J Biol Chem, Vol. 274, Issue 38, 26783-26788, September 17, 1999
The Targeted Disruption of Both Alleles of RAR
2 in
F9 Cells Results in the Loss of Retinoic Acid-associated Growth
Arrest*
Teresa N.
Faria
,
Cathy
Mendelsohn§,
Pierre
Chambon¶, and
Lorraine J.
Gudas
From the Department of Pharmacology, Weill Medical
College of Cornell University, New York, New York 10021, the
§ Department of Urology, Columbia-Presbyterian Medical
Center, New York, New York 10032, and the ¶ Institut de
Genetique et de Biologie Moleculaire et Cellulaire, College de France,
BP 163, 67404 Illkirch Cedex, France
 |
ABSTRACT |
F9 teratocarcinoma cell lines, carrying one or
two disrupted alleles of the RAR
2 gene, were
generated by homologous recombination to study the role of
RAR2 in mediating the effects of retinoids on cell
growth and differentiation. Retinoic acid (RA) does not induce growth
arrest of the RAR2
/ cells, whereas the F9 WT and
RAR2+/ heterozygote lines undergo RA-induced growth
arrest. The RAR2+/ lines also exhibit a faster cell
cycle transit time in the absence of RA. The RAR2/
stem cells exhibit an altered morphology when compared with the F9 WT
parent line, and after RA treatment, the RAR2/ cells
do not exhibit a fully differentiated cell morphology. As compared with
F9 WT cells, the RAR/ cells exhibited a markedly lower induction
of several early RA-responsive genes and no induction of laminin B1, a
late response gene. The induction of RA metabolism in the F9
RAR2/ cells following differentiation was not
impaired. The research presented here, and prior research suggest that
RAR is required for RA-induced growth arrest in a variety of cell
types and that RAR also functions in mediating late responses to RA.
These findings are significant in view of the reduced expression of
RAR transcripts in a number of different types of human carcinomas.
| |
INTRODUCTION |
All-trans-retinoic acid
(RA)1 is one of the most
biologically active retinoids. RA exerts its effects, in part, by
acting through two types of nuclear receptors, the retinoic acid
receptors (RARs) and the retinoid X receptors (RXRs) (1-3), both of
which are members of the nuclear receptor superfamily. Each of these
RAR and RXR receptors has three isotypes (
, , and 
), which are encoded by separate genes. In addition, for each RAR isotype, there are
several isoforms, generated by differential promoter usage and
alternative splicing (1, 3).
RAR is one of the subtypes of the retinoic acid receptors. The
RAR gene has four isoforms: 1, 2,
3, and 4. 2 is the most
abundant RAR isoform (3-6). There is a very high affinity retinoic
acid-responsive element in the promoter of the RAR2 and
RAR4 isoforms (4, 7-9), which is associated with the rapid transcriptional activation of RAR2 by RA in a
variety of cells (4, 7, 8, 10-12). RAR exhibits a restricted
pattern of expression during development as well as in the mature
organism (4, 6, 13, 14). This pattern of expression is different from
those of the other RARs and suggests that RAR performs specific functions distinct from those of RAR and RAR.
F9 teratocarcinoma cells (15) express all known RA receptors, but
RAR mRNA is only present in high amounts after RA addition (10),
consistent with the fact that RAR2 is the predominant RAR isoform expressed in F9 cells (4, 6). F9 cells have recently
been used in our laboratories to inactivate the RAR and RAR genes
by homologous recombination (16, 17). Both F9 RAR and RAR null
cell lines exhibit marked modulation of a variety of genes when
compared with the F9 wild-type cells. This gene knockout approach has
allowed the identification of a series of genes that are direct or
indirect targets of RAR or RAR, such as the "homeobox" genes
of the Hoxb and Hoxa clusters, and the genes encoding the extracellular
matrix proteins laminin and collagen IV(1) (16, 17).
