From the Cancer Research Laboratories and the
Departments of § Pathology,
Biochemistry, and
§§ Medicine, Queen's University, Kingston,
Ontario K7L 3N6, Canada and ** Institut de Génétique et de
Biologie Moléculaire et Cellulaire,
CNRS/INSERM/Université Louis Pasteur, Collège de France,
BP 163, 67404 Illkirch-Cédex, France
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ABSTRACT |
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We have cloned a mouse cDNA homolog of
P450RAI, a cytochrome P450 belonging to a new family (CYP26), which has
previously been isolated from zebrafish and human cDNAs and found
to encode a retinoic acid-inducible retinoic acid hydroxylase activity. The cross-species conservation of the amino acid sequence is high, particularly between the mouse and the human enzymes, in which it is
over 90%. Like its human and zibrafish counterparts, the mouse P450RAI
cDNA catalyzes metabolism of retinoic acid into 4-OH-retinoic acid,
4-oxo-retinoic acid, 18-OH-retinoic acid, and unidentified
water-soluble metabolites when transfected into COS-1 cells. Retinoic
acid-inducible retinoic acid metabolism has previously been observed in
F9 murine embryonal carcinoma cells and some derivatives lacking
retinoid receptors. We were interested in determining whether P450RAI
could be responsible for retinoic acid metabolism in F9 cells and in
studying the effect of retinoid receptor ablation on P450RAI
expression. In wild-type F9 cells and derivatives lacking RAR,
RAR
, and/or RXR
, we observed a direct relationship between the
level of retinoic acid metabolic activity and retinoic acid-induced
P450RAI mRNA. These experiments, as well as others using synthetic
receptor subtype-specific retinoids, suggest that the RAR
and RXR
receptors mediate the effects of retinoic acid on the expression of the
P450RAI gene.
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INTRODUCTION |
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Vitamin A, through its active metabolite retinoic acid (RA)1 and possibly other derivatives (1-4), is believed to play a fundamental role in vertebrate embryogenesis and development, limb and tail regeneration (5, 6), and maintenance of adult epithelial tissues (7). It also has a dramatic effect on growth and differentiation of cells in culture (8, 9) and has been shown to be effective in prevention and treatment of various human cancers, including leukemias (10), secondary tumors of the head and neck (11), and cervical cancer (12). The importance of vitamin A levels in mammals is manifested in conditions of vitamin A depletion or excess. Vitamin A deficiency during embryonic development results in congenital malformations or lethality, and in adults, it results in disturbances in vision and reproduction and metaplasia of epithelial tissues. Vitamin A excess during embryonic development results in malformations in neural tube-derived structures and limb duplications (13). Because the developmental consequences of retinoid deficiency, as well as toxicity, are severe, the mechanisms that regulate RA levels in tissues are critical.
In vertebrates, the effects of RA are mediated primarily through gene
transcription, via several members of the steroid/thyroid/vitamin D
superfamily of nuclear receptors: the retinoic acid receptors (RARs)
and retinoid X receptors (RXRs), which each comprise three different
subtypes (,
, and
) and which bind RA and 9-cis-RA, respectively, with high affinity. RARs and RXRs have been shown to bind
as heterodimers to RA response elements of RA target genes (14).
Genetic and biochemical evidence suggests that the nuclear receptor
heterodimers are essential for RAR-mediated RA signaling (15-17).
Other high-affinity retinoid binding proteins, the retinol binding
proteins (CRBP-I and CRPB-II) (18), and the retinoic acid binding
proteins (CRABP-I and CRABP-II) (18), may be involved in RA synthesis
and metabolism; however, their role is unclear (19, 20).
Although much progress has been made in understanding how RA mediates its biological effects, the functions of RA synthesis and metabolism in RA signaling remain unclear. RA homeostasis is maintained by regulation of its rate of synthesis from retinol (21, 22) and by controlling its rate of degradation through oxidative pathways that give rise to 4-OH-RA and 4-oxo-RA or to 18-OH-RA (23). The catabolic activities have been studied for many years in microsomal preparations (see, e.g. Ref. 24), and have been found to have properties characteristic of the cytochrome P450 family, such as dependence on NADPH and oxygen, inhibition by carbon monoxide, and inhibition by the general P450 inhibitors ketoconazole and liarozole (25, 26).
Recently, we cloned and characterized a RA-inducible cytochrome P450 that specifically metabolizes RA to hydroxylated products, including 4-OH-RA and 4-oxo-RA, and is expressed in the wound epithelium of zibrafish regenerating caudal fin (27). The human P450RAI counterpart has been identified, and its expression has been studied in a variety of human cell lines (28). In the breast cancer-derived epithelial cell line MCF-7, metabolic activity and P450RAI mRNA are closely correlated at various times after RA treatment, whereas the nonmalignant, nontransformed breast epithelial cell line MCF-10A has neither P450RAI expression nor RA metabolic activity following RA treatment (28). These findings strongly implicate P450RAI as a key participant in RA-inducible metabolism, which may act in limiting cellular RA levels.
To address the role of RA metabolism in RA signaling and cell
differentiation, we cloned the murine P450RAI counterpart,
assessed its metabolic activity by expressing it in COS-1 cells, and
studied its expression in the F9 murine embryonal carcinoma cell line, which differentiates into primitive, parietal, or visceral endoderm depending on the RA treatment conditions and is known to have RA-inducible RA metabolic activity (29-31). Previous work (32) using
F9-derived RAR knockout lines, has implicated RAR and RAR
in the
regulation of RA metabolism. In our present study, we used F9
wild-type, RAR
/
, RAR
/
, RXR
/
,
RXR
/
/RAR
/
, and RXR
/
/RAR
/
cell lines and a
number of receptor-specific synthetic retinoids to establish whether
P450RAI was responsible for RA-induced RA metabolism and which
receptors were involved in its regulation.
