transgenic mice, the PML NBs are
disrupted into a microspeckled pattern.3-5,13-15 Based on
these findings it has been suggested that the integrity of PML NBs is
important for normal myeloid differentiation and that PML/RAR may
cause leukemia by interfering with either the RAR- or PML-dependent
differentiation pathways.3-5,16
APL is a unique model for differentiation therapy, as indicated by the
fact that RA induces terminal differentiation of PML/RAR-expressing cells both in vivo and in vitro.1,2,16,17 Treatment with RA
induces downregulation of the fusion protein, disappearance of the
PML/RAR microspeckles, reorganization of PML NBs pattern, and
relocalization of PML to its physiological site, therefore suggesting
that the main effect of RA in APL cells is to release the dominant
negative effect of PML/RAR on wild-type PML.3-5,18,19 However, expression of PML/RAR into different myeloid cell lines increases RA sensitivity and alterations of PML/RAR correlates with
RA resistance in APL-derived NB4 cell lines.11,20-22 These findings would indicate that the fusion protein is actively involved in
conferring RA sensitivity and that the integrity of the PML NBs plays
no role.
Downregulation of PML/RAR by RA occurs at the posttranslational
level18 and is prevented by proteasome
inhibitors.19 Recent findings show that PML is associated
with ubiquitin-related proteins,23,24 and a possible role
for the proteasome pathway in the RA-induced degradation of PML/RAR
has been suggested.19 The proteasome is also involved in
cellular pathways leading to caspase activation and
apoptosis.25,26 Caspases are a family of cysteine proteases
with aspartic acid substrate specificity, thought to be key effectors
of cellular apoptosis in multicellular organisms.27,28
Here, we show that caspases mediate the RA-induced degradation of
PML/RAR. Using different caspase inhibitor peptides, we were able to
prevent RA-induced PML/RAR degradation and test directly the
contribution of PML/RAR degradation and PML NBs reorganization to RA
response of APL cells.
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MATERIALS AND METHODS |
Chemicals, antibodies, and plasmids.
All-trans-retinoic acid was obtained from Sigma (Milano,
Italy). z-Al-Ala-DL-Asp-fluorometylketone (zVAD),
Ac-Asp-Glu-Val-aspartic acid aldehyde (DEVD), and
Ac-Tyr-Val-Ala-Asp-chloromethylketone (YVAD) were obtained from Bachem,
Switzerland. RAR antibody was obtained from Dr P. Chambon
(Strasbourg, France)29 and from Santa Cruz Biotechnology
(Santa Cruz, CA). PG-M3 and -PML3 are anti-PML
antibodies.12,30
PML/RAR and the mutants  C P/R, H P/R, and HA-tagged PML/RAR
were previously described.11,12 The pSG5/H4-RAR mutant was
constructed by polymerase chain reaction (PCR) amplification and fusion
of the PML heptad region with the RAR moiety of the PML/RAR
fusion protein (F. Grignani and P.G. Pelicci, manuscript in preparation). MBP-PML/RAR construct was generated by
PCR/subcloning in the pMal vector (New England Biolabs,
Beverly, MA) and expressed in Escherichia coli B21 cells by
induction with isopropyl-1-thio- -D-galactopyranoside (IPTG) for 1 to
3 hours at 23°C. Poly(ADP-ribose) polymerase (PARP) cDNA31 was subcloned into a pBluescript vector (Stratagene, La Jolla, CA). [35S]-PARP, -PML/RAR, and -mutant
proteins were prepared by in vitro transcription-translation using the
TNT T7 coupled reticulocyte lysate system (Promega, Madison, WI).
[35S]-methionine was purchased from Amersham (Arlington
Heights, IL).
Cell culture and differentiation.
The human APL cell line NB4 was obtained from Dr M. Lanotte, INSERM,
Paris, France32; the U937/PR9 and the U937/HA-PR1 are
described subclones of the U937 promonocytic cell
line.11,12 Leukemic cells from informed, newly diagnosed
APL patients were isolated and cultured as previously described.33 Cells were maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS). Cell differentiation was
evaluated as described.11,12
Immunofluorescence staining and immunoblot analysis.
