Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France BP 163, 67404 ILLKIRCH Cedex CU de Strasbourg, France
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ABSTRACT |
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RA exerts its pleiotropic effects through two classes of nuclear
ligand-dependent transregulators: the retinoic acid receptors (RAR,
RARß, and RAR
isotypes and their isoforms) activated by either
all-trans-RA or its 9-cis-isomer and the retinoid
X receptors (RXR
, RXRß and RXR
) activated by
9-cis-RA only (811). F9 cells express all RARs and RXRs
(1214), and two strategies have been used to investigate their roles
in the response of F9 EC cells to RA treatment. Firstly, using
homologous recombination, we engineered F9 cells in which either the
RAR
, the RAR
, or the RXR
gene, both the RAR
and RXR
genes or both the RAR
and RXR
genes, are knocked out (6, 7, 15,
16). Secondly, wild-type (WT) and mutant F9 cells were treated with
pan-RXR- and RAR isotype (
, ß, or
)-selective retinoids (7,
1720). It was established that RAR
is indispensible for RA-induced
differentiation of F9 EC cells into primitive endoderm-like cells,
whereas RAR
is additionally required for efficient parietal
endodermal differentiation that occurs in the presence of RA and cAMP
(20), and for which primitive endodermal differentiation is a
prerequisite. These studies also demonstrated that RAR/RXR heterodimers
are the functional units transducing the retinoid signal in
vivo (7, 19).
RARs and RXRs possess two transcriptional activation functions (AFs):
AF-1, located in the N-terminal A/B region, and AF-2, associated with
the ligand binding domain (LBD) (region E) and activated by agonistic
ligands (8, 2124). The integrity of a conserved amphipatic -helix,
referred to as the AF-2AD core, is required for AF-2 activity (23,
2528, and references therein). The AF-2AD core is located in the
C-terminal
helix of the LBD (helix 12) that is indispensible for
the formation of the coactivator-binding surface generated during the
LBD transconformation triggered by ligand binding (8, 2931).
In addition, RARs and RXRs are phosphorylated in their AF-1 domain that
contains sites for proline- directed kinases (20, 32, 33). In
RAR1, the phospho-rylated residue in the AF-1domain has been
identified as serine 77 (32). Furthermore, RAR
1 can be
phosphorylated by PKA at serine 369 that is located in the LBD/AF-2
domain (34). Interestingly, RAR
1 is phosphorylated at these two
residues in F9 cells, as well as in transfected COS cells (20, 34).
Similar phospho-rylation sites are present in other RARs and have
been shown to be phosphorylated in RAR
2, which is the major RAR
isoform in F9 cells (34, 35). The role of RAR
phosphorylation in
retinoid-induced events has been studied in
RAR
-/- F9 cells, by establishing rescue cell
lines reexpressing either RAR
WT or RAR
mutated at its
phosphorylation sites (20). The RAR
AF-1 domain and the
proline-directed phosphorylation sites located in this domain were
found to be required for rescuing the differentiation of F9 cells into
primitive endoderm-like cells, whereas the PKA phosphorylation site was
dispensible (20). As we had previously established that overexpression
of RAR
in RAR
-/- cells could also restore
their primitive and parietal endodermal differentiation (14), the role
of RAR
phosphorylation was similarly studied using overexpressed
RAR
mutants. We concluded that both the RAR
AF-1 and AF-2
phosphorylation sites were not required for allowing overexpressed
RAR
to functionally replace RAR
for primitive endodermal
differentiation, but they were apparently mandatory for parietal
differentiation (20).
Thus, RAR and its phosphorylation sites could be selectively
required for parietal endodermal differentiation. However, an
unequivocal demonstration of this RAR
-selective function requires to
establish rescue cell lines reexpressing RAR
(either WT or mutated)
in RAR
-/- F9 EC cells that can still
differentiate into primitive endodermal cells (16) but whose parietal
endodermal differentiation is greatly delayed (20), thus providing a
model for directly analyzing the contribution of RAR
AF-1 and AF-2
and of their phospho-rylation sites in parietal endodermal
differentiation. Furthermore, such lines also offer the possibility to
study which RAR
mutations may generate dominant negatives,
preventing endogenous RAR
from mediating primitive endodermal
differentiation. Rescue lines were therefore established with RAR
mutated at either one of its phosphorylation sites, as well as with
AF-1 or AF-2AD core deletion mutants. We demonstrate here that the PKA
phosphorylation site and the AF-2AD core (helix 12 of the LBD) of
RAR
are required for parietal endodermal differentiation, whereas
the AF-1 proline-directed kinase phosphorylation site is dispensible.
