From the a Unité INSERM 417, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75012 Paris, France, the Departments of d Pathology and e Medicine and the f Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the h BASF Bioresearch Corporation, Worcester, Massachusetts 01605, and the i Department of Microbiology/Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5541
Received for publication, December 26, 2000, and in revised form, March 21, 2001
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ABSTRACT |
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The t(15;17) translocation, found in
95% of acute promyelocytic leukemia, encodes a promyelocytic
leukemia (PML)-retinoic acid receptor In human acute promyelocytic leukemia
(APL),1 a specific
translocation, t(15;17) (1), creates a promyelocytic leukemia
(PML)-retinoic acid receptor Antigens binding to T and B cell receptors initiate
intracellular signaling events leading to clonal expansion of reactive cells. Unlike growth factor receptors, T cell receptor and B cell receptor lack intrinsic tyrosine kinase activities. Instead, antigen receptor engagement results in activation of cytoplasmic protein tyrosine kinases (PTK). Among the substrates of the antigen receptor PTKs are members of a class of proteins that mediate protein-protein interactions, known as adaptors (18, 19). With multiple binding sites
and the potential to create combinations of multiprotein complexes,
adaptors are well suited to integrate signals from surface receptors.
Little is known about adaptor proteins in myeloid cells (18).
Like APL patient blasts, NB4 leukemic cells carry the t(15;17) and
undergo ATRA-induced granulocytic differentiation (20). By applying a
subtraction cloning approach to these cells, we identified a novel
ATRA-inducible gene designated PRAM-1
(PML-RAR Cell Lines, Culture, and Differentiation--
NB4 (20), NB4.306
(23), and U937-PR9 and U937-MT cell lines (17) were cultured in RPMI
1640 medium with 10% fetal bovine serum (Life Technologies, Inc.) and
2 mM L-glutamine (Life Technologies, Inc.).
COS-7 cells were grown in 9-cm Petri dishes in Dulbecco's modified
Eagle's medium containing 5% fetal bovine serum. Cell viability was
estimated using standard trypan blue dye exclusion. Cells were grown at
37 °C in a humidified atmosphere of 5% CO2.
Exponentially growing NB4, NB4.306, and U937-derived cells ( Human Cell Preparation and Liquid Culture--
Cord blood cells
were obtained with informed consent from healthy donors at the end of
full term deliveries at the Saint-Vincent de Paul Hospital (Paris,
France). Sample preparation (achieving a purity of 95 ± 2%
CD34+) and CD34+ cell culture were conducted as
described (25). Primary APL cells expressing the PML-RAR PRAM-1 cDNA Cloning--
A cDNA fragment isolated
from an ATRA-treated NB4 cDNA
library2 contained
1063 bp corresponding to the 3' end of PRAM-1 cDNA. This fragment
was used to search expressed sequence tag data bases (GenBankTM/EMBL and Incyte Genomics Lifeseq®) and, in
combination with RACE-PCR reactions using HL-60 RACE-ready cDNA
(CLONTECH), allowed us to generate 1800 bp of
continuous open reading frame sequence. A 494-bp 5' cDNA sequence
was amplified by RACE-PCR from ATRA-treated NB4 RNA (with 5 × 10 Sequencing and Computer Analysis--
Plasmid DNA was purified
through Nucleobond columns (Macherey-Nagel), and double-stranded DNA
templates were sequenced using pBluescript and internal primers.
Sequence analysis and alignments were obtained using the GCG programs
(Wisconsin Package, version 10.0-UNIX, Genetic Computer Group, Inc.).
Sequence homologies were identified using the FastA and BLAST programs
by searching data banks as above and, in the case of translated
sequences, by searching Swiss-Prot, PIR/NBRF, and SPTREMBL data banks.
Plasmid Constructs--
The PRAM-1 coding sequence was subcloned
into a pCB6+-derived expression vector (26) under the control of the
zinc-inducible sheep metallothionein promoter from the pMT010 vector
(27), resulting in the pMT-PRAM-1 vector. The PRAM-1 coding sequence was also inserted into the pAT4 vector (28), to direct the expression of PRAM-1 tagged with the F domain of the human estrogen receptor at
its amino terminus. SLP-76 and SKAP-HOM cDNAs were inserted into
the pEF-BOS expression vector (29, 30), at a site located 3' to the
sequences encoding the Flag epitope.
Northern Blots--
Total RNA extraction and hybridization were
as described (25). Human RNA Master Blot and human immune system
multiple tissue Northern blot II were from
CLONTECH. The PRAM-1 probe corresponded to the 3'
end (1063 bp) of the PRAM-1 cDNA. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a 574-bp PCR product obtained using 5'-ATCACCATCTTCCAGGA-3' sense and 5'-CCTGCTTCACCACCTTCTTG-3' antisense primers. Radioactivity was detected and quantified using a
Storm 860 phosphorimaging system (Amersham Pharmacia
Biotech).