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EXPERIMENTAL PROCEDURES |
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Cloning and Expression of Mouse P450RAI--
The mouse homolog
of P450RAI was isolated from a RA-induced P19
teratocarcinoma cDNA library in Unizap XR (Stratagene). A total
of 106 independent plaques were plated and screened using a
32P-labeled random-primed full-length human
P450RAI probe. Hybridization conditions were as described
previously (28). pBluescript-containing colonies were generated using
the in vivo excision protocol (Stratagene) and plated onto
LB-ampicillin plates. DNA was prepared using the Qiaprep Mini-Plasmid
kit (Qiagen). Sequencing of the cDNA clone was done using an
Applied Biosystems model 373A automated DNA sequencer. For expression
in cultured cells, full-length cDNA was cut out of pBluescript
using the ApaI and NotI sites and subcloned into
pRC/CMV expression plasmid (Invitrogen) cut with the same restriction
enzymes.
Cell Culture--
All F9 cell lines (wild-type, RAR/
(32), RAR
/
(19), RXR
/
(33),
RXR
/
/RAR
/
,2 and
RXR
/
/RAR
/
2) were maintained as described
previously (29). In brief, cells were grown in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with glucose
(4.5 g/liter), 10% fetal bovine serum, penicillin, streptomycin,
fungizone, and gentamycin. For general maintenance, cells were split
every other day, seeded into gelatinized tissue culture plates, and
incubated in a humidified, 37 °C atmosphere of 5% CO2.
For experiments involving treatment with RA, cells were plated at
1.3 × 106 cells per 100-mm tissue culture plate,
grown overnight, and treated with all-trans-RA at 1 µM in dimethyl sulfoxide or with 0.1% dimethyl sulfoxide
only. RA treatments were done under reduced light conditions. For
removal of RA from the medium, cells were washed twice with phosphate-buffered saline followed by the addition of fresh medium.
Northern Blot Analysis--
Following cell treatment, total RNA
was extracted using TRIzol Reagent (Life Technologies, Inc.) as
outlined by the manufacturer. Gel electrophoresis and Northern blotting
onto nitrocellulose (Hybond C, Amersham Corp.) were performed as
described by White et al. (27) using 20 µg of total RNA.
Radiolabeled mouse P450RAI cDNA probes were prepared by random
priming of cDNA using the Stratagene Prime-It kit. Following
hybridization in Quick-Hyb (Stratagene) and membrane washings as
described by the manufacturer, blots were exposed to Kodak X-OMAT AR or
BIOMAX MR film at 70 °C. To control for quantitation between RNA
samples, blots were probed with 32P-labeled 36B4 cDNA,
a ribosomal phosphoprotein, the expression of which is not altered by
treatment with RA (32).
Analysis of RA Metabolic Activity of Mouse P450RAI and HPLC-- COS-1 cells were transfected with either mouse P450RAI in expression vector pRC/CMV or empty vector control DNA, along with ferridoxin and ferridoxin reductase expression vectors, as described previously (27, 28). Extraction of the products from cell culture media and HPLC analysis of the products from COS-1 cells or F9 cells expressing P450RAI treated with unlabeled or 3H-labeled RA substrates have been previously described (28). Briefly, media from treated cells incubated with 575 pM all-trans-[11,12-3H]RA (23.1 Ci/mmol) for 4 h were acidified to pH 6.1 with glacial acetic acid (0.1%). Lipid-soluble and aqueous-soluble metabolites in the media were separated using a modified Bligh and Dyer total lipid extraction (35). Conversion of all-trans-[11,12-3H]RA to total aqueous-soluble metabolites was measured by liquid scintillation counting of the aqueous-soluble extract. Lipid-soluble extracts were evaporated to dryness under a stream of nitrogen and resuspended in 93.5:5:1:0.5, hexane/isopropyl alcohol/methanol/acetic acid. Metabolites were separated by HPLC using Zorbax-CN (6 µm, 4.6 mm inner diameter × 25 cm) column eluted with a solvent system of 93.5:5:1:0.5, hexane/isopropyl alcohol/methanol/acetic acid at a flow rate of 1 ml/min.
RT-PCR Analysis--
RNA preparation, RT-PCR, and Southern
blotting were performed as described previously (19, 32,
33).2 In brief, RT-PCR was performed under standard
conditions using avian myeloblastosis virus reverse transcriptase and
Taq polymerase with 2 µg of RNA and 50 pmol of each
primer. The PCR primers were as follows: 36B4,
5CAGCTCTGGAGAAACTGCTG-3
(nucleotides 290-309) and
5
GTGTACTCAGTCTCCACAGA-3
(nucleotides 826-845) (36); mouse P450RAI,
5
CTCCTGATTGAGCACTCGTG-3
(nucleotides 841-860) and
5
GGATCTGCTATCCATTCAGCTC-3
(nucleotides 1168-1189). The
amplifications were done for 17 or 19 cycles, aliquots were
electrophoresed on a 1.5% agarose gel, and samples were transferred
onto Hybond N membranes. The blots were hybridized to the corresponding
random primed mouse P450RAI- or 36B4-labeled cDNA probes.
Transcript levels were quantified using a BAS 2000 BioImaging analyzer
and normalized to corresponding 36B4 mRNA levels. The mRNA
levels were expressed relative to the amount present in wild-type cells
treated with 1 µM all-trans-RA for 96 h.
Synthetic Retinoids--
Retinoids selective for