None of the corresponding mutants behaved as dominant negatives,
preventing primitive endodermal differentiation, whereas deletion of
the AF-1 activating domain (the A/B region) generated such a dominant
negative mutant. The present study also shows that RAR
AF-1 and AF-2
are differentially implicated in the induction of RA-responsive genes,
whereas their phosphorylation sites are not involved.
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RESULTS |
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Role of RAR AF-1 and AF-2 and of Their Phosphorylation Sites in
F9 Cell Primitive and Parietal Endodermal Differentiation
When grown as monolayers in the presence of
all-trans-RA alone, WT F9 cells differentiate at 96 h
into primitive endoderm-like cells exhibiting a characteristic flat
triangular morphology (4, 20). Addition of cAMP along with RA, results
in the formation of parietal endoderm-like cells that have a rounded
and refractile appearance (1, 20).
WT F9 cells also differentiated into primitive endoderm-like cells when
treated for 96 h with a combination of a RAR agonist (BMS961)
and a pan-RXR agonist (BMS649) (36) (Fig. 2b
). Parietal endodermal differentiation
occurred upon subsequent addition at 48 h of the RAR
agonist
BMS753 (36), the pan-RXR agonist BMS649, and cAMP (Fig. 2c
). No
parietal differentiation occurred at 96 h (or even later at
120 h) in the absence of BMS753, confirming that RAR
is
required for parietal endodermal differentiation of WT F9 cells (Ref.
20 and data not shown).
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Reexpression of RARWT (RAR
WT cell line) restored parietal
endoderm differentiation at 96 h (Fig. 2i
). The two RAR
S74/77A
lines also differentiated efficiently into parietal endoderm-like cells
(Fig. 2l
, and data not shown), indicating that RAR
can efficiently
mediate this differentiation in the absence of AF-1 phosphorylation. In
contrast, the RAR
S369A rescue line did not differentiate into
parietal endoderm at 96 h (Fig. 2o
). As in the case of the
parental RAR
-/- cell line, parietal
endodermal differentiation was delayed and occurred only at 120 h
(data not shown). The same observation was made with the RAR
AF2
rescue line (Fig. 2u
). Interestingly, the RAR
AB rescue line
retained a stem cell morphology in response to addition of BMS961 and
BMS649. Only a few differentiated cells were observed upon subsequent
addition of BMS753, BMS649, and cAMP (Fig. 2
, pr), thus suggesting
that deletion of the A/B region generates a RAR
dominant negative
mutant that prevents endogenous RAR
from mediating primitive
endodermal differentiation and subsequently suppresses parietal
endodermal differentiation.
The extent of differentiation of the various rescue lines was also
assessed by the expression of transcripts of two markers of primitive
endodermal differentiation, collagen IV (1), and laminin B1 (4, 37),
and of one marker specific for parietal endodermal differentiation,
thrombomodulin (2, 38). Semiquantitative RT-PCR analysis showed that
the induction of collagen IV and laminin B1, which was not affected in
RAR
-/- cells (Fig. 3A
, lanes 46), was similar in all
rescue cell lines, except the RAR
AB line where it was
significantly reduced (Fig. 3A
, lanes 1618), in agreement with the
absence of obvious morphological differentiation (see above).