Semi-quantitative Reverse Transcription-PCR--
Total RNA from
CD34+ cells induced to differentiate toward the myeloid
lineage was extracted at different times using an Rneasy mini kit
(Qiagen). Reverse transcription was performed using the AdvantageTM system (CLONTECH).
Expression of the PRAM-1 and control Antibodies--
An amino-terminal peptide (ESHQDFRSIKAKFQA) was
synthesized according to the deduced amino acid sequence of the PRAM-1
protein and coupled to keyhole limpet hemocyanin through a cysteine
residue added to the carboxyl-terminal amino acid of the peptide
(Eurogentec). Rabbit sera (1PNP) were collected before (pre-immune) and
3 months after the initial injection and three booster immunizations
(Eurogentec). A polyclonal sheep antiserum (Elmira Biologicals) was
raised against the carboxyl-terminal 134 amino acids of human PRAM-1
fused with glutathione S-transferase (generated by cloning
the corresponding PRAM-1 coding sequence into the pGEX vector (Amersham
Pharmacia Biotech)). The monoclonal antibody directed against the F
domain of the human estrogen receptor was as described (28).
Anti-Tyr(P) monoclonal antibody 4G10 was purchased from Upstate
Biotechnologies, Inc., anti-Flag monoclonal antibody M2 was purchased
from International Biotechnologies, Inc., and anti-lyn was purchased
from Santa Cruz Biotechnology, Inc. Anti-SLP-76 sheep antiserum 0083 and anti-SKAP-HOM rabbit antiserum were as described (29, 30).
In Vivo Expression and Protein Extracts--
COS-7 cells were
transfected using calcium phosphate coprecipitation (31) of 0.5-µg
DNA vectors (adjusted to 14 µg per 9-cm Petri dish with pBluescript
carrier DNA). The medium was changed after 16 h, and for
zinc-induced expression of PRAM-1, 75 µM
ZnSO4 was added to the medium. Thirty-six h after
transfection, COS-7 cells were harvested, washed once in
phosphate-buffered saline, and resuspended in lysis buffer (0.4 M KCl, 20 mM Tris-HCl, pH 7.9, 20% glycerol, 5 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and a protease inhibitor mixture
(Sigma)). ATRA-treated NB4 cells were washed once in phosphate-buffered saline and resuspended in lysis buffer. After two freeze-thaw cycles in
liquid nitrogen, the resulting cell lysates were cleared by a 15-min
10,000 × g centrifugation at 4 °C.
Immunoprecipitation--
After one-step preclearing (30 min,
4 °C) with GammaBind G-Sepharose (Amersham Pharmacia Biotech),
antibodies (1 µl of antiserum or 1 µl of ascites fluid) were added
to the cell protein extract, in a binding buffer adjusted to 20 mM Tris-HCl, pH 7.5, 0.25 M NaCl, and 0.1%
Nonidet P-40. After 90 min at 4 °C, 15 µl of GammaBind G-Sepharose
suspension were added for an additional hour. Sepharose beads were then
washed three times in 0.5 ml of the binding buffer. After 5 min of
boiling in Laemmli buffer, samples were resolved by SDS-polyacrylamide
gel electrophoresis (PAGE) and transferred onto polyvinylidene
difluoride membranes (PerkinElmer Life Sciences). Background
immunoreactivity was reduced by pre-incubating membranes with
phosphate-buffered saline containing 5% nonfat dry milk and 0.1%
Tween 20 for 1 h. Western analysis was carried out with antisera diluted 1:5000 in phosphate-buffered saline containing 0.1% Tween 20. Detection of antibody binding was achieved with horseradish peroxidase-conjugated secondary antibodies (Jackson
Laboratories). Enzymatic activity was detected using the
chemiluminescence reagent plus kit (PerkinElmer Life Sciences) and autoradiography.
PRAM-1 mRNA Is Regulated during ATRA-induced Granulocytic
Differentiation of NB4 Cells--
PRAM-1 mRNA expression
was increased 4 h after exposure of NB4 cells to ATRA, with
maximum expression at 96 h (Fig.
