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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 793-802
Overexpression of Wild-Type Retinoic Acid Receptor  (RAR)
Recapitulates Retinoic Acid-Sensitive Transformation of Primary Myeloid
Progenitors by Acute Promyelocytic Leukemia RAR-Fusion Genes
By
Changchun Du,
Robert L. Redner,
Michael P. Cooke, and
Catherine Lavau
From Systemix Inc, Palo Alto, CA; and the University of Pittsburgh
Medical Center, Pittsburg, PA.
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ABSTRACT |
Retinoic acid receptor  (RAR) is the target of several
chromosomal translocations associated with acute promyelocytic
leukemias (APLs). These rearrangements fuse RAR to different partner
genes creating the chimeric proteins: PML-RAR, PLZF-RAR, and
NPM-RAR. Although the vast majority of APLs respond to retinoic acid
therapy, those associated with PLZF-RAR are resistant. We have used
retroviruses to express PML-RAR, PLZF-RAR, NPM-RAR, RAR403
(a dominant negative mutant of RAR), and wild-type RAR in murine
bone marrow progenitors and found that all of these constructs blocked
differentiation and led to the immortalization of myeloid progenitors.
This cellular transformation is specific to an alteration of the RAR
pathway because overexpression of RAR , RAR , or RXR did not
result in similar growth perturbations. Pharmacological doses of RA
induced differentiation and inhibited proliferation of cells
transformed with either of the APL fusion genes, including PLZF-RAR,
whereas physiological retinoic acid concentrations were sufficient to reverse the phenotype of cells transformed with wild-type RAR. The
cellular responses to retinoic acid were accompanied by a sharp
decrease in the amount of the RAR-fusion proteins expressed in the
cells. Our findings suggest that the oncogenicity of RAR-fusion proteins results from their nature to behave as unliganded RAR in
the presence of physiological concentrations of retinoic acid.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ACUTE PROMYELOCYTIC leukemias
(APLs) are characterized by an early block in myeloid maturation and
are associated with chromosomal translocations that disrupt the
retinoic acid receptor  (RAR) gene located on chromosome 17 (q21). RAR is a member of the retinoic acid receptor (RAR) family
that comprises two other isotypes, RAR and RAR. These receptors
share marked structural similarity in their ligand and DNA binding
domains but display a distinct tissue-specific pattern of
expression1,2 and control the expression of different
subsets of target genes.3 When dimerized with a retinoid X
receptor (RXR), RARs bind to specific promoter sequences of downstream
genes and activate their transcription in the presence of all
trans retinoic acid (RA) or 9-cis RA. In the absence of
ligand, RAR/RXR heterodimers interact with nuclear receptor
corepressors (termed SMRT and N-CoR) that recruit histone deacetylases
which induce chromatin modifications and repression of
transcription.4 RA has a broad range of effects during
embryogenesis as well as on adult tissues and, in the case of the
hematopoietic compartment, RAR appears to be the principal mediator
of RA's activity. For example, a mutation in RAR was shown to block
the granulocytic differentiation of the HL-60 promyelocytic cell line in response to RA,5 and the expression of a dominant
negative mutant, RAR403, in murine bone marrow cells leads to the
immortalization of a multipotent hematopoietic progenitor.6
The vast majority of APLs are associated with a t(15;17) chromosomal
translocation that fuses RAR to the PML (promyelocytic leukemia)
gene. These leukemias are characterized by their sensitivity to RA
therapy. PML is a ubiquitous phosphoprotein whose expression is induced
by interferons and acts as a growth suppressor in several cell
lines.7,8 It is concentrated within poorly understood, discrete, nuclear compartments called nuclear bodies or PODs (PML oncogenic domains) that are disrupted in APLs associated with the
t(15;17) translocation. The chimeric protein PML-RAR retains the
DNA-binding and ligand-binding domains of RAR and the amino-terminal portion of PML. Transfection studies have shown that PML-RAR can
antagonize the transactivation function of wild-type RAR on
RA-inducible promoters.9-11 Moreover, PML-RAR can
heterodimerize with RXRs and PML, and this could lead to an
interference with the normal function of these proteins as well.
Three variant chromosomal translocations have been identified in APLs
that result in the fusion of RAR with PLZF, NPM, or NuMA.12-14 All of the different fusion proteins retain the
same portion of RAR. The PLZF (promyelocytic leukemia zinc finger) gene located on band 11q23 encodes a protein that binds to specific DNA
sequences and represses transcription by interacting with the
corepressor SMRT or N-CoR.11,15-17 PLZF shares some common features with PML: it can inhibit proliferation of cell
lines18 and it localizes in nuclear structures similar to
PML nuclear bodies.19 Unlike all other APLs, those
associated with the PLZF-RAR fusion protein respond poorly to RA
therapy. The two other RAR fusion partners that have been cloned in
APLs present little similarities with PML or PLZF; NPM (nucleophosmin)
is a ubiquitously expressed RNA-binding nucleolar phosphoprotein and
NuMA is a nuclear mitotic apparatus protein. The only apparent common
denominators among these four partners are their nuclear localization
and their ability to participate in protein-protein interactions.