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Effect of RAR AF-1 and AF-2 Activating Domains and of Their
Phosphorylation Sites on Expression of RA-Responsive Genes
Whereas knockout of the RAR gene in F9 cells results in a
marked reduction of the expression of several RA-responsive genes such
as Hoxa-1, HNF3
, HNF1ß, Stra6, and Stra8 (19, 20), knockout of
RAR
had no effect on their expression (Fig. 4
, compare lanes 2 and 4; Fig. 5
, compare lanes 6 and 7 and Fig. 6
, lane 2). In fact, only Hoxb-1 and
CRABP II gene expression was significantly reduced in
RAR
-/- cells (Fig. 4
, compare lanes 2 and 4;
Fig. 5
, compare lanes 6 and 7 and Fig. 6
, lane 2) (16, 19). The
expression of these RA-responsive genes was investigated in the various
rescue cell lines using semiquantitative RT-PCR (for each gene, in the
linear range of the PCR amplification reaction) after treatment for
24 h with 100 nM RA or a synthetic retinoid selective
for RAR
(BMS961) at 100 nM. The results that are
presented in Figs. 4
and 5
are summarized in Fig. 6
.
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We previously concluded that the retinoid-induced expression of Hoxb-1
and CRABPII could be mediated by both RAR-RXR and RAR
-RXR
heterodimers (19). However, the expression of both genes, in WT F9
cells, was less efficiently induced by a retinoid selective for RAR
(BMS961) than by RA (Fig. 5
, compare lanes 6 and 11 and Fig. 6
, compare
lanes 1 and 8). Interestingly, this latter retinoid was more efficient
in RAR
-/- cells (Fig. 6
, compare lanes 8 and
9), indicating that the presence of RAR
was to some extent
preventing RAR
to mediate the induction of Hoxb-1 (19), and CRABPII.
Note the down-effect of reexpressing RAR
WT (Fig. 6
, lane 10), which
was not observed with the two deletion mutants (Fig. 6
, lanes 11 and
12).
Genes whose expression was not affected in
RAR-/- cells, e.g. Hoxa-1,
HNF3
, HNF1ß, Stra6, and Stra8, were also analyzed in the rescue
lines. Their induction was not significantly affected in RAR
WT,
RAR
S74/77A, and RAR
S369A rescue lines (Fig. 6
, lanes 35). Note
that HNF3
expression was mildly reduced in the RAR
S74/77A line
corresponding to clone 14 (Fig. 4
, lane 10), which expressed higher
RAR
levels than the WT rescue line (Fig. 1B
, lane 5). The
RAR
AB and RAR
AF2 deletion mutants did not affect the
RA-induced expression of Hoxa-1 but inhibited markedly those of HNF3
and HNF1ß (Fig. 6
, lanes 6 and 7). On the other hand, only
RAR
AF2 mildly decreased the RA-inducibility of Stra6 and Stra8
(Fig. 6
, lanes 6 and 7).
The RAR selective agonist (BMS961) was almost as efficient as
RA in inducing the expression of Hoxa-1, HNF3
and Stra8 in WT and
RAR
-/- cells (Fig. 5
, compare lanes 6 and
11; Fig. 6
, compare lanes 1 and 8). Whereas Hoxa-1 expression was
unaffected by reexpressed RAR
AB and RAR
AF2, that of HNF3
was reduced and that of Stra8 was reduced only by RAR
AF2 (Fig. 6
, lanes 11 and 12). Note that the basal level of expression of Stra8 was
also decreased by this mutant (Fig. 5
, lane 5). In contrast, the
RAR
-selective retinoid was more efficient in
RAR
-/- cells than in WT F9 cells for
inducing not only the expression of Stra6 (19), but also that of
HNF1ß (Fig. 6
, lanes 8 and 9), confirming that maximal induction of
some genes by RAR
may be competed for by RAR
(see above). Note,
in that respect, the effect of reexpressing RAR
WT (Fig. 6
, lane 10).
Note also that in some experiments, the basal levels of HNF1ß
expression were mildly up-regulated in
RAR
-/- cells (Fig. 5
, lane 2). RAR
AF2
was as efficient as RAR
WT in preventing maximal induction of Stra6
by RAR
but completely abrogated the activation of HNF1ß by BMS961
(Fig. 6
, lane 12). RAR
AB also abrogated the activation of HNF1ß
but did not inhibit the activation of Stra6 by the RAR
agonist (Fig. 6
, lane 11).
We also analyzed the expression of another RA-inducible gene,
CYP26, whose expression has been shown to be mediated by RAR/RXR
heterodimers (40). Accordingly, it was only slightly decreased in
RAR
-/- cells (Fig. 7
, compare lanes 14). The induction of
CYP26, which was maximal at 96 h, was not affected in the various
rescue lines with the exception of the RAR
AB line where it was
markedly reduced (Fig. 7
, lane 10).