1A). Sequential treatment of
NB4 cells with cycloheximide and ATRA indicated that ATRA-induced
up-regulation of the PRAM-1 mRNA does not require de
novo protein synthesis (Fig. 1A). As expected, ATRA did
not affect growth and differentiation (Fig. 1B, a
and b, respectively) of NB4.306, an ATRA-resistant NB4
subclone that has lost the capacity to encode an intact ATRA-binding
PML-RAR PRAM-1 Has the Structural Features of a Novel Adaptor
Protein--
A 2356-bp PRAM-1 cDNA was obtained from ATRA-treated
NB4 cells (Fig. 1C). The sequence surrounding a 5' ATG codon
is in agreement with the consensus initiation sequence (32). The 3'
untranslated region ends with a ATAAA polyadenylation signal followed
by a poly(A) tail (33). The complete open reading frame of PRAM-1 consists of 2154 bp translated into a predicted protein of 718 amino
acids. Residues 70-165 comprise a series of eight proline-rich repeats
with the consensus sequence KPP(P/Q)P(E/Q)(V/A/ F)TDLPK. Several
proline residues are clustered as type I
(RXPXXP) or type II
(PXXPXR) SH3 recognition motifs (34). In the
carboxyl-terminal region of PRAM-1, two tyrosine-based motifs have
sequences conforming to the general motif preferentially selected by
Group I SH2 domains (35). PRAM-1 shares homologies with the
hematopoietic-specific adaptor molecule SLAP-130/fyb (21, 22), with a
57% similarity concentrated in the SH3-like domains in the extreme
carboxyl termini of the two proteins. Both PRAM-1 and SLAP-130/fyb
contain a central, proline-rich region. Together, these data suggest
that PRAM-1 is a novel adaptor protein.
PRAM-1 mRNA Is Expressed and Regulated in Primary Hematopoietic
Tissues--
PRAM-1 mRNA was mainly expressed in bone marrow and
peripheral blood leukocytes (Fig.
2A), in freshly purified human
granulocytes and monocytes, as well as, albeit to a much lesser extent,
in lymphocytes (Fig. 2B). PRAM-1 mRNA was not detected
in non-hematopoietic tissues, except in lung, which is known to contain
a number of myelo-monocytic cells (data not shown). Purified
CD34-positive human cord blood cells expressed low levels of the PRAM-1
mRNA (Fig. 2C, b, lane 1), which
increased gradually during myeloid differentiation, with maximal
expression at the promyelocytic stage (Fig. 2C,
b, lane 6). Analysis of leukemia cell lines
assigned to specific hematopoietic lineages supported the notion that
PRAM-1 is mainly expressed in myeloid cells (data not shown). Together, these results indicate that PRAM-1 mRNA is expressed and regulated during normal myelopoiesis.
PML-RAR
PRAM-1 mRNA was highly induced by in vitro treatment
with ATRA of blasts freshly obtained from peripheral blood of newly
diagnosed patients with APL (Fig. 3C) expressing the
PML-RAR PRAM-1 Is an Adaptor Molecule--
Using COS-7 cells transfected
with a PRAM-1-expressing vector, anti-PRAM-1 polyclonal
antibodies detected a specific protein migrating with an
apparent molecular mass of 97 kDa (Fig.
4A, a). The
discrepancy between the predicted molecular mass of 79 kDa and its
migration on SDS-PAGE was probably due to numerous prolines and charged
residues in PRAM-1, as also noted for the SLP-76 (37) and SLAP-130/fyb
(21) adaptor proteins. In ATRA-treated NB4 cells, endogenous PRAM-1
migrated at the same rate as ectopically expressed PRAM-1 in COS-7
cells (Fig. 4A, a), and was
tyrosine-phosphorylated upon pervanadate treatment (Fig. 4A,
b). In kinetic studies, PRAM-1 protein expression was
induced after 6 h of ATRA treatment and persisted through 6 days
(Fig. 4B).
PRAM-1 shares sequence homologies with the adaptor protein
SLAP-130/fyb, which can associate with SLP-76 (21, 22) and SKAP-HOM
(also known as SKAP55-R) in T cells (29, 38). Upon finding that SLP-76
was expressed constitutively and that SKAP-HOM was induced by ATRA in
NB4 cells (Fig. 4B), we investigated whether PRAM-1
associates with these proteins. First, we documented the association of
SLP-76 and SKAP-HOM with PRAM-1 by coexpressing PRAM-1 tagged with the
F domain of the human estrogen receptor (F-PRAM-1) with Flag-tagged
SLP-76 or Flag-tagged SKAP-HOM in COS-7 cells. Fig. 4C,
a shows that in these cells Flag-SLP-76 and F-PRAM-1, as
well as Flag-SKAP-HOM and F-PRAM-1 proteins, were detected in
anti-F immunoprecipitates. We next asked whether PRAM-1 can associate
with SKAP-HOM and SLP-76 in leukemic cell lines. As shown in Fig.
4C, b, PRAM-1 and SKAP-HOM from ATRA-treated NB4
cell lysates were detected in SKAP-HOM and PRAM-1 immunoprecipitates, respectively. We also observed that PRAM-1 was detected in SLP-76 immunoprecipitates, and SLP-76 was found in PRAM-1 immunoprecipitates from lysates of NB4 cells treated with both ATRA and pervanadate (Fig.