Cells were collected, cytocentrifuged, and fixed in 100% methanol at
room temperature for 5 minutes, followed by acetone for 2 minutes at
 20°C. PML staining was performed with the PGM3 or with the
anti-HA monoclonal antibodies as described.12,30 The immunofluorescence was detected using an Olympus BX-60 fluoromicroscope equipped with a chilled 3CCD digital color camera (C5810 Hamamatsu Photonics, Hamamatsu City, Japan).
Immunoblot analysis was performed on total cell homogenates as
previously described.12,18 Immunoreactivity was determined using the ECL method (Amersham).
In vitro cleavage assays.
Cells were collected by centrifugation and resuspended in 100 µL of a
lysis buffer containing 50 mmol/L NaCl, 2 mmol/L MgCl2, 40 mmol/L -glycerophosphate, 5 mmol/L EGTA, and 10 mmol/L HEPES pH 7.0. Cleavage reactions were performed in a volume of 36 µL containing 25 mmol/L HEPES, pH 7.5, 100 mmol/L NaCl, 2 mmol/L MgCl2, 5 mmol/L dithiotreitol, 0.1% Triton X, 1 mmol/L phenylmethylsulfonyl fluoride, and 2 µg/mL of each aprotinin, leupeptin, and pepstatin with 3 µL of [35S] methionine-labeled PARP and 15 µL
of the cell lysates. Each reaction was incubated for 1 hour at
37°C. To map the PML/RAR caspase cleavage site, cell lysates
were substituted with bacterially expressed GST or GST caspase1, or
GST-caspase 3 fusion proteins.31 The reactions were
fractionated on sodium dodecyl sulfate (SDS)-PAGE. The gels were fixed
(acetic acid 60%, methanol 40%, and glycerol 5%), treated with the
Amplify solution (Amersham), and dried. The cleavage products were
visualized after an overnight exposure at 80°C or by
immunoblotting as described above.
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RESULTS |
Inhibition of caspases prevents RA-induced PML/RAR degradation in
vivo.
To investigate the involvement of caspases in the RA-induced
degradation of PML/RAR, NB4 cells, fresh APL blasts, and the U937/PR9 cell line, a subclone of the promonocytic leukemia cell line
U937 stably transfected with the PML/RAR cDNA under the control of a
Zinc (Zn)-inducible promoter were treated with RA, in the
presence or absence of the broad spectrum caspase inhibitor oligopeptide zVAD,34 and analyzed for
PML/RAR expression by immunoblotting using the anti-RAR RP(F)
antibody.29 As shown in Fig 1A,
RA-induced degradation of PML/RAR was inhibited by the addition of
100 µmol/L zVAD. Densitometric analysis of the Western blots shown in
Fig 1A revealed that zVAD is able to inhibit about 40% to 50% of the
RA-induced PML/RAR degradation in NB4 cells and in PML/RAR
expressing U937/PR9 cells, whereas in APL blasts inhibition by zVAD is
87%. In addition, treatment of NB4 and APL blasts with zVAD in the
absence of RA often resulted into increased levels of PML/RAR. This
effect of zVAD was not detectable in PML/RAR expressing AML cells
(U937/PR9 cells), suggesting that caspases are involved in the
steady-state regulation of PML/RAR stability in APL cells.
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| Fig 1.
Effect of the caspase inhibitors zVAD, DEVD, and YVAD in
preventing RA-induced PML/RAR degradation. (A) Cells were treated
with 1 µmol/L RA for 96 hours. U937/PR9 cells were induced to express
PML/RAR by treatment with 100 µmol/L Zn for 16 hours. zVAD (100 µmol/L) was added for 1 hour before RA treatment. (B) Zn-induced
U937/PR9 cells were treated with the indicated concentrations of DEVD
or YVAD. Immunoblot analysis was performed on total cellular proteins
(50 µg) using the anti-RAR RP(F) antibody. Immunoreactivity to
-tubulin was used for loading control.
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Caspase inhibitors containing the peptide-recognition elements of
endogenous substrates may reveal the involvement of specific classes of
caspases.27,28 The tetrapeptide YVAD is a potent inhibitor
of the caspase 1-like subfamily and a poor inhibitor of CED-3-like
caspases. Conversely, the tetrapeptide DEVD effectively inhibits
caspase 3-like activities. Increasing concentrations of YVAD or DEVD
were added for 1 hour, before 1 µmol/L RA treatment for 20 hours.