RAR mRNA expression has been reported to be greatly reduced in
breast cancer cells (18, 19), oral and epidermal squamous cell
carcinoma (cell lines and tissues) (20-24), and lung carcinoma lines
and tissues (25-28). In breast cancer cell lines, RA is not able to
induce RAR mRNA expression, even though these cells can transcriptionally activate an exogenous RAR RARE (retinoic acid response element) (19). When the expression of the RAR gene is
restored in breast cancer cell lines via an exogenous RAR cDNA
expression vector, the cells acquire sensitivity to RA-mediated apoptosis and growth arrest (29-31). Transfection of a human
epidermoid lung cancer cell line with RAR causes decreased
tumorigenicity (32). In addition, overexpression of RAR2
in HeLa cells induces growth inhibition (33), while expression of
RAR antisense mRNA decreases RA sensitivity in responsive cell
lines (31) and causes an increased frequency of carcinomas in
transgenic mice (34). The loss of RAR may be an essential step in
neoplastic progression, since there is evidence of a progressive
decrease in RAR mRNA expression during breast carcinogenesis
(35) and greatly reduced RAR mRNA expression in morphologically
normal tissue adjacent to breast carcinomas (36). Therefore,
characterizing the role of RAR in the control of RA-mediated
differentiation and growth arrest may lead to a greater understanding
of the mechanisms underlying the development and progression of cancer.
We have generated RAR2 knockout F9 cells by homologous
recombination in order to study the role of RAR2 in
mediating the effects of retinoids on cell growth and differentiation.
RA is incapable of inducing growth arrest in these
RAR2/ cells, indicating that in F9 cells
RAR2 is required for the growth inhibitory actions of
RA. This finding is significant in light of the reduced expression of
RAR transcripts in a number of carcinomas, and it suggests that
RAR could regulate RA-induced growth arrest in a variety of cell types.
| |
EXPERIMENTAL PROCEDURES |
The RAR2 disruption vector has been described in
detail previously (37). F9 WT cells were cultured under standard
conditions, and electroporation was performed as described previously
(16, 17, 38). The cells were treated with 1 µM
all-trans-RA and all-trans-ROL (Sigma),
all-trans-4-oxoROL (prepared as described previously (39,
40)), all-trans-4-oxoRA (Hoffman-LaRoche, Nutley, NJ), 4-HPR
(fenretinide, R. W. Johnson Pharmaceutical Institute, Raritan,
NJ), BMS 188,649 (Bristol-Myers Squibb Pharmaceutical Research
Institute, Buffalo, NY) and 9-cis-RA (Hoffman-LaRoche, Nutley, NJ). [3H]RA was purchased from NEN Life Science
Products. Southern and Northern analyses were performed as described
(41). For Northern analyses, 10 µg of total mRNA were loaded per
lane and the signals were quantitated by phosphorimaging (Molecular
Dynamics). Reverse transcriptase-polymerase chain reaction
(RT-PCR) was performed under standard conditions as described (42)
using the primers described previously (37) for 30 cycles. The probes
for the Northern blots have been described previously (16, 17), except for the murine p450RAI (CYP26) probe, which was purchased from Genome
Systems, St. Louis, MO (Expressed Sequence Tag Database accession no.
AA 239785). Western blot analysis was performed as described (42, 43).
Cleared lysates were analyzed for protein content by the Bradford
method (Bio-Rad), and 150 µg of protein was loaded per lane. A rabbit
polyclonal antibody (1:1000) directed against RAR (RP(F)2; Ref.
44) or a mouse monoclonal antibody (1:1000) directed against RXR
(4RX3A2; Ref. 45) were used. Immune complexes were detected with a
horseradish peroxidase-conjugated anti-rabbit or anti-mouse serum
(1:2000), respectively, and the Super-Signal Ultra Chemiluminescent
Substrate (Pierce). Extraction of retinoids and high performance liquid
chromatography (HPLC) were performed as described (39, 46).
| |
RESULTS |
Generation of Targeted Disruptions in the RAR Gene--
The
RAR2 isoform was inactivated with a disruption vector
described previously (37). The neomycin-resistance gene was inserted into the exon 4 of the RAR gene, which encodes the 5'-untranslated and the A2 region sequences of the RAR2 isoform. The
insertion of the neomycin resistance gene in this region disrupts only
the RAR2 isoform of the RAR gene. F9 WT cells were
electroporated and selected in G418. Resistant colonies were isolated
and screened by Southern analysis for the expected change in size of
the 6.5-kb wild-type (WT) KpnI genomic fragment (the mutated
allele is 4.3 kb), using a probe derived from DNA sequence located 5'
of the sequence contained in the targeting construct. Two heterozygous lines were generated, F9 WT-2-291 and F9 WT-2-270, out of a total of 800 colonies screened (Fig.
1A). To target the second allele, the heterozygous lines were selected in high concentrations of
G418 for 21 days (16, 17, 38). One clone was isolated, out of 300 surviving colonies (F9 WT-2-270-110), as a result of a second
recombination event in which the second WT allele was disrupted (Fig.
1A). This clone was then subcloned, and eight subclone lines
were generated that showed similar behavior (data not shown).