4C, c). Together, these data indicate that the
PRAM-1 adaptor protein can associate with SLP-76 and SKAP-HOM in
ATRA-treated NB4 cells. Because PTK have been shown to be induced in
RA-treated HL-60 cells (39), we investigated whether PRAM-1 and lyn can associate in ATRA-treated NB4 cells. As shown in Fig. 4D,
both PRAM-1 and lyn from ATRA-treated NB4 cell lysate were detected in
PRAM-1 immunoprecipitate. Altogether, our results strongly suggest that
PRAM-1 is an adaptor protein interacting with proteins involved in an
ATRA-signaling pathway.
We identified a novel adaptor protein, PRAM-1, which is induced by
ATRA in human leukemia promyelocytes and is expressed and regulated
during normal myelopoiesis. That PML-RAR Adaptor proteins have achieved increasing prominence in recent years
because of recognition of their roles in numerous cellular processes
(40-42). Much evidence supports the hypothesis that these proteins
exert their effects upon differentiation through assembly of
cytoplasmic signaling complexes that are required to modulate transcription. PRAM-1 shares homologies with SLAP-130/fyb, an adaptor
protein initially identified through associations with SLP-76 (22) and
fyn (21). The SLAP-130 proline-rich region mediates binding to the SH3
domains of two related proteins, SKAP-HOM (38) and SKAP55 (43).
Phosphorylated tyrosines amino-terminal to the SLAP-130 SH3-like motif
mediate binding to SLP-76 (44, 45). Similar domains within PRAM-1 may
mediate the association between PRAM-1 and SLP-76 or PRAM-1 and
SKAP-HOM. Given the observations of shared structure and binding
partners for PRAM-1 and SLAP-130, the two molecules may occupy a
similar functional niche. Myeloid cells express SLAP-130 (21), and
several observations suggest that it plays a role in myeloid signal
transduction through Fc receptors (46) by coupling Fc
receptor-stimulated protein tyrosine kinases to downstream signaling
events. Furthermore, in murine bone marrow-derived macrophages,
SLAP-130 is phosphorylated and inducibly associated with SLP-76 after
exposure to antibody-coated sheep red blood
cells.3 Our data showing that
PRAM-1 is structurally similar to SLAP-130 and that it inducibly
associates with SLP-76 thus reinforces the view that it may also
regulate signaling through Fc receptors. SKAP-HOM, which can associate
with PTK fyn (29), forms a complex with SLAP-130, which associates with
the macrophage inhibitory receptor SHPS-1 (47). Our observation that
PRAM-1 associates with SKAP-HOM raises the possibility that PRAM-1
might also participate in signaling through additional myeloid
receptors. Furthermore, SKAP-HOM can associate with PTK lyn (48), which
is induced and tyrosine-phosphorylated in RA-treated myeloid leukemia
cells. Coordinate induction of PRAM-1, SKAP-HOM, and lyn protein
expression by ATRA in NB4 cells, coupled with capacity of the two
adaptors to associate together and with lyn, suggest that PRAM-1,
SKAP-HOM, and lyn may be functionally linked in a common
ATRA-dependent signaling pathway. The PTK lyn prevents
apoptosis to promote RA-induced differentiation of HL-60 cells (39).
Whether PRAM-1 is involved in this process remains to be determined.
Much attention has been given recently to cytosolic signalings in APL
cell differentiation (11). PML-RAR (RAR
) fusion protein.
Complete remission of acute promyelocytic leukemia can be obtained by
treating patients with all-trans retinoic acid, and
PML-RAR
plays a major role in mediating retinoic acid effects in
leukemia cells. A main model proposed for acute promyelocytic leukemia
is that PML-RAR
exerts its oncogenic effects by repressing the
expression of retinoic acid-inducible genes critical to myeloid
differentiation. By applying subtraction cloning to acute promyelocytic
leukemia cells, we identified a retinoic acid-induced gene, PRAM-1
(PML-RAR
target gene encoding an
Adaptor Molecule-1), which encodes a novel
adaptor protein sharing structural homologies with the SLAP-130/fyb
adaptor. PRAM-1 is expressed and regulated during normal human
myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is
inhibited by expression of PML-RAR
in the absence of ligand and
de novo superinduced by retinoic acid. PRAM-1 associates
with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and
with protein tyrosine kinase lyn. By providing the first evidence that
PML-RAR
dysregulates expression of an adaptor protein, our data open
new insights into signaling events that are disrupted during
transformation by PML-RAR
and induced by retinoic acid during
de novo differentiation of acute promyelocytic leukemia cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(RAR
) fusion between the
amino-terminal region of the PML locus (2-5) and the
carboxyl terminus of the RAR
(6, 7). The causal role of the
PML-RAR
fusion protein in APL has been demonstrated in transgenic
mice (8), although its effects do not appear to confer a fully
malignant phenotype in murine promyelocytes (9). Leukemic
transformation is likely to initiate in a subpopulation of
hematopoietic stem/progenitor cells (10). In these cells, PML-RAR
confers resistance to growth factor starvation (10), presumably
exerting its oncogenic effect through repression of the genetic program
normally leading to full myeloid differentiation. Because external
signals such as cytokines regulate limited expansion and maturation of
hematopoietic progenitor cells in the bone marrow, PML-RAR
may
initiate leukemia transformation in these cells by disrupting
differentiation signaling pathways. However, though cytosolic
signalings in APL cell differentiation are getting increasing attention
(11), genes whose expression is repressed by PML-RAR
remain poorly
identified (9). Complete remission of APL can be obtained by treating
patients with all-trans retinoic acid (ATRA) (12-15), which
induces full differentiation of leukemic cells (15). This suggests that
pharmacological levels of ligand can reverse the oncogenic effects of
PML-RAR
. It is likely that PML-RAR
mediates the sensitivity of
APL blasts to ATRA-induced differentiation. Indeed, ligand binding to
the fusion protein induces the release of transcriptional repression
(16), and PML-RAR
may have an ATRA-dependent activator
function (17). However, little is known about specific signaling
through which ATRA induces differentiation.