Degradation of PML/RAR was almost completely blocked by DEVD (60%
to 80%), whereas YVAD was less effective (Fig 1B). Similar results
were obtained in NB4 cells (not shown). These results suggested that a
caspase 3-like protease is preferentially involved in the degradation
of PML/RAR by RA in vivo.
RA induces caspase 3-like activity in NB4 cells.
To evaluate whether caspase 3-like activity was induced by RA
treatment, we tested NB4 cellular lysates for their capacity to induce
cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), an
endogenous substrate for caspase 3-like proteases.28 Incubation of in vitro-translated PARP with cell lysates from untreated NB4 cells resulted in a partial PARP cleavage, as shown by
the appearance of the 85-kD cleavage product
(Fig 2, arrow). However, PARP cleavage was
greatly enhanced by lysates from RA-treated NB4 cells (Fig 2A). In both
cases, this cleavage was prevented by DEVD in vitro, the inhibitor
peptide mimicking the PARP cleavage site (Fig 2A). These results
indicate that caspase 3-like activity is endogenously present in NB4
cells and that it is further enhanced after RA treatment.
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| Fig 2.
Cleavage of in vitro-translated PARP.
[35S]-PARP was incubated for 1 hour at 37°C with cell
lysates from untreated or from 1µmol/L RA-treated (A) NB4 and (B)
U937/PR9 cells induced (+) or not () by 100 µmol/L Zn to
express PML/RAR. One hundred micromoles per liter DEVD was added as
indicated. Arrow indicates the 85-kD PARP cleavage product.
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RA-induced caspase 3-like activity requires PML/RAR expression.
We investigated the role of PML/RAR on the induction of RA-dependent
PARP cleavage activity using cell lysates from the U937/PR9 clone. In
these cells treatment with 100 µmol/L Zn results in the expression of
the PML/RAR protein.11 Cell extracts prepared from
untreated U937/PR9 cells, or from cells treated with RA or Zn, failed
to cleave in vitro-translated PARP. However, PARP cleavage was induced
by lysates from U937/PR9 cells treated with Zn and RA. PARP cleavage
was prevented by DEVD (Fig 2B). Simultaneous treatment of parental U937
cells with Zn and RA did not induce caspase 3 activity (not shown).
Therefore, PML/RAR expression is required for the induction of
caspase 3-like activity that follows RA treatment.
Mapping of a major caspase 3 cleavage site within the -helix
region of PML.
We then directly tested whether PML/RAR could be cleaved by caspase
3 in vitro. Incubation of in vitro-translated or bacterially expressed
(MBP-PML/RAR; see Materials and Methods) PML/RAR with recombinant
GST caspase 3 for 1 hour induced the formation of ~50 kD proteolytic
fragment (Fig 3B and C). This cleavage
product was specifically induced by caspase 3 because it was blocked in vitro by DEVD and was not observed using GST or GST caspase 1 (Fig 3B
and C and data not shown). Anti-RAR RP(F) antibody specifically reacted with p50 (Fig 3B), thereby suggesting that GST caspase 3 induces a p50 proteolytic fragment that contains the C-terminal F
region of RAR.
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| Fig 3.
(A) Schematic representation of the amino acid sequences
of PML/RAR and mutants. P, proline-rich region; R, RING finger
domain; B1 and B2, B-boxes; C.C., coiled-coil region; -H, -helix
region; SP, the -helix serine and proline-rich region; BP,
PML/RAR junction. (B through F) RAR functional regions. (B)
MBP-PML/RAR (MBP-P/R) or (C) in vitro-translated
[35S]-PML/RAR (P/R) or (D) [35S]- C
P/R, (E) [35S]-H P/R, and (F)
[35S]-H4-RAR were incubated with GST or GST caspase 3 for
1 hour at 37°C. One hundred micromoles per liter DEVD was added
where indicated. Samples were analyzed by discontinous 8% to 15%
SDS-PAGE and visualized by autoradiography or by immunoblotting (B)
using an anti-RAR antibody (Santa Cruz Biotechnology). Molecular
weight markers are indicated. Arrows indicate the p50 proteolytic
fragment.
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Because in vitro-translated RAR was not cleaved by GST caspase 3 or
1 (data not shown) and the RAR component of the fusion protein is
~46 kD, the caspase 3 cleavage site was expected to map within the
C-terminal portion of the PML component of PML/RAR fusion product.