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Fig. 1.
Generation of F9
RAR2/ cell lines.
A, Southern blot analysis demonstrating the successful
disruption of the RAR gene. Wild type locus, 6.5 kb; mutated locus,
4.3 kb. B, detection of wild-type RAR2
alleles. The levels of RAR2 transcripts were monitored
in F9 WT, 270 RAR2+/ and RAR2/
lines by RT-PCR analysis using the conditions outlined under
"Experimental Procedures." C, retinoid receptor levels
expression in F9 WT, 270 RAR2+/, and
RAR2/ cells. The levels of retinoid receptors were
monitored by Northern or Western analysis using the conditions outlined
under "Experimental Procedures." D, RAR protein
levels in F9 WT, 270 RAR2+/, and
RAR2/ cells. The levels of RAR protein were
measured by Western blot analysis using the conditions outlined in
under "Experimental Procedures." These experiments were all
performed two or three times with identical results.
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|
In order to show that the RAR2/ line lacked
detectable RAR2 mRNA, RT-PCR analysis was performed
(Fig. 1B). Whereas bands of the appropriate size for
RAR2 were detected in the F9 WT and RAR2+/ 270 lines, no bands of that size were detected
in the RAR2/ line. Thus, the
RAR2/ cells do not express the RAR2 transcripts which are expressed in the F9 WT cells. The expression of
retinoid receptors was evaluated by Northern and Western analysis (Fig.
1C). As expected, there is no detectable induction of RAR transcripts upon treatment of the F9 RAR2/ cells
with 1 µM RA. In contrast, in the heterozygous lines and
the F9 WT line, there was a large (
20-fold) induction of the RAR
transcripts after RA treatment. The expression of RAR and RXR
mRNA was similar in all the lines. The expression of RAR
mRNA increased slightly (approximately) 2-fold after RA treatment
of the F9 RAR2/ cells, in contrast to the decrease
in RAR mRNA observed in RA-treated F9 WT and
RAR2+/ cells. The levels of RXR protein (Fig.
1C) were lower in the RAR2+/ and
RAR2/ than in the wild type cells. In summary, the
RAR2/ line has a lower level of RXR protein and
(after RA treatment) a higher amount of RAR mRNA. The other
retinoid receptors are present at levels similar to those seen in F9 WT
cells (Fig. 1C).
The levels of the RAR protein in the F9 WT, F9
RAR2+/, and F9 RAR2/ cell lines
were evaluated by Western analysis (Fig. 1D). After 48 h of RA treatment, the RAR protein is induced in the F9 WT cells and
is induced to a lesser extent in the F9 RAR2+/ cells.
No RAR protein was detected in the F9 RAR2/ cells
(Fig. 1D).
Analysis of the Growth of the F9 RAR2/
Cells--
The responsiveness of the F9 RAR2/ line
to RA-induced growth arrest was evaluated (Fig.
2). Unlike the F9 WT line and the heterozygote lines, the F9 RAR2/ line did not growth
arrest in response to RA. Other retinoids and retinoid agonists were tested, including a RXR pan-agonist (BMS 188,649), and these were also
not able to induce growth arrest in the F9 RAR2/
cell line (Fig. 3), although they were
effective to varying degrees in arresting the growth of the parent F9
WT cell line. This suggests that RAR2 is required for
all of these compounds to exert their growth inhibitory effects in F9
cells.
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Fig. 2.
Comparison of the growth of F9 WT,
RAR2+/ 270, RAR2+/ 291, and
RAR2/ lines. The cells
were plated in duplicate wells at a density of 3000 cells/well in the
absence or in the presence of 1 µM RA, and cells were
counted on the indicated days. The results are plotted as cell number
versus day of culture. Note the different y axis
scales in each of the four graphs. Data points, means of
triplicate samples; bars, S.E. This experiment was repeated
three times with similar results.
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Fig. 3.
Effects of various retinoids on the growth
rates of F9 WT and F9 RAR2/
cells. The cells were plated in triplicate wells at a density of
1000 cells/well in the presence of RA, ROL, 4-oxoROL, 4-oxoRA, BMS
188,649 (RXR pan-agonist), 9-cis-RA, or fenretinide (all
drugs were 1 µM) and counted after 6 days in culture. The
results are plotted as cell number versus treatment.
Data points, means of triplicate samples; bars,
S.E. This experiment was performed two times with similar
results.