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DISCUSSION |
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The RAR AF-2 Activating Domain and Its PKA Phosphorylation Site
Are Required for Efficient Parietal Endodermal Differentiation
The AF-2AD core motif of RARs belongs to the C-terminal
-helix of nuclear receptor ligand binding domains (helix 12), which
is indispensible for the formation of the coactivator-binding surface
during the LBD transconformation triggered by ligand binding, thus
generating transcriptionally active receptors (8, 31). Numerous studies
have demonstrated that the integrity of the AF-2AD core is required for
efficient ligand-dependent interactions with coactivators in
vitro (for reviews, see Refs. 8, 41, 42). In the present
study, we demonstrate that the AF-2AD core of RAR
and therefore its
AF-2 activation function, is required to mediate the effects of RA and
cAMP on F9 cells differentiation in vivo, as RAR
deleted
for this domain is unable to restore parietal endodermal
differentiation. Note that this AF-2 activation function was also found
to be required for RAR
and overexpressed RAR
to restore
primitive endodermal differentiation in
RAR
-/- cells (see Table 1
).
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Collectively these results demonstrate that the contribution of RAR
to parietal endodermal differentiation of F9 cells requires the
integrity of the AF-2 surface interacting with coactivators, as well as
the integrity of the PKA phosphorylation site located in the AF-2
domain. The effect of this cAMP-dependent RAR
LBD phosphorylation is
unknown, but it may modulate the efficiency of ligand-dependent
coactivator/corepressor binding to RAR
, and/or its dimerization with
RXR and thus its DNA binding properties to RA-response elements (34).
Interestingly, PKA-mediated phosphorylation of certain coactivators may
also modulate their interaction with RAR/RXR heterodimers (Refs. 41, 42, 43, 44
and references therein).
Phosphorylation of the AF-1 Activating Domain Is Not Required for
Parietal Endodermal Differentiation, but Deletion of this Domain
Generates a Dominant Negative Mutant for F9 Cell Differentiation
When reexpressed in RAR-/- cells,
RAR
mutated at its proline-directed kinase phosphorylation site in
the N-terminal AF-1 domain (RAR
S74/77A) is as efficient as RAR
WT
at restoring parietal endodermal differentiation, indicating that
phosphorylation of the RAR
AF-1 domain is not required for this F9
cells differentiation. In contrast, we concluded in our previous study
(see Table 1
) that although the RAR
AF-1 phosphorylation site was
apparently not required to allow overexpressed RAR
to restore, at
the morphological level, the primitive endodermal differentiation of
RAR
-/- cells, it was indispensible for
subsequent parietal endodermal differentiation. However, the expression
of several RA-responsive genes was not restored to WT levels in
RAR
-/- cells rescued with RAR
S74/77A (see
Table 1
). It is therefore possible that the phosphorylation of RAR
AF-1 is in fact indispensible in the RAR
-/-
rescued cells to efficiently rescue primitive endodermal
differentiation at the molecular level, which is a prerequisite for
subsequent parietal differentiation, whereas this phosphorylation is
not required for parietal differentiation per se.
Expression of RAR lacking the A/B region in
RAR
-/- cells (RAR
AB rescue line)
abrogates their differentiation into primitive endoderm-like cells.
This must reflect a ligand-independent (see Fig. 2
) dn effect of
RAR
AB on the endogenous RAR
, which is required for primitive
endodermal differentiation. This dn effect of RAR
AB was also
exerted on the expression of certain RA-responsive genes (see below and
Fig. 6
). Note in this respect that we have previously shown (Table 1
)
that RAR
AB overexpressed in RAR
-/-
cells could not restore RA-induced primitive endodermal differentiation
and expression of most RA-induced genes, and thus could not
functionally replace RAR
.
We conclude that the RAR AF-1 proline-directed phosphorylation
site is dispensible for parietal endodermal differentiation of F9
cells, whereas deletion of the A/B region (see Fig. 2
, RAR
AB),
but not of the AF-2AD core (RAR
AF2), results in a dominant
negative receptor that blocks the RAR
-mediated primitive endodermal
differentiation.