target gene encoding an
Adaptor Molecule-1). The predicted PRAM-1 protein contains proline-rich and SH3-like domains and shares structural homologies with the adaptor SLAP-130/fyb (21, 22). Ectopic
expression of PML-RAR
in U937 myeloid precursor cells inhibits
PRAM-1 expression, which is superinduced upon treatment with ATRA.
Furthermore, PRAM-1 is expressed and regulated early during in
vitro differentiation of primary human hematopoietic stem/progenitor cells. Finally, PRAM-1 interacts with known partner molecules of SLAP-130/fyb, such as SLP-76 and SKAP-HOM, as well as with
the PTK lyn. These observations point to PRAM-1 as an adaptor protein
with potentially important roles in leukemogenesis and myeloid differentiation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 × 106 cells/ml) were seeded at 2 × 105
cells/ml 16 h prior to ATRA treatment. U937-PR9 and U937-MT cells were used for induction of PML-RAR
expression or as control, respectively. Cells were cultured either with or without varying concentrations of ZnSO4 (Sigma) and ATRA (Sigma).
Differentiation was assessed by (i) the percentage of cells with nitro
blue tetrazolium (Sigma) deposits, as described (24), and (ii) cell
morphology under light microscopy on cytospin slides stained with
May-Grünwald-Giemsa (Sigma).
gene were
obtained from patients diagnosed at Saint-Louis Hospital (Paris,
France) and kindly provided by C. Chomienne. Cells were purified and
cultivated with 10
7 M ATRA as
described (14). 5 days after treatment, cells were tested for
differentiation (
80% nitro blue tetrazolium-positive). Mononuclear
cells and granulocytes were isolated from peripheral blood using a
Ficoll Paque (Amersham Pharmacia Biotech) density gradient. Monocytes
were further purified from lymphocytes using an indirect labeling
system followed by a magnetic separation on a macs column (Myltenyi
Biotec). Cell purity assessed by May-Grünwald-Giemsa was
95%.
7 M for 48 h) using the
Marathon cDNA amplification kit
(CLON- TECH) with a
5'-CCTTGGAAACGGAGTGGCCCCCAGC-3' primer. Sequence homologies were
identified using the FastA and BLAST programs by searching
GenBankTM, EMBL, and the Incyte Genomics Lifeseq® data
base. Sequence information from many different Incyte Genomics data
base clones was used to confirm and extend the results of RACE-PCR
experiments. For example, clone 3418957H1 is 100% identical to the 5'
terminal end of the final PRAM-1 RACE product clone. Using the Marathon cDNA and the Advantage-GC polymerase
(CLONTECH), a contiguous cDNA was generated by
reverse transcription-PCR amplification of PRAM-1 from ATRA-treated NB4
RNA using 5'-GGCCCCAGCTCGGGTCCCACTCATC-3' and
5'-TGGCTGTCCTGGCCCCACGCCTACC-3' primers. PCR products were subcloned into pT-Adv (CLONTECH) and sequenced. All
PCR-generated cDNAs were confirmed by multiple, independent
derivations from RNA templates.
-actin mRNA in
differentiating cells was analyzed by semi-quantitative PCR using a
GeneAmp PCR System 9600 (PerkinElmer Life Sciences) with
the following primers corresponding to distinct exon sequences: sense
(5'-CCTCAGTTCAGCAAGCCGCCAGGAG-3') and antisense
(5'-CCAGGGGGAGTGGTTGGTTTTCCAG-3') for PRAM-1; sense
(5'-CCTCGCCTTTGCCGATCC-3') and antisense
(5'-GGATCTTCATGAGGTAGTCAGTC-3') for
-actin. PCR cycles were
conducted with (i) denaturation for 45 s at 94 °C, (ii)
annealing for 45 s at 60 °C, and (iii) extension for 1 min at
72 °C, followed by an extension final step at 72 °C for 7 min.