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Interestingly, both of the independently generated F9
RAR2+/ heterozygote lines grow faster that the WT F9
cells in the absence of RA (Fig. 2, note the different
y axis scales). The mean doubling time for the parent WT
cell line was 22 h, whereas the F9 RAR2+/ 270 heterozygote line had a mean doubling time of 17 h, indicating
that reduced levels of RAR2 protein are associated with
a decrease in the cell cycle transit time.
Morphological Characterization of the F9 RAR2/
Cells--
The morphology of the RAR2/ cells is
markedly different from that of the parent F9 WT cells (Fig.
4). In the absence of RA, there are
noticeable differences between the F9 WT cells and the F9
RAR2/ cells; the latter clump together in cell
aggregates, which do not exhibit the irregular borders characteristic
of F9 WT cells. Following RA treatment, F9 WT cells form long cellular processes that are absent in the RAR2/ cells, which
look like undifferentiated cells (Fig. 4). F9 WT cells exhibit even
more dramatic morphological alterations after the addition of cAMP and
theophylline (47), unlike the RAR2/ cells. It should be noted that it is possible to observe a few morphological changes occurring in the F9 RAR2/ cells after treatment with
RA; the RA-treated F9 RAR2/ cells become more
flattened and exhibit more vacuoles. The F9 RAR2+/
cells exhibit a similar morphology to F9 WT cells in both the absence
and presence of RA (data not shown), indicating that both alleles of
RAR2 must be inactivated in order to observe alterations
in the morphological phenotype.
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Fig. 4.
Morphology of the F9 WT and
RAR2/ lines in culture.
The cells were plated and left untreated, or treated with 1 µM RA or with RA plus 250 µM dibutyryl cAMP
and 500 µM theophylline (RACT). The cells were
photographed in phase contrast after 4 days in culture.
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Effects of RA on Gene Expression in F9 RAR2+/ and
F9 RAR2/ Lines--
The F9 WT, F9
RAR2+/, and F9 RAR2/ cell lines
were cultured in the presence of 1 µM
all-trans-RA for the indicated times, and the expression of
several genes which are transcriptionally activated by RA was evaluated
(Fig. 5, A and B).
In the heterozygous F9 RAR2+/ lines, the induction of
laminin B1, CRABP II, and P450RAI (CYP26) by RA was similar to or
slightly lower than that in the F9 WT cells. The expression of the
REX-1 gene, which in F9 WT cells was reduced by ~2-fold after 48 h of RA treatment, was reduced similarly in the RAR2+/
cells (2.4-fold). The greatest difference between F9 WT and F9
RAR2+/ cells was that the Hoxa-1 gene was always
induced to a greater extent at earlier time points in the heterozygote
lines than in the F9 WT cells. In the experiment shown, there was an
18-fold induction of Hoxa-1 mRNA in the heterozygote lines cultured
in the presence of RA for 48 h versus a 4.7-fold induction in F9 WT cells cultured under the same conditions (Fig. 5,
A and B). The increased expression of Hoxa-1 in
the heterozygote lines was transient, and after 96 h of RA
treatment, the levels of Hoxa-1 mRNA were comparable in the F9 WT
and the RAR2+/ lines (7.3- and 6.5-fold
induction).
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Fig. 5.
Effects of RA on the expression of
differentiation specific genes in the F9 WT and the F9
RAR2/ cells. F9 WT, 270 RAR2+/, and RAR2/ cells were
treated with 1 µM RA, and RNA was harvested at the
indicated times and analyzed on Northern blots (A). 10 µg
of total RNA were loaded in each lane. The expression of CRABP II,
laminin B1, Hoxa-1, p450RAI, and REX-1 mRNAs was evaluated, and
bands were quantitated by phosphorimaging (B). Actin
mRNA was used as a control for loading. This experiment was
performed three times with similar results.
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In the F9 RAR2/ line, the expression of early
response genes such as p450RAI and CRABP II was RA-responsive, although
the magnitude of the induction of these mRNAs was much lower in the F9 RAR2/ cells than in the F9 WT cells. In contrast,
the induction of the Hoxa-1 message after 48 h of RA treatment was
only 2-fold lower in the RAR2/ cells than in the F9
WT cells. The reduction in the levels of REX-1 message after 24 h
of RA treatment was comparable in the RAR2/ cells
and the F9 WT cells (Fig. 5, A and B).
Interestingly, the reduced REX-1 mRNA levels were not maintained in
the RAR2/ cells, and by 96 h REX-1 levels were the same as those in the untreated cells (see Fig. 5B for
normalized data). Laminin B1, a late response gene, was not induced in
the RAR2/ cells (Fig. 5, A and
B). In summary, the F9 RAR2/ cells
exhibited a reduction in the magnitude of the induction of a number of
RA-responsive genes following RA treatment.