To directly show it, we tested the stability of various in
vitro-translated PML/RAR deletion mutants (shown in Fig 3A) in the
presence of GST caspase 3. C P/R is a PML/RAR deletion mutant in
which the N-terminal RING and B1+ B2 regions were
deleted.12 In the H P/R mutant, the coiled-coil region was deleted.12 The two regions deleted in C P/R and H
P/R are adjacent in PML. Figure 3D and E shows that p50 is released following the incubation of GST caspase 3 with in vitro-translated C P/R and H P/R mutants, thereby suggesting that the caspase 3 cleavage site maps downstream to PML coil-coiled region, within the
-helix region. Notably, recombinant caspase 3 was not able to cleave
the H4-RAR mutant obtained by fusing the PML heptad repeats directly to
the RAR portion of PML/RAR (Fig 3F). Thus, a caspase 3 cleavage
site maps within the -helix region of PML retained in both C P/R
and H P/R mutants but not in H4-RAR. This region contains only one
aspartic acid (Asp) at position 522, within a sequence (PHLD:GP)
permissive to caspases cleavage. The predicted molecular weight for the
434 amino acids proteolytic product (~49.9 kD) is
consistent with the observed p50 fragment and with Asp 522 being the
cleaved residue.
Inhibition of PML/RAR degradation does not prevent RA-induced
myeloid differentiation.
To investigate the role of PML/RAR degradation in RA-induced
terminal differentiation, we took advantage of the possibility to
prevent PML/RAR downregulation using caspase inhibitors. NB4 and
Zn-induced U937/PR9 cells were pretreated with caspase inhibitors (DEVD
or zVAD) or control medium for 1 hour before the addition of 1 µmol/L
RA for 4 days. Immunoblot analysis revealed a marked effect of caspase
inhibitor DEVD on the stability of PML/RAR in both NB4
(Fig 4A) and U937/PR9 (Fig 4B) cells.
Treatment with RA and caspase inhibitors, either DEVD or zVAD, induced
differentiation of both NB4 and Zn-induced U937/PR9 cells, as shown by
the nitroblue tetrazolium (NBT) dye reduction test (Fig
4C) and the quantitative fluorescence-activated cell sorter (FACS)
analysis of the expression of the CD11a surface differentiation antigen
(Fig 4D). In control experiments performed in the absence of RA,
caspase inhibitors (DEVD or zVAD) had no effect on the differentiation
properties of these cells (not shown). Comparison of differentiation in
cells treated with RA or RA plus caspase inhibitors revealed a slight, but consistent, synergistic effect of caspase inhibitors (Fig 4C and
D). Remarkably, similar results were obtained using leukemic blasts
from peripheral blood of newly diagnosed APL patients
(Fig 5).
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| Fig 4.
Time-dependent inhibition of 1 µmol/L RA-induced
PML/RAR degradation by 100 µmol/L DEVD in (A) NB4 cells and (B)
Zn-induced U937/PR9 cells. Immunoblot analysis was performed on total
cellular proteins using an anti-RAR antibody (Santa Cruz
Biotechnology). -tubulin antibody was used for loading control. (C
and D) Effect of DEVD and zVAD on cell differentiation in cells
grown in the absence (black bars) or in the presence of 1 µmol/L RA
for 96 hours (diagonal bars) evaluated by NBT reduction test (C) or
quantitative expression of CD11a antigen (D).
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| Fig 5.
Leukemic cells isolated from a newly diagnosed APL
patient were treated for 96 hours with either 1 µmol/L RA or 100 µmol/L zVAD or with the combination of both agents as indicated. (A)
Immunoblot analysis performed using the anti-RAR RP(F) antibody;
(B) percentage of differentiated cells determined by NBT reduction
assay.
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Inhibition of PML/RAR degradation by caspase inhibitors prevents
reorganization of PML NBs.
RA treatment of APL blasts induces progressive disappearance of
PML/RAR microspeckles, reorganization of PML NBs, and in consequence, relocalization of PML to its physiological site. Whether
these phenomena are involved in RA differentiation and the role of
PML/RAR degradation is not clear. Therefore, we analyzed the effect
of DEVD on the subcellular localization of PML and PML/RAR in NB4
and in U937/PR9 cells by indirect immunofluorescence using the PG-M3
anti-PML monoclonal antibody.30 NB4 and Zn-treated U937/PR9
cells showed the microspeckled pattern of PML and PML/RAR expression
typical of APL cells (Fig 6a and d). RA
treatment restored the speckled pattern of PML NBs in both cell lines
(Fig 6b and e). However, this effect was blocked by caspase 3 inhibitor
DEVD (Fig 6c and f). In fact, cells treated with both RA and DEVD
revealed a microspeckled pattern of anti-PML staining (Fig 6c and f).