The number of PCR cycles was determined for PRAM-1 (35 cycles) and
-actin (25 cycles) to ensure quantification of the PCR products in a
linear range of amplification. The
-actin PCR reaction was performed
using cDNA prepared at each day of the kinetic to determine
the dilutions needed to provide equal amounts of reverse transcription
products for all samples. Aliquots of each PCR reaction were
electrophoresed, transferred onto nylon filters (Hybond-N,
Amersham Pharmacia Biotech), and hybridized with the PRAM-1
(5'-CGGCACAGCATCTCCTCATTG-3') and
-actin
(5'-ACCCCGTGCTGCTGACCGAGG-3') 32P-labeled probes as
described (25). Radioactivity was quantified using a Storm 860 phosphorimaging system (Amersham Pharmacia Biotech).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
fusion protein (23). In these cells, no induction of PRAM-1
mRNA was observed (Fig. 1B, c). In contrast,
expression of PRAM-1 mRNA was strongly up-regulated in NB4 cells
induced to growth arrest and differentiation by ATRA (Fig.
1B, a and b, respectively). These data
strongly suggest that increased PRAM-1 mRNA expression correlates
with the capacity of leukemia cells to undergo granulocytic
differentiation when treated with ATRA.
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Fig. 1.
PRAM-1 is induced in ATRA-treated NB4 cells.
A, time course in response to 5 × 10 7 M ATRA in NB4 cells. Where
indicated, de novo protein synthesis was inhibited by
pretreating cells for 30 min with 10 µg/ml cycloheximide
(CHX) followed by an additional 8 h in the absence
(0) or presence (8) of ATRA. B, PRAM-1
expression correlates with the capacity of NB4 cells to differentiate.
NB4 and NB4.306 cells cultured with 10
6
M ATRA were harvested after 0, 24, and 48 h. The
percent of viable (a) and nitro blue tetrazolium-positive
(b) cells is shown, as well as the level of PRAM-1 mRNA
expression (c). Northern blots were performed using 5 µg
(A) or 3 µg (B) of total RNA. Top
and bottom panels show signal obtained with PRAM-1 and GAPDH
(to control for RNA quantities) probes. C, cDNA
and deduced amino acid sequences of the human PRAM-1. Nucleotide
residues are numbered in the 5' to 3' orientation, and amino acids in
the reading frame are designated by the one-letter code. Kozak and
poly(A) addition sequences are underlined, and the
asterisk indicates the stop codon. The eight proline-rich
repeats are underlined in bold. Proline residues
clustered as either a type I or type II SH3 recognition motif are
underlined with dotted lines. The
SH3-like carboxyl-terminal domain is boxed. Tyrosine
residues within motifs that bind SH2 domains are
shaded.
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Fig. 2.
Expression of PRAM-1 mRNA in primary
hematopoietic tissues. A and B,
autoradiograms of PRAM-1 mRNA expression in immune tissues and
peripheral blood cells. Northern blots were obtained from
CLONTECH (A) or were performed using 5 µg of total RNA from granulocytes, monocytes, and lymphocytes
(B). In A and B, GAPDH was used as a
probe for assessment of RNA quantities in each lane.
PBL, peripheral blood leucocytes. C,
PRAM-1 mRNA is up-regulated during myeloid differentiation of human
hematopoietic stem/progenitor cells. a, morphological
differentiation of CD34+ cells toward the myeloid lineage.
b, Southern blot analysis (upper panels) and
relative expression (lower panel) of reverse
transcripton-PCR products from CD34+ cells differentiating
toward the myeloid lineage. Expression of -actin mRNA was
assessed as a control. Error bars indicate standard deviations from the
results of three independent PCRs. This experiment was performed a
second time with independent cell cultures, with similar results. A
parallel PCR was conducted without addition of cDNA as a negative
control (NC) or cDNA obtained from ATRA-treated NB4
cells as a positive control (PC).
Inhibits, and ATRA Subsequently Superinduces, Expression
of PRAM-1 mRNA in U937 Cells--
In contrast to NB4 cells, PRAM-1
mRNA was not induced in NB4.306 cells, suggesting that an intact
PML-RAR
is required for ATRA to regulate PRAM-1 mRNA expression.