Metabolism of RA in the F9 RAR2/
Cells--
Since we observed that the induction of p450RAI message
after RA exposure was lower in the RAR2/ cells than
in the F9 WT cells (Fig. 5, A and B), we compared
the amounts of RA metabolized by the F9 WT and F9
RAR2/ lines. The two cell lines were treated for
72 h with vehicle or with 1 µM RA and were then
cultured in the presence of [3H]all-trans-RA
for 1 h or for 3 h, after which both the intracellular and
the extracellular levels of the polar metabolites of
[3H]all-trans-RA were quantitated by
reverse-phase HPLC (Fig. 6). Like the F9
WT cells, the F9 RAR2/ cells were able to metabolize RA and the levels of RA metabolites produced were dramatically increased after RA-induced differentiation. Since the levels of metabolites produced from [3H]RA were similar in the F9
WT and the RAR2/ cell lines, the lower induction of
the p450RAI transcripts after RA in the RAR2/ cells
(Fig. 5) was not associated with an impairment of the ability to
metabolize [3H]RA under the conditions of our
assays.
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Fig. 6.
Metabolism of
[3H]all-trans-RA in F9 WT and
RAR2/ lines. Cells were
first cultured in the presence or absence of 1 µM RA for
72 h, followed by culture in the presence of 50 nM
[3H] RA for the indicated times;
[3H]retinoids were then extracted and analyzed by HPLC.
The levels of radiolabeled polar RA derivatives were quantitated as
described under "Experimental Procedures." The total amounts of
polar RA derivatives (in the cells and in the media) are shown,
expressed as nM/million cells. This experiment was
performed two times with very similar results.
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| |
DISCUSSION |
We have previously reported the generation of F9 teratocarcinoma
cell lines carrying targeted disruptions of the RAR and RAR genes
(16, 17). We now continue the characterization of the roles of the RARs
in mediating RA-induced F9 cell differentiation by generating cell
lines carrying one or two disrupted alleles of the RAR2
gene. The RAR2 disruption completely abrogates the growth arrest that is one of the hallmarks of RA action in a variety of
cell types. The F9 RAR2/ cells did not growth arrest
in response to RA or a variety of other retinoids (Figs. 2 and 3). Thus, the data presented in this report, together with prior data from
various F9 RAR and RXR knockout lines, strongly argue for a critical
role for RAR2 in mediating growth arrest in response to
RA in F9 cells (Figs. 2 and 3). Whereas in the absence of
RAR2 no growth arrest in response to RA is
observed in the F9 RAR2/ cells (Fig. 2), the
RAR/ and RAR/ lines growth arrest like WT cells, and the
RXR/ and RXR/ RAR/ lines partially growth arrest
after RA addition (16, 17, 48-50). Only the RXR/ RAR/
line shows no growth arrest in response to RA. Although in this line
RAR2 mRNA induction is decreased by 2-3-fold (49), this relatively small impairment of RAR2 expression
probably does not contribute to the observed lack of growth arrest of
the RXR/ RAR/ cells (50). Since the
RAR2/ line has a lower level of RXR protein than
F9 WT cells (Fig. 1C), it is possible that in these
RAR2/ cells the RXR:RAR heterodimer is
involved in mediating the RA-induced growth arrest.
The mechanism by which RA induces growth arrest is not known, although
several reports have linked RAR to growth regulation and apoptosis
(see Introduction). Interestingly, the two independent F9
RAR2+/ heterozygote lines, which arrested their growth
normally in response to RA, showed a faster cell cycle transit time in the absence of RA (Fig. 2). At present we do not have an
explanation for this observation. The finding that two independently
derived RAR+/ lines grow faster than the F9 WT cells in the
absence of RA (Fig. 2) is particularly intriguing in light of the
greatly reduced expression of RAR in a variety of human cancers and
preneoplastic cells (see Introduction). Our data support the hypothesis
that reduced levels of RAR confer a selective growth advantage to preneoplastic cells and, therefore, that the loss of one allele of
RAR may be a key step in neoplastic progression.