The causal role of PML-RAR and PLZF-RAR in the pathogenesis of
APLs has been demonstrated in several transgenic mouse
models.16,20-22 However, these in vivo studies are
impractical to analyze a large series of mutants, and most of the work
performed to address the molecular mechanisms of cellular
transformation by PML-RAR and PLZF-RAR has relied upon the U937
promonocytic cell line that can be induced to differentiate in culture.
In this cellular system, both fusion genes are capable of blocking
differentiation, but whereas the antidifferentiative effect of
PML-RAR is abolished in the presence of pharmacological doses of RA,
the action of PLZF-RAR is resistant to RA.23,24 Analyses
of mutants of PML-RAR in U937 cells have provided important insights
into the structure-function relationships of this
oncogene.25,26 However, the U937 cell line that requires
vitamin D3 and transforming growth factor to differentiate is a
somewhat artificial system and the abnormal growth of these
immortalized cells may interfere with the biologic activities of the
RAR-fusion proteins. This raises the question of the relevance of
such a cellular model to elucidate the oncogenic processes that take
place in APLs. To meet the need of a more physiological system, we have
expressed RAR-fusion genes in normal murine bone marrow cells using
retroviral vectors and characterized the effect of these genes on the
differentiation and the proliferation of myeloid progenitors in the
presence or absence or RA. We have compared PML-RAR, PLZF-RAR,
NPM-RAR, the dominant negative mutant RAR403 (a truncated version
of RAR that is interrupted in the C-terminal ligand-binding domain),
and wild-type RAR in murine hematopoietic progenitors. We provide
here the first experimental demonstration of the transforming
properties of the NPM-RAR fusion protein in primary cells. Our
findings also point to the central role of RAR deregulation in the
pathogenesis of APLs and support the hypothesis that the main
contribution of the PML, PLZF, and NPM partner genes may be to abolish
the sensitivity of the resulting fusion proteins to physiological
concentrations of RA.
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MATERIALS AND METHODS |
Design and production of retroviral constructs.
All cDNAs were cloned in the poly-linker upstream of the PGK-neo
cassette of the MSCVneoEB retroviral vector.27 The 1.7-kb EcoRI fragment encoding human RAR was derived from the
pSG5-RAR vector. The RAR403 mutant was generated by inserting a
universal termination Xba I linker (Biolabs, Beverly, MA)
between the Sma I sites in the human RAR cDNA. The 3087 nucleotide (nt) EcoRI-Sca I PML-RAR fragment was
derived from the pSG5-PML-RARL vector.9 A 2,980-nt
EcoRI fragment encoding PLZF-RAR was generated from the pGEM
vector. The integrity of the first 700 nt of the PLZF-RAR coding
sequence was confirmed by Dye Terminator (PE Applied
Biosystems, Foster City, CA) sequencing reactions performed and
processed on an ABI 377 automated sequencer (PE Applied Biosystems)
using an oligo upstream of the cloning site in the MSCV
vector and PLZF oligos (5'-cctcttccaccgcaatag-3';
5'-gcctccgtgtcattgtcg-3'; and 5'-ggaagtccaaagtatagtgttgac-3'). The NPM-RAR construct
was based on the NPML-RAR cDNA encoded by a 2.0-kb
BamHI fragment derived from a pSG5 vector.13 In
pilot studies, we compared the transforming properties of
NPMS-RAR and NPML-RAR, short and long form,
respectively,13 but found that these were very similar, so
we only included the NPML-RAR form in our subsequent
work, which is referred to as NPM-RAR in this report. A partially
digested EcoRI-BamHI 1.4-kb fragment encoding human
RAR was derived from pCOD20 plasmid.28 A 1.9-kb
EcoRI fragment encoding murine RAR was derived from a pSG5
vector. A 1.7-kb EcoRI fragment encoding human RXR was derived from a PTZ18 plasmid. The MIE vector was engineered by exchanging the NFGR cDNA with the EGFP (enhanced green fluorescent protein) cDNA (Clontech, Palo Alto, CA) in the MIN
vector.29 Retroviral supernatants were produced from
transiently transfected Bosc23 ecotropic packaging cells,30
as described.31
Infection of primary myeloid progenitors and methylcellulose
colony-forming assays.
Enrichment of primitive progenitors from bone marrow of
5-fluorouracil-treated BA.1 mice by magnetic bead depletion was
performed as described.29 The panel of lineage antibodies
was directed against the following antigens: CD5, CD8a, CD11b (Mac-1),
Gr-1, and B220 (Pharmingen Inc, San Diego, CA). Methods used to
transduce the progenitors and methylcellulose culture conditions were
described previously.31 Between 500 and 15,000 cells were
plated per dish, depending on the viral vector used (a higher number of
cells transduced with PML-RAR were seeded to compensate for the
lower titer generated with that construct). Where indicated, RA (1 µmol/L, 0.1 µmol/L, 10 nmol/L, or 1 nmol/L; Sigma, St Louis, MO)
and/or trichostatin A (TSA; 20 ng/mL; Wako Bioproducts, Richmond,
VA) were added. We chose to use TSA at a concentration of
20 ng/mL, because we found that higher doses were toxic to the cell
lines immortalized by the different RAR fusion genes. All
methylcellulose cultures were set up in triplicates, and each data
point corresponds to the mean number of colonies scored after 7 days of culture.
Establishment of immortalized cell lines in liquid culture.
Cells from third passage methylcellulose dishes containing 50 to 300 colonies were harvested and seeded in RPMI 1640 medium containing 20%
horse serum plus supplements (50 U/mL penicillin G, 50 µg/mL
streptomycin, 2 mmol/L L-glutamine, and 0.05 mmol/L 2-mercaptoethanol)
in the presence of recombinant murine interleukin-3 (IL-3; 10 ng/mL;
R&D Systems, Minneapolis, MN) and stem cell factor (100 ng/mL;
Systemix, Palo Alto, CA). Nonadherent cells were split into fresh media approximately once per week and were maintained for
more than 12 months.
Assessment of myeloid differentiation.
For morphological analysis, cells in liquid culture were spun in a
cytocentrifuge onto glass slides and stained with Wright-Giemsa. Immunophenotypic analysis of cells harvested on day 7 of
methylcellulose culture in the presence of G418 was performed by
staining with phycoerythrin (PE)-conjugated anti-Gr-1 and fluoresceine
isothyocyanate (FITC)-conjugated anti-Mac-1 antibodies (Pharmingen
Inc), as described.29 Cells were labeled with isotype
controls IgG2b-PE and IgG2b-FITC to set the
quadrants delimiting the positive populations.
Western blot analysis of protein expression.
Whole cell extracts were made from G418-resistant primary
methylcellulose colonies pooled after 8 to 10 days of culture or from
immortalized cells grown in suspension. Protein quantification was
performed using the BCA reagent (Pierce, Rockford, IL). Protein samples
(20 µg per lane) were separated on 4% to 12% polyacrylamide precast
gels (Novex, San Diego, CA). After electrophoretic transfer, the
nitrocellulose membranes were blocked with 10% skimmed milk and
incubated with rabbit polyclonal serum raised against human RAR,
RAR, RAR, or RXR (Santa Cruz Biotechnologies, Santa Cruz, CA).
Proteins were detected with horseradish peroxidase-conjugated protein A
(Amersham, Arlington Heights, IL) using a standard chemiluminescence Western blotting protocol (ECL system; Amersham).
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RESULTS |
Expression of RAR fusion genes in primary murine myeloid progenitors
transduced with retroviral vectors.
To determine the transforming properties of APL-derived RAR fusion
genes, we cloned PML-RAR, PLZF-RAR, and NPM-RAR cDNAs in the
MSCV (murine stem cell virus) retroviral vector27 under the
control of the viral long terminal repeat (LTR) and upstream of an
internal phospho glycerate kinase (PGK)-neomycine cassette. High-titer
retroviral supernatants were generated by transient transfection of the
Bosc23 packaging cells.30 This method of viral production
does not require the selection of stably transfected packaging cells
and therefore minimizes the risk of generating retroviral vectors with
acquired mutations. This is crucial when the encoded genes have toxic
properties, as is the case for PML-RAR.32-34 As a
positive control, we transduced cells with the dominant negative mutant
RAR403,35 which can immortalize primary murine bone marrow cells.6 We also compared in our assay the
overexpression of wild-type RAR, RAR, RAR, and RXR. With
the exception of PML-RAR, the recombinant retroviral supernatants
generated with the different transgenes were similar in their
efficiency to transduce hematopoietic progenitors. The percentage of
G418-resistant colonies was in the range of 10% to 15%, with the
PML-RAR encoding vector, whereas it reached 70% to 100% with all
of the other vectors. The inferior titer of the PML-RAR vector is
most likely due to the toxic properties of PML-RAR that may hinder
the growth of most of the PML-RAR-transduced colony-forming cells.