Differential Promoter Context-Dependent Effect of the AF-1 and AF-2
Activating Domains of RAR and of Their Phosphorylation Sites on
Expression of RA-Responsive Genes
Among the F9 cell RA-responsive genes that we have studied
here and previously (16, 19), the expression of only two of them
(Hoxb-1 and CRABPII) is reduced in RAR-/-
cells, whereas that of the others is not affected. In the present
study, we have investigated the role played by RAR
AF-1 and AF-2
activating domains and their phosphorylation sites in the expression of
these RA-responsive genes. Our results, summarized in Fig. 6
and in
Table 1
, lead to the following conclusions.
Firstly, the PKA site located in the AF-2 domain and the
proline-directed kinase site located in the AF-1 domain are not
required for restoring the RA-induced expression of Hoxb-1 and CRABPII
genes, which is decreased in RAR-/- cells.
This is in contrast with our previous observation showing that in
RAR
-/- cells overexpressing RAR
, the
phosphorylation of RAR
1 AF-1 (RAR
S74/77A) is required for
RA-induced expression of some other RA-responsive genes (see Table 1
).
Note in this respect that phosphorylation of RAR
AF-1
(RAR
S66/68A) is also required for RA-induced expression of a subset
of responsive genes (see Table 1
).
Secondly, the AF-1 domain and the AF-2AD core of RAR are
differentially required for RA-induced expression of target genes.
Indeed, RAR
deleted for either of these two domains restores the
induction of Hoxb-1, but not of CRABPII. Note that a similar
promoter-context dependent requirement of the AF-1 activating domains
of RAR
and RAR
was also reported for RA-induced expression of
other responsive genes in RAR
-/- rescue
cells (see Table 1
).
Thirdly, both AF-1 and AF-2 deletion mutants of RAR behave as
dominant negatives in a promoter context-dependent manner, as the
induction of RA-responsive genes whose expression is not altered in
RAR
-/- cells (Hoxa-1, HNF3
, HNF1ß,
Stra6, Stra8, and CYP26) was differentially decreased upon reexpression
of RAR
mutants deleted for either the A/B region or the AF-2AD core
(see RAR
AB and RAR
AF2 in Fig. 6
). Note that promoter
context-dependent dn effects have also been observed with
reexpressed RAR
AF2 and overexpressed RAR
AF2 in
RAR
-/- cells (see Table 1
and Ref. 46), in
keeping with the previous suggestion that AF-2 deletion mutants may
behave as dominant negatives for the transcription of RA-responsive
genes (23, 45).
Fourthly, depending also on the promoter context, the presence of
RAR differentially hinders the potential ability of RAR
to
mediate RA-induced expression of a number of responsive genes. Indeed,
in several cases, the RAR
-selective agonist BMS961 was found to be a
more efficient inducing ligand in RAR
-/-
cells than in F9 WT cells (Fig. 6
). These observations confirm that
functional redundancies between RARs can be artefactually generated
upon gene knockout (18, 20), and furthermore clearly show that the
extent of these redundancies is variable, being dependent on the
promoter context of the RA-responsive genes.
Conclusion
Using the cell autonomous RA-responsive F9 EC cell
differentiation system, we have demonstrated in our present and
previous studies (see Table 1) that the AF-1 and AF-2 activation
functions of both RAR
and RAR
, as well as their proline-directed
kinase and PKA phosphorylated forms, play important and distinct roles
in primitive and parietal endodermal differentiation and RA-induced
expression of responsive genes. Moreover, our data show that RAR
and
RAR
AF-1 and AF-2, and their phosphorylated forms exhibit distinct
functions that are differentially required, in a promoter
context-dependent manner, to elicit the induction of RA-responsive
genes expression. Interestingly, the dominant negative effect of some
AF-1 and AF-2 deletion mutants on RAR
- and RAR
-mediated induction
of expression of RA-responsive genes also exhibits a promoter-context
dependency. Finally, a similar promoter-context dependency is also
characteristic of the artefactual functional redundancy generated
between RAR
and RAR
upon RAR
knockout in F9 cells. All of
these promoter-context dependencies certainly reflect the diversity of
the responsive complexes that can be combinatorially assembled on
polymorphic control regions with different receptor isoforms that are
differentially phosphorylated, interact with different coactivators,
and synergize with a variety of transregulators (41, 42).