To explore further the role of PML-RAR
in the expression of PRAM-1
mRNA, we used cell lines generated by stable transfection into
human U937 myeloid precursor cells of a zinc-inducible vector without
an exogenous cDNA (U937-MT) or driving the PML-RAR
cDNA
(U937-PR9) (17). Zinc-induced expression of the PML-RAR
protein in
U937-PR9 cells resulted in an 8-fold decrease of PRAM-1 mRNA
expression (Fig. 3A,
lane 2 versus lane 1). Subsequent ATRA
treatment of these cells resulted in a 60-fold increase of PRAM-1
mRNA expression at 24 h (Fig. 3A, lane 6 versus lane 2). No repression was observed in
zinc-treated U937-MT cells (Fig. 3A, lane 8 versus lane 7), and a 3-fold increase was
observed in these cells with ATRA treatment (Fig. 3A,
lane 12 versus lane 7), as expected,
because U937 cells express an active retinoic acid receptor (36).
U937-PR9 and U937-MT cells were then left untreated or treated with
increasing concentrations of ZnSO4 with or without ATRA
(Fig. 3B). This dose-response experiment indicates that
repression and ATRA inducibility of PRAM-1 correlates with the amount
of the PML-RAR
expressed. Together, these results suggest that
PRAM-1 is a PML-RAR
target gene.
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Fig. 3.
PRAM-1 mRNA expression is dysregulated by
PML-RAR . A, PRAM-1 mRNA
expression is down-regulated by ectopic expression of PML-RAR
in
U937 cells and markedly increased following ATRA treatment.
Autoradiogram (upper panels) and relative expression
(lower panel) of the PRAM-1 mRNA in U937-PR9 and U937-MT
cells either untreated (
) or treated (+) with
100 µM ZnSO4 for 12 h, followed by
exposure to 5 × 10
7 M ATRA
for different times. B, autoradiogram (left
panels) and relative expression (right panel) of PRAM-1
mRNA in U937-PR9 and U937-MT cells treated with increasing
concentrations of ZnSO4. Cells were either untreated or
treated with 5 × 10
7 M ATRA
(
ATRA and +ATRA, respectively) for 12 h.
The signal was quantified using densitometric analysis of
phosphorimaging data. PRAM-1 mRNA quantities were adjusted for
GAPDH expression. In A (lower panel) and B
(right panel), mean values and standard errors from two
independent experiments are shown. C, PRAM-1 mRNA is
induced in ATRA-treated primary APL cells. Cells were purified from
three untreated patients cultivated in the absence (
) or
presence (+) of 10
7 M
ATRA for 5 (APL#2) or 6 (APL#1 and
APL#3) days. Northern blots were performed using 8 µg
(A and B) or 2 µg (C) of total RNA.
Hybridization with a GAPDH probe controlled for RNA quantities in each
lane.
gene. This induction correlated with ATRA-induced
differentiation of these cells, as assessed morphologically and by
nitro blue tetrazolium reduction assay (data not shown).
View larger version (35K):
[in a new window]
Fig. 4.
PRAM-1 is an adaptor molecule.
A, PRAM-1 protein is induced upon ATRA treatment in NB4
cells and is tyrosine-phosphorylated. a, COS-7 cells
transfected with pMT-PRAM-1 were either untreated ( ) or
treated (Zn) with 75 µM ZnSO4 for
24 h. NB4 cells were either untreated (
) or treated
(RA) with 5 × 10
7
M ATRA for 12 h. COS-7 cell lysates (10 µg) and
protein extracts from 105 NB4 cells were separated by
SDS-PAGE and immunoblotted for PRAM-1. b, NB4 cells were
treated with 10
6 M ATRA for
72 h and either left unstimulated (
) or stimulated
with pervanadate (PV) for 5 min. Cells were then lysed,
subjected to immunoprecipitation with anti-PRAM-1 antiserum, and then
assessed for phosphotyrosine (4G10) or PRAM-1. B,
expression of PRAM-1, SKAP-HOM, and SLP-76 during ATRA-induced
differentiation of NB4 cells. NB4 cells were treated with 5 × 10
7 M ATRA for different times.
Protein extracts corresponding to 105 cells were analyzed
by Western blot, using anti-PRAM-1, -SKAP-HOM, and -SLP-76 polyclonal
antibodies. C, PRAM-1 associates with SKAP-HOM and SLP-76.
a, vectors encoding F-PRAM-1, Flag-SLP-76, and Flag-SKAP-HOM
were transfected (+) into COS-7 cells. 25-µg aliquots of
each protein extract were immunoprecipitated (IP) with
monoclonal anti-F antibodies. Immunoprecipitated proteins (right
panels), as well as a 10-µg aliquot of the protein extracts
(left panels) were separated by SDS-PAGE and analyzed for
PRAM-1, SLP-76, and SKAP-HOM (Western blot, WB).
b, protein extracts from ATRA-treated NB4 cells were
immunoprecipitated (IP) with anti-PRAM-1 or anti-SKAP-HOM
polyclonal antibodies or with control PRAM-1 pre-immune serum
(control antibody, Control Ab).
Immunoprecipitated proteins were separated by SDS-PAGE and transferred
onto nitrocellulose. After Western blotting, membranes were hybridized
with anti-PRAM-1 or anti-SKAP-HOM polyclonal antibodies. The
arrowhead indicates the heavy chain of immunoglobulins
(Ig (H)). c, ATRA-treated NB4 cells were either
unstimulated (
) or stimulated (+) with
pervanadate for 5 min. Lysates were subjected to immunoprecipitation
with anti-SLP-76 or anti-PRAM-1 antisera or with the corresponding
pre-immune serum. The resulting complexes were resolved by SDS-PAGE and
analyzed for SLP-76 or PRAM-1. The asterisk indicates a
nonspecific band. D, PRAM-1 associates with lyn in
ATRA-treated NB4 cells. 120-µg aliquots of the ATRA-treated NB4 cell
extracts were immunoprecipitated with anti-PRAM-1 polyclonal antibodies
or with control PRAM-1 pre-immune serum (Control Ab).
Immunoprecipitated proteins (right panels), as well as
40-µg aliquots of untreated or ATRA-treated protein extracts
(left panels), were separated by SDS-PAGE and transferred
onto polyvinylidene difluoride. Membranes were hybridized with
anti-PRAM-1 or anti-lyn antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
down-regulates expression
of PRAM-1 mRNA in the absence of ligand indicates that PRAM-1 may
be an oncogenic target of the fusion protein. Furthermore, superinduction of PRAM-1 by ATRA when PML-RAR
is expressed suggests that this adaptor may be an important component of a signaling pathway
through which ATRA induces differentiation in leukemia promyelocytes.
confers marked resistance to
hematopoietic growth factor starvation to hematopoietic stem/progenitor
cells (10). Therefore, dysregulation of PML-RAR
target genes might
result in abnormal signaling, thereby freeing pre-leukemic cells from
controls that normally induce the onset of differentiation. This, in
association with other genetic dysregulations, may contribute to full
leukemic transformation. It is also likely that treatment of APL cells
by ATRA induces de novo up-regulation of the same genes that
are dominantly repressed by PML-RAR
and whose expression is required
for reactivation of the differentiation program (16). To date, the
myeloid transcription factor CCAAT/enhancer-binding protein
is the only example of such a gene (49). PRAM-1 is a first example of
an adaptor molecule whose expression is inhibited and superinduced when
PML-RAR
is expressed alone and in the presence of ATRA,
respectively. The evidence that PRAM-1 mRNA expression is inhibited
by PML-RAR
, together with the association of PRAM-1 with other
RA-induced proteins including lyn, suggest that the fusion protein may
indirectly dysregulate the organization of molecular complexes
comprising scaffold proteins and therefore disrupt signaling events
important for ATRA-induced differentiation.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. P. G. Pelicci for generous gifts of U937-PR9 and U937-MT cell lines, C. Nervi for the NB4.306 cell line, M. Lanotte for NB4 cells, C. Chomienne and N. Balitrand for ATRA-treated APL cells, B. Schraven for the SKAP-HOM-expressing vector and anti-SKAP-HOM antibodies, and V. Witko-Sarsat for help in human blood cell purifications. We thank S. Axisa-Coon and F. C. Guibal for their interest in the work. Sequencing was conducted by C. Cruaud at the Center National de Séquençage (Evry, France) and by the Nucleic Acid/Protein Research Core Facility at the Children's Hospital of Philadelphia.
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FOOTNOTES |
---|
* This work was supported in part by INSERM and by grants from the Fondation de France, the Ligue Nationale Contre le Cancer, the Association Combattre La Leucémie, the Association pour la Recherche sur le Cancer, the Lady Tata Memorial Trust, and the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the EMBL/GenBankTM/EBI Data Bank with accession number AJ272324.
b These authors contributed equally to this work.
c A fellow of the Leukemia Research Foundation.
g Supported by a Mentored Clinician Scientist Development Award from the National Institutes of Health.
j These authors should be considered as equal last authors.
k To whom correspondence should be addressed. Tel.: 33149284613; Fax: 33143406837; E-mail: cayre@st-antoine.inserm.fr.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M011683200
2 C. Moog-Lutz, unpublished results.
3 P. Myung and G. Koretzky, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
APL, acute
promyelocytic leukemia;
PML, promyelocytic leukemia;
RA, retinoic acid;
RAR, RA receptor
;
ATRA, all-trans retinoic acid;
PTK, protein tyrosine kinase;
SH, Src homology domain;
bp, base pair(s);
RACE, rapid amplification of cDNA ends;
PCR, polymerase chain
reaction;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
PAGE, polyacrylamide gel electrophoresis.
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