The RAR2 disruption alters the morphology of these cells
as compared with the F9 WT parent line (Fig. 4). In the absence of RA,
this difference in morphology may result from the presence of low
levels of RAR2 transcripts in the F9 WT cells and in the RAR2+/ heterozygote cells, but not in the
RAR2/ line. After RA treatment, the F9
RAR2 / cells do not exhibit a typically differentiated morphology. A partial or complete lack of morphological differentiation following RA treatment has been previously observed in
the F9 RAR/ lines (16), the F9 RXR/ lines (48), and in
the double mutant lines F9 RXR/RAR/ and F9
RXR/RAR/ (50).
There was a markedly lower induction of several RA-responsive genes in
the F9 RAR2/ cells when compared with F9 WT cells (Fig. 5). In contrast, in the F9 RAR/ and RAR/ lines, the inactivation of RAR and RAR, respectively, caused a severely impaired induction of particular RA-responsive genes, whereas the
induction of other RA-responsive genes was like that in F9 WT cells
(16, 17). The modulation of gene expression in response to RA in the
RAR2/ cells was actually strongest at early time points when compared with the F9 WT cells. This RA induction of gene
expression diminished at later times, and laminin B1, a late response
gene, was not induced in the F9 RAR2/ cells. Our
data suggests that in F9 WT cells the initial response to RA is
mediated via the RAR and RAR receptors, but that the large
increase in RAR2 receptors after RA treatment (which
occurs after 16-24 h) is required for the maximal expression of
RA-responsive genes at later times after RA addition. Alternatively,
the lack of growth arrest in response to RA observed in the
RAR2 / cells may contribute to a reduction in the
magnitude of the differentiation response in these cells. That the
initial RA response with respect to transcriptional activation of
RA-responsive genes such as Hoxa1 is present in the F9
RAR2/ cells is consistent with prior data indicating that RXR and either RAR or RAR are the essential receptor
pairs for these early responses during the RA-induced conversion of F9
stem cells to primitive endoderm cells (49). Collectively, the research
presented here as well as prior research (48-52) argue against a role
for RAR in the initial events in the RA-induced differentiation process.
The RA-inducible P450RAI has been implicated in the metabolism of RA in
a variety of cell types (53, 54). Previous data suggested that the
RAR·RXR heterodimer is responsible for the induction of p450RAI
in F9 cells (17, 54). Although in RAR2/ cells the
levels of p450RAI transcripts attained after RA treatment were much
lower than in the F9 WT cells (Fig. 5), there was no corresponding
impairment in the ability of the RAR2/ cells to
metabolize [3H]RA (Fig. 6). Therefore, it is likely that
the lower level of p450RAI mRNA present in RAR2/
cells is sufficient to metabolize 50 nM
[3H]RA at the same rate as that seen in F9 WT cells. In
summary, our data indicate that RAR2 is not required for
the induction of RA metabolism in F9 cells.
Since most of the genes evaluated (with the exception of laminin B1)
were induced in the F9 RAR2/ cells following RA
treatment, albeit at reduced levels, it is possible that in F9 cells
RAR does not activate the transcription of the early response genes tested, but instead is required to achieve the maximum induction of
these RA-responsive genes. This result, together with the high degree
of functional redundancy among the different RARs in the context of
mouse knockouts (55) may explain why mice carrying two disrupted
alleles of RAR2 were phenotypically normal (37) and why
mice in which all isoforms of RAR were disrupted (56, 57) presented
a relatively mild phenotype when compared with the phenotypes of other
RAR knockouts (58, 59). This may also account for the observations that
RAR/ mice, as well as RAR/ mice knocked out for the p53
gene or carrying a MMTV-ras transgene, did not exhibit any increase in
tumor formation over a period of at least 14 months.2
| |
ACKNOWLEDGEMENTS |
We are grateful to P. R. Reczek for the
gift of BMS 188,649. We thank the members of the Gudas laboratory for
helpful comments and James Thompson, Daniel Metzger, and Norbert
Ghyselinck for critically reading this manuscript.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant RO1CA43796 (to L. J. G.), National Institutes of Health
Training Grant CA62948 (to T. F.), and grants from the CNRS,
INSERM and College de France (to P. C.).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.
To whom correspondence should be addressed. Tel.:
212-746-6250; Fax: 212-746-8858; E-mail:
ljgudas@mail.med.cornell.edu.
2
N. Ghyselinck, K. Niederreither and P. Chambon,
unpublished observations.
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ABBREVIATIONS |
The abbreviations used are:
RA, all-trans-retinoic acid;
HPLC, high performance liquid
chromatography;
RAR, retinoic acid receptor;
RXR, retinoid X receptor;
ROL, all-trans-retinol;
WT, wild type;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
kb, kilobase(s).
| |
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ABSTRACT
INTRODUCTION