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MATERIALS AND METHODS |
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Cell Culture and Establishment of Stable Rescue Lines
Fig 9 cells were cultured as monolayers on gelatinized surfaces
as described (15). For differentiation studies,
105 cells were cultured in a 10-cm dish, and
treated with RA (100 nM) alone or in combination with cAMP
(250 µM) for 96 h, with a change of media after
48 h. Cells were also treated with a combination of a RAR
agonist (BMS961, 100 nM) and a pan-RXR agonist (BMS649, 1
µM), followed by the subsequent addition at 48 h of
a RAR
agonist (BMS753, 100 nM), the pan-RXR agonist and
cAMP. Control cells were treated with vehicle alone (0.1% ethanol
final concentration). To establish rescue lines,
RAR
-/- cells were electroporated with each
of the constructs displayed in Fig. 1A
along with a plasmid conferring
resistance to puromycin (pD503; gift of D. Lohnes), in a ratio of 10:1.
After 2436 h, cells were selected with 0.8 µg/ml of puromycin for
10 days as described (14) and analyzed for the presence and expression
of the transgene by Southern and Western blotting.
Cell Extracts, Immunoprecipitation and Immunoblotting of
RAR
Whole cell extracts were prepared (48) from rescue lines grown
as monolayers in the absence of RA, and RAR was immunoprecipitated
with Protein A-Sepharose cross-linked with monoclonal antibodies
directed to the F region of RAR
[(Ab9
(F)] (49). Proteins were
resolved by SDS-PAGE (10% acrylamide), electrotransferred onto
nitrocellulose filters and immunoprobed with polyclonal antibodies
against the F region of RAR
[RP
(F)] (49), followed by
peroxidase-labeled protein A and chemiluminescence detection, according
to manufacturers protocol (Amersham Pharmacia Biotech Europe GmbH, Saclay, France).
RNA Isolation and RT-PCR
RNA was isolated using the guanidinium thiocyanate method and
conditions for semiquantitative RT-PCR were as described (5). The
quantity of RNA used for RT-PCR in each reaction (2 µg) was
normalized with 36B4 transcripts (5, 50) which are unresponsive to
retinoid treatment. The RT-PCR oligonucleotides used for Hoxa-1,
HNF3, HNF1ß, Stra8, laminin B1, collagen type IV (
1), Hoxb-1,
CRABPII, and 36B4 were as described (6, 20). The RT-PCR primers for
throm-bomodulin (51) were as follows: 5'-TGGAGCATGAGTGCTTCGC-3' and
5'-GGTGTTGTAGGTACTAGAGA-3'. The primers for CYP26 (52) were
5'-GCTCAAGCTCTGGGACCTGT-3' and 5'-CGATCACGAGCACGTAGCAC-3'. The
amplifications were done for 13 cycles (collagen IV), 15 cycles
(laminin B1, Stra8, and 36B4), 18 cycles (Hoxb-1, Hoxa-1, and HNF3
),
20 cycles (thrombomodulin, CRABPII, Stra6, and CYP26) or 24 cycles
(HNF1ß). Aliquots were electrophoresed on a 1.5% agarose gel and
samples were transferred onto Hybond N membranes. The RT-PCR blots were
probed with cognate [32P]-labeled cDNA
fragments or with end-labeled oligonucleotide probes for thrombomodulin
and CYP26. The signals were quantified using a Bio-Imaging Analyzer
(BAS 2000, Fuji Photo Film Co., Ltd., Tokyo, Japan).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by funds from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer (ARC), the Comité Départemental du Haut-Rhin de la Ligue Nationale Contre le Cancer, and Bristol-Myers Squibb.
1 Present address: Department of Biochemistry and Molecular Biology,
Box 1126, Mount Sinai School of Medicine, New York, New York
10029-6574.
Received for publication March 27, 2000. Revision received June 2, 2000. Accepted for publication June 8, 2000.
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REFERENCES |
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