It appears that inhibition of PML/RAR degradation prevents the
reorganization of PML NBs. These results imply that PML/RAR
localization is not sensitive to RA and that restructuring of the PML
NBs induced by RA might be the consequence of the degradation of
PML/RAR within the microspeckles, rather than its relocalization to
PML NBs. To directly test this hypothesis, we analyzed the effects of
RA treatment independently on the localization of PML and PML/RAR using an U937 clone expressing the HA-tagged PML/RAR protein (clone
HA-PR1), where PML/RAR and the endogenous PML stainings can be
recognized by double immunostaining using -HA and -PML3 antibodies, respectively. The anti-PML3 polyclonal antibody recognizes a carboxyterminal, isoform-specific PML3 epitope not retained within
PML/RAR.12 Immunofluorescence analysis performed in untreated U937-HA-PR1 cells showed that both PML and PML/RAR are
expressed into a microspeckled pattern (Fig
7a and b). After RA treatment, the immunofluorescence analysis of
HA-PML/RAR-expressing cells using the -HA antibody revealed a
progressive disappearance of the labeling (Fig 7c). Immunostaining of
the same cells with the -PML3 antibody demonstrated the simultaneous
reformation of the PML NBs pattern (compare the microspeckled pattern
of staining in control cells with the speckled pattern in RA-treated
cells, Fig 7b and d, respectively). These data confirm that PML/RAR expression is downregulated by RA and indicate that RA-induced PML/RAR degradation takes place into the microspeckles and that there is minimal, if any, RA-induced PML/RAR relocalization to the
PML NBs.
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| Fig 6.
Immunofluorescence analysis in NB4 (a through c) and
Zn-induced U937/PR9 cells (e through g) using the PG-M3 anti-PML
monoclonal antibody. Control cells (a and d) and cells treated for 96 hours with 1 µmol/L RA (b and e) or 1 µmol/L RA + 100 µmol/L
DEVD (c and f).
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| Fig 7.
Immunofluorescence analysis in Zn-induced U937/HA-PR1
cells analyzed after 24 hours in the absence (a and b) or in the
presence (c and d) of 1 µmol/L RA using the monoclonal anti-HA (a and
c) or the polyclonal anti-PML3 antibodies (b and d).
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DISCUSSION |
It has been recently proposed that the PML/RAR fusion protein is
degraded through the proteasome during RA treatment of APL cells and
that this event is required for the differentiative activity of
RA.16,18,19 Recent studies show that a proteasomal pathway
can be upstream to caspase activation in thymocytes and neurons induced
by different stimuli,25,26 suggesting a possible linkage
between these two pathways.
In this study, we have elucidated the molecular mechanism and the
biological significance of PML/RAR degradation by RA. We first show
that a member of the caspase 3 family is responsible for PML/RAR
proteolysis during RA treatment of APL cells. In fact, RA-induced
degradation of PML/RAR can be prevented in vivo with the broad
spectrum caspase inhibitor zVAD and the caspase 3 selective inhibitor
tetrapeptide DEVD. Degradation of the fusion protein can be
reconstituted in a cell-free system by using in vitro-translated
PML/RAR and recombinant caspases. One major caspase cleavage site
maps within the PML component of the fusion protein (-helix; Asp
522), and the proteolytic event leaves the RAR component intact and
potentially able to mediate RA-dependent transcriptional events.
Notably, Asp 522 is not retained in a shorter variant of PML/RAR
(bcr3-PML/RAR isoform)1,2 found in approximately
35% of patients.35,36 This short PML/RAR isoform is
indeed resistant to RA-induced degradation.37
The mechanisms involved in the triggering of caspase-mediated
degradation of PML/RAR by RA treatment remain unclear. A caspase 3-like activity responsible for the proteolysis of PARP was detectable after treatment with RA in NB4 cells as well as in U937/PR9 cells induced to express PML/RAR. However, caspase activation also depends
on PML/RAR expression, as shown by the finding that RA induces
caspase activation in U937 cells only when the fusion protein is
expressed. These findings are in agreement with recent data that shows
that PARP polypeptide and its enzymatic activity decline dramatically
during RA-induced differentiation in NB4 cells but not in HL-60 cells,
a myeloblastic cell line that does not express
PML/RAR.38 It appears, therefore, that the PML/RAR target protein is also required for RA-induced caspase activation in
APL cells.