To study the biological effects of these chimeric genes in primary
murine hematopoietic cells, we used a myeloid transformation assay
similar to that which we developed to dissect the transforming properties of another leukemic fusion gene, HRX-ENL.29,31
The experimental strategy is shown in Fig
1A. In brief, bone marrow cells from mice treated with 5-fluorouracil
and further enriched in primitive progenitors by depletion of cells
expressing markers of terminal differentiation were transduced with the
recombinant retroviral supernatants and seeded in methylcellulose
myeloid cultures in the presence of G418. The primary colonies were
scored after 7 days of growth, and the cells from pooled colonies were harvested and seeded into secondary methylcellulose cultures. The
cultures were serially replated in a total of four successive rounds
and the proliferative potential of the transduced cells was assessed by
scoring the number of colonies formed at each passage. To determine how
the transduced genes affected myeloid maturation, we analyzed the
expression of the differentiation markers Mac-1 and Gr-1 on
G418-selected cells harvested from the primary colonies using flow
cytometry.
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| Fig 1.
Experimental strategy and protein expression in primary
hematopoietic progenitors.(A) Scheme of the experimental approach used
to study the effect of RAR-fusion genes on the proliferation and
myeloid differentiation of the target cells and to evaluate the
responsiveness of the transformed cells to retinoic acid. (B) Western
blot analysis of cells transduced with the different vectors after 8 days of culture in methylcellulose (20 µg of protein was loaded per
lane). When indicated, the cells were grown in the presence of RA (1 µmol/L). Arrows point to exogenously expressed proteins detected with
the specific antihuman retinoid receptor antibodies indicated above
each panel.
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The expression of the appropriate proteins was shown by Western
analysis performed on cells from pooled G418-resistant colonies (see
Fig 1B). The lack of antibodies directed against epitopes conserved in
the RAR403 mutant precluded protein expression analysis of cells
transduced with this construct. We noted that cells transduced with the
RAR or the NPM-RAR constructs expressed consistently much higher
levels of the transgene than cells transduced with the PML-RAR or
the PLZF-RAR fusion genes. Because this cannot solely be accounted
for by a difference in viral titer, it must also reflect a toxicity of
PML-RAR and PLZF-RAR or variations in expression at the RNA
and/or protein level.
RAR and RAR mutant genes affect the differentiation and
proliferation of myeloid progenitors.
The methylcellulose cultures were performed in the presence of IL-3,
IL-6, stem cell factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF); a combination of cytokines that stimulates myeloid cell growth and differentiation resulting in the formation of granulocytic and/or macrophagic colonies. Under these conditions, cells
that are initially negative for the expression of lineage markers will
mature and generate colonies primarily composed of differentiated
cells. Thus, after 7 days of culture, more than 95% of the cells
mock-transduced (not shown) or infected with the MSCVneo control vector
expressed Mac-1 and/or Gr-1. We observed that this myeloid maturation
was greatly impaired when progenitors were transduced with vectors
encoding RAR or either of the RAR mutants
(Fig 2). The block in differentiation was
most striking in cells transduced with PLZF-RAR, RAR403, or
RAR, with less than 30% of the cells expressing Mac-1 or Gr-1.
PML-RAR and NPM-RAR had a milder effect, with an average of 40%
to 50% mature myeloid cells present in the culture. This impairment of
differentiation was not observed in cells transduced with RAR,
RAR, or RXR, which were indistinguishable from the control
MSCVneo-infected cells.
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| Fig 2.
RAR and RAR403 and RAR-fusion genes inhibit
myeloid differentiation of primary progenitors. The bars represent the
percentages of cells transduced with the different vectors that express
Mac-1 and Gr-1 after 7 days of methylcellulose cultures. Each value is
the average of 5 to 7 independent transduction experiments (±SEM).
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To compare the effect of the different transgenes on proliferation, we
measured the colony-forming ability of the transduced cells along
serial passages in methylcellulose culture. The cloning efficiency
observed upon tertiary plating is represented in
Fig 3. As previously observed, cells
transduced with the control MSCVneo vector had limited replating
ability and very few colonies of more than 100 cells were generated in
tertiary methylcellulose cultures.29 The progenitors
transduced with RAR, RAR, and RXR similarly exhausted their
proliferative potential upon serial replating. In sharp contrast,
retroviral transfer of RAR or the RAR mutants enabled the cells
to remain highly proliferative. The progenitors transduced with RAR,
RAR403, or PML-RAR had the highest cloning efficiency, generating
200 to 300 colonies per 10,000 seeded cells; progenitors transduced
with PLZF-RAR had a significantly lower plating efficiency,
generating about 130 colonies per 10,000 cells, and
NPM-RAR-transduced progenitors formed only about 50 colonies per
10,000 cells. Furthermore, the colonies generated with NPM-RAR
transduced progenitors were smaller than those obtained with the other
constructs (data not shown). This sustained replatability in semisolid
culture correlated with an unlimited growth potential in vitro, because
the cells transduced with RAR or either of the RAR mutant genes
could be transferred from pooled third passage methylcellulose colonies
into suspension cultures and maintained for more than 1 year in liquid
media supplemented with IL-3 and stem cell factor.
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| Fig 3.
RAR and RAR403 and RAR-fusion genes induce the
proliferation in vitro of primary myeloid progenitors. Each bar
represents the number of colonies per 10,000 input cells in a third
passage of methylcellulose culture (mean of 4 to 9 independent
experiments ± SEM)
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Effects of RA on cells transformed by RAR or RAR mutants.
We first studied the effect of RA on the growth and differentiation of
the immortalized cell lines established with RAR or the various
RAR mutants. In all of the cell lines, RA treatment (1 µmol/L)
strongly inhibited cellular growth (data not shown). This was
accompanied by a loss of viability of the cells expressing RAR,
RAR403 and NPM-RAR, and in the cells transduced with PML-RAR or PLZF-RAR, a clear induction of granulocytic differentiation was
observed (Fig 4).
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| Fig 4.
Cells immortalized by PML-RAR or PLZF-RAR undergo
granulocytic maturation in response to RA. Wright Giemsa stain of
cytospin preparations made from cells grown in the absence (left) or in
the presence (right) of RA (1 µmol/L) for 5 days. Bar = 20 µm.
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To further characterize the effect of RA on myeloid cell growth and
differentiation, we studied its effects on freshly transduced primary
progenitors in methylcellulose culture. After retroviral transduction,
lineage-depleted bone marrow cells were split in G418-containing
methylcellulose cultures in the absence or presence of RA (1 or 100 nmol/L). The effect of RA on myeloid differentiation was assessed by
analyzing the profile of Mac-1 and Gr-1 expression in cells harvested
from the primary colonies. The results of a representative experiment
are shown in Fig 5. RA treatment of cells
transduced with the control MSCVneo vector did not significantly alter
the percentage of cells expressing Mac-1/Gr-1, although an increase in
the intensity of Gr-1 expression was noted. Similar results were found
in cells expressing RAR, RAR, or RXR (data not shown).
RAR403-transduced cells exposed to RA at 100 nmol/L concentration
underwent a moderate degree of differentiation, with more than 50% of
the cells expressing Gr-1. On the other hand, RA-treatment (100 nmol/L)
of the cells transduced with RAR, PML-RAR, PLZF-RAR, or
NPM-RAR induced an almost complete differentiative response
accompanied by the expression of Mac-1 on more than 85% of the cells
and an upregulation of Gr-1. Remarkably, the maturation of the
PLZF-RAR-expressing cells induced by 100 nmol/L RA was comparable
to that seen in cells expressing the other RAR-fusion genes, and in
the presence of 1 µmol/L RA, the Mac-1/Gr-1 profile of the
PLZF-RAR-cells was indistinguishable from that of the cells
expressing the other fusion genes (data not shown). At a dose of 1 nmol/L of RA, a concentration within the physiological range, the cells
expressing RAR403 or either of the fusion proteins remained blocked
at an immature myeloid stage, whereas the differentiation block of the
RAR-transformed cells was essentially abolished (compare 47% of
Mac-1 /Gr-1 cells v 14%
of Mac-1/Gr-1 cells in the
presence of 1 nmol/L RA on Fig 5).
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| Fig 5.
RA triggers the myeloid maturation of progenitors
transformed by RAR, PML-RAR, PLZF-RAR, or NPM-RAR. Analysis
of the expression of Mac-1 (horizontal axis) and Gr-1 (vertical axis)
by flow cytometry in cells harvested after 7 days of methylcellulose
culture in the absence of exogenous RA (left column) or in the presence
of 1 nmol/L RA (middle column) or 100 nmol/L RA (right column). These
are the results of a representative analysis; similar findings were
obtained in three independent transductions experiments.
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The effect of RA on the proliferation of the myeloid progenitors
transduced with the different vectors was determined by scoring the
number of primary colonies formed in the presence of RA (1 µmol/L)
relative to the number of colonies formed in its absence (Fig 6). The clonogenicity of the cells
transduced with the empty MSCVneo vector was substantially diminished
by RA, with a 35% decrease in the number of colonies formed. The
negative effect of RA on cellular proliferation was much more
pronounced in cells transduced with RAR or either of the
RAR-fusion genes, because the inhibition in the colony formation
potential reached approximately 70% (PML-RAR and PLZF-RAR) or
more than 95% (RAR and NPM-RAR) in the presence of RA. On the
other hand, the proliferative potential of the cells transduced with
RAR403 was not significantly affected by the addition of RA as the
reduction in plating efficiency was less than 5%. This indicates that
RAR403 actually had protective properties against the antigrowth
effect of RA in myeloid progenitors. The overexpression of RAR,
RAR, or RXR increased the cells' sensitivity to the
antiproliferative effect of RA to comparable degrees as did RAR or
the RAR-fusion mutants. Similar results were obtained in the
presence of 100 nmol/L of RA (data not shown). In the presence of
physiological concentrations of RA (1 nmol/L), no significant reduction
in the number of colonies was observed in culture of cells transduced
by either RAR403 or the RAR-fusion genes. On the other hand,
cells overexpressing wild-type RAR generated on average twofold less
colonies in the presence of 1 nmol/L RA relative to the colonies formed
without exogenous RA (data not shown).
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| Fig 6.
Effect of RA and TSA on the clonal proliferation of
primary myeloid progenitors transduced with RAR, RAR403, RAR,
RAR, RXR, or RAR-fusion genes. The number of primary colonies
formed in the presence of RA ( ) or TSA ( ) is represented as a
percentage of the numbers of colonies formed in the absence of both RA
or TSA (mean of 3 experiments ± SEM).
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Our finding that RA reverses the transforming effects of PLZF-RAR in
bone marrow cells appears to be in contradiction to previous reports on
the lack of response of U937 cells or transgenic mice expressing
PLZF-RAR.16,24 To rule out the possibility that this
discrepancy may be the result of mutations in our cDNA, we sequenced a
region of the MSCV-PLZF-RAR vector that encompasses the POZ domain
of PLZF (see Materials and Methods) that mediates interaction with
corepressors17 and confirmed that all of the nucleotides
were conserved.
Reports that inhibitors of histone deacetylases, such as TSA, could
enhance the differentiative effect of RA in cells transformed by
PML-RAR or PLZF-RAR by relieving the repressive effect of the
fusion proteins on transcription11,16,26 prompted us to explore the effect of TSA in our assay. After retroviral infection, the
cells were seeded in methylcellulose media with or without the addition
of TSA with or without RA, and the cellular proliferation and
differentiation were monitored. TSA alone inhibited the proliferation of all of the cells, regardless of the nature of the genes encoded by
the MSCV vector (Fig 6), and resulted in a reduction of colony formation of approximately twofold. However, the antigrowth effect on
cells transduced with RAR or either of the RAR-fusion genes was
more pronounced (between 70% and 90% reduction in colony formation). The combined treatment of TSA and RA (at either 1 µmol/L or 100 nmol/L) further reduced the cloning potential of the cells (data not
shown). To determine if TSA induced the differentiation of the cells
transduced with the different constructs, we monitored the effect of
TSA on the expression of Mac-1 and Gr-1 but found that TSA did not
induce myeloid maturation or enhance the differentiative effect of RA
(data not shown). Because inhibitors of histone deacetylases have been
reported to enhance expression of integrated retroviral sequences,36 we were concerned that this phenomenon may be
occurring and contributing to the lack of differentiative activity of
TSA in our system. To test this, we transduced myeloid progenitors with
a MSCV vector encoding the green fluorescent protein (MIE vector, see
Materials and Methods) and examined by flow cytometry how the
expression of this marker was affected by TSA. With this sensitive
technique, we did not detect any upregulation of the transduced gene
(data not shown), making it unlikely that variations in retroviral
expression mask the effects of TSA on cellular differentiation per se.
To determine whether the reversal of the transformed phenotype induced
by RA (ie, the reduction in the proliferative potential and the
induction of myeloid differentiation) was associated with a variation
in the expression of RAR or of the RAR-fusion proteins, we
performed Western blot analyses on the transduced cells grown in the
absence or presence of RA. As depicted in
Fig 7A, the levels of PLZF-RAR,
PML-RAR, and NPM-RAR proteins were reduced in cells grown in
methylcellulose containing RA (1 µmol/L) compared with cells cultured
without exogenous RA. However, in cells overexpressing wild-type
RAR, no overt alteration in protein level was induced by RA
exposure. We also examined the effect of RA on the protein-expression levels of RAR, RAR, and RXR and found that the level of RAR was reduced in cells treated with RA, whereas the levels of RAR or
RXR remained constant (see Fig 1B). The reduction of the
RAR-fusion gene products observed in the transduced population could
result from a selective antigrowth effect of RA towards the cells
expressing higher amounts of the transgene. To explore this
possibility, we examined the effect of RA on the stable cell lines
immortalized with either RAR or the RAR fusion mutants, which can
be assumed to express uniform levels of the transgene as these cell
lines were found to be monoclonal based on their proviral integration pattern (data not shown). We analyzed the protein levels in the cell
lines 40 hours after supplementing the suspension cultures with 1 µmol/L RA and found the same pattern as that displayed by freshly
transduced bone marrow (Fig 7B). Altogether, our results show that the
inhibition of growth and the induction of myeloid differentiation
observed in primary progenitors transformed with RAR-fusion genes,
in response to RA treatment, are accompanied by a reduction in the
expression levels of the RAR-fusion proteins.
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| Fig 7.
RA induces the degradation of PML-RAR, PLZF-RAR,
and NPM-RAR. (A) Western blot analysis of progenitors cultured in
methylcellulose for 8 days in the absence or presence of RA (1 µmol/L) after transduction with RAR (15 µg of protein per lane),
PML-RAR (30 µg of protein per lane), PLZF-RAR (40 µg of
protein per lane), or NPM-RAR (20 µg of protein per lane). (B)
Western blot analysis of cell lines established with RAR or
RAR-fusion gene treated or not treated with RA (1 µmol/L) for 40 hours (20 µg of protein per lane). The transduced proteins are
designated by the white arrows.
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DISCUSSION |
Most of the studies performed to unravel the oncogenic processes
involved in APL have relied on stable clones derived from U937 cells
transfected with PML-RAR or PLZF-RAR.23,24,26 This
cellular tool has been exploited to conduct structure-function analyses
with a series of deletion mutants of PML-RAR and has yielded
important information. However, studies in a biologically more relevant
system are required before conclusions can be drawn on the mechanisms
at play in the pathogenesis of APLs. Recently, several laboratories
have generated PML-RAR transgenic mice that develop a
myeloproliferative disease that progresses to acute myeloid leukemia
reactive to RA therapy.20-22 These works have provided
formal evidence of the decisive role of PML-RAR in the development
of APLs; however, only a minority of mice developed the disease and
generally after a long latency, indicating that other events were
necessary for full transformation in these models. The experimental
approach we present here offers several advantages over those systems.
Because we are working with primary bone marrow cells that have not
been exposed to other transforming agents, we are able to study the
effect of the introduced genes not only on the differentiation, but
also on the proliferation potential ex vivo of the transduced
progenitors. Furthermore, the target cells are analyzed immediately
after transduction, making this system a one-step transformation assay
in which the occurrence of other confounding events is very unlikely.
Finally, our assay lends itself to structure-function analyses in which
series of mutants can be compared with respect to their effects on
myeloid differentiation, cellular proliferation, and sensitivity to RA as well as to other potential therapeutic agents.
The aim of our study was to analyze the proliferation and
differentiation potential of primary myeloid progenitors expressing RAR-mutants or wild-type RAR and RXR proteins. The major finding in the
present work is that overexpression of wild-type RAR can alter
myeloid cell growth and differentiation in vitro. This somewhat surprising result has been reported previously. Onodera et
al37 showed that transduction of RAR in murine bone
marrow blocked maturation at the promyelocyte stage in vitro, and
transfection studies performed in U937 cells also showed that RAR
could partially impair the differentiation of this cell
line.25 The novelty of our study resides in the important
finding that this effect of wild-type RAR is abolished in the
presence of physiological concentrations of RA. This indicates that
this phenomenon is dependent on the experimental conditions used,
implying that, in contrast to RAR mutants, overexpression of
wild-type RAR in vivo would unlikely be associated with abnormal
myeloid growth. But it also sheds light on its molecular basis,
suggesting that the transforming potential of RAR coincides with its
function as a transcriptional repressor. Interestingly, a recent study
has shown that, in media depleted of T3 hormone, overexpression of
wild-type thyroid hormone receptor (c-ErbA/TR) promotes
proliferation and arrests differentiation of primary avian
erythroblasts similarly to that observed with the oncogene
v-ErbA.38 Thus, it appears that a common mechanism for
oncogenic transformation by altered nuclear receptors may be to mimic
the activity of their normal counterpart in the nonliganded state.
Our observation that, unlike RAR, overexpression of RAR or RAR
did not affect differentiation or proliferation in our assay reinforces
the view that these events are predominantly regulated by target genes
of RAR in myeloid cells. However, this repartition is not absolute,
because overexpression of RAR conferred a high sensitivity to the
antiproliferative effect of RA (Fig 6), which was corroborated by the
detection of a smaller amount of the RAR protein in the population
of transduced cells selected in the presence of RA (Fig 1B).
The fact that RAR and RAR403 are as potent as RAR-fusion
proteins in blocking differentiation and immortalizing primary myeloid
progenitors argues that the pathogenesis of APLs resides primarily in
an alteration of the RAR signalling pathway and not from disruption
of the normal function of the fusion partners. In particular, the
absence of a significant proliferative advantage of the cells
expressing PML-RAR or PLZF-RAR compared with cells overexpressing
RAR (see Fig 3) undermines the hypothesis that a suppression of the
antigrowth effects of PML or PLZF by a dominant negative effect of the
fusion mutants contributes to the expansion of myeloid progenitors. The
diversity among the four fusion partners of RAR cloned in APLs was a
first indication that a repression in their normal function resulting
from the haplo-insufficiency in their gene copy and/or a dominant
negative effect of the fusion protein played little, if any, role in
leukemogenesis. The recent finding that PML may be a mediator of RA
signalling also suggests that a deregulation of the RA signalling
pathway is critical in the genesis of APLs.39
Several mechanisms have been proposed to explain how RAR-fusion
proteins disrupt the RA signalling pathway. The chimeras may titrate
out RXR molecules and interfere with RAR/RXR signalling (this could
also affect other pathways requiring the participation of RXR such as
the one mediated by the vitamin D3 receptor/RXR heterodimer); the
fusion proteins may bind to the promoter of RA-responsive genes and
behave like constitutive repressors; the target gene specificity of the
fusion proteins may be altered and result in the aberrant
transactivation of proto-oncogenes40; the fusion genes may
sequester proteins of the N-CoR or SMRT corepressor complexes that are
also required for other inhibitors of cell growth that function through
repression of transcription such as the MAD/MAX complex, the
retinoblastoma protein, PLZF, or other yet to be discovered growth
suppressors. Our study provides elements that help sort out which of
these molecular scenarios are more likely involved in the genesis of
APLs. The fact that, unlike RAR, overexpression of RAR and RAR
does not transform the cells argues against the possibility that the
titration of RXR molecules is the key event in transformation, because
RAR and RAR should heterodimerize with RXR with similar affinity
as RAR. For the same reason, it seems improbable that squelching of
corepressors by RAR/RXR or RAR mutant/RXR heterodimer is
essential, although we cannot rule out that, in myeloid progenitors,
RAR/RXR dimers interact with corepressors in a distinct manner
compared with RAR/RXR or RAR/RXR dimers.41
Transformation by RAR also indicates that leukemogenesis does not
require the transactivation of aberrant target genes by the fusion
proteins. Our results are compatible with the hypothesis that, like
cells overexpressing RAR in the quasi-absence of RA, the
transformation of cells expressing RAR-fusion proteins results from
a transcriptional repression of RAR target genes. However, definite
confirmation of this hypothesis would require additional study, such as
using our assay to test mutant fusion proteins that can no longer bind
to DNA or to corepressors.
The behavior of PML-RAR, PLZF-RAR, and RAR403 as constitutive
transcriptional repressors of RA-target genes in the presence of
physiologic concentrations of RA has been documented and has been
attributed to the persistent binding of the mutant proteins to
transcriptional corepressors.11,15,16,26,42 However, these
studies showed that, unlike what was observed with PML-RAR, the
repressing activity of PLZF-RAR persisted in the presence of
pharmacological concentrations of RA. This was paralleled by the lack
of differentiative response of PLZF-RAR-transformed U937 or
transgenic mouse bone marrow cells to RA stimulation16,24; simultaneous RA and TSA treatment was necessary to trigger
differentiation of these cells. In contrast, we found that
PLZF-RAR-expressing cells were as responsive to the differentiative
and antigrowth effects of RA as were cells expressing PML-RAR. This
was observed both on stable PLZF-RAR cell lines in liquid culture (Fig
4) and on freshly transduced progenitors in methylcellulose (Fig 5). It
is possible that this discrepancy between our findings and previous
reports is due to disparities between experimental models. However, our
approach is similar to the transgenic model in that both are based on
murine hematopoietic precursors and RA did have some therapeutic
activity in the PLZF-RAR mice when used at high dosage.16 It is noteworthy, nevertheless, that the
leukemias developing in the PLZF-RAR transgenic mice have a more
mature phenotype (they express high levels of Mac-1 and Gr-1) compared with our PLZF-RAR-transduced primary cells (judging by the profiles of Mac-1 and Gr-1 expression, Figs 2 and 5). This difference in the
degree of myeloid maturation may account for distinct sensitivities |
TOP
ABSTRACT
INTRODUCTION