Caspase activation is a complex and still obscure process that is
triggered by apoptotic signals.28,39 Bcl2 family members control the mitochondrial APAFs and the activation of caspase 3.28,39-41 Downregulation of Bcl2 by RA has been reported
for some myeloid cells,42-44 and it is a constant and more
pronounced phenomenon in PML/RAR-expressing
cells.37,42,45 Therefore, Bcl2 might be a direct molecular
target of PML/RAR, and its RA-induced downregulation might
contribute to caspase activation and consequent PML/RAR degradation.
Notably, RA-induced downregulation of Bcl2 and caspase activation is
not followed by apoptotic cell death in APL cells,33,45-47
thereby suggesting that additional molecular events are required to
complete the apoptotic program.
Finally, these results show that APL cell differentiation might occur
despite the persistence of PML/RAR expression. Oligopeptides that
are selective caspase inhibitors could effectively prevent RA-induced
PML/RAR degradation without impairing RA-induced differentiation in
NB4 cells, in U937/PR9 cells, and in cells derived from APL patients,
as evaluated by morphology, NBT reduction, and quantitative FACS
analysis of the CD11a surface antigen expression. Indeed, in the
presence of caspase inhibitors, we observed increased expression of the
fusion protein and a parallel increased differentiative effect of RA,
thereby suggesting an active contribution of PML/RAR to RA-induced
differentiation. This is supported by the fact that RA induces cell
differentiation and clinical remission also in APL patients expressing
the short isoform of PML/RAR (bcr3),35,36 which
appears to be resistant to RA-induced degradation.37
Inhibition of PML/RAR degradation largely maintained the
microspeckled localization of the fusion protein preventing the reorganization of the PML NBs. Thus, in APL cells, RA-induced reassembly of NBs seems to be the consequence of the disappearance of
PML/RAR within microspeckles and may not be required for myeloid differentiation. Taken together these data suggest that RA might convert PML/RAR to an active inducer of myeloid differentiation, possibly by triggering its trascriptional activator function on specific RA-target genes.
It has been recently shown that unligated nuclear receptors, including
RARs, are associated with coregulatory proteins such as N-CoR and SMRT
that can act as transcriptional corepressors. SMRT and N-CoR associate
with the corepressor Sin3 and the histone deacetylase protein to form a
transcriptional repressor complex.48,49 RA binding releases
this complex and recruits the multisubunit activation complex, which
possesses histone acetyltransferase activity.48 PML/RAR
has been found to be associated with transcriptional corepressors that
can dissociate from the fusion products at much higher RA
concentrations than from wild-type RAR.50-54 In
agreement with these findings, we propose that in APL cells, RA binding to PML/RAR induces allosteric changes of the fusion protein that release the corepressors and recruit the activator complex resulting in
histone acetylation, modification of gene expression, and cell differentiation. The same allosteric changes in PML/RAR conformation might also allow specific PML/RAR amino acid sequences to be cleaved
by caspases.
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FOOTNOTES |
Submitted May 11, 1998;
accepted July 10, 1998.
Supported by grants from the Associazione Italiana per la Ricerca sul
Cancro (AIRC), Ministero Università e Ricerca Scientifica e
Tecnologica (MURST), CNR Biotechnology and European Community (Biomed).
Address reprint requests to Clara Nervi, MD, PhD, Dipartimento di
Istologia ed Embriologia Medica, University of Rome "La Sapienza," Via A. Scarpa 14, 00161 Rome, Italy; e-mail:
nervi{at}axrma.uniroma1.it.
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 gratefully acknowledge Wilson Miller, Giulio Cossu, and Angelika
Rosenauer for helpful suggestions and discussions. We also thank P. Chambon, M. Lanotte, K. Schulze-Osthoff, R. Beyaert, and Sara Droetto
for providing reagents.
| |
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Abstract
Introduction
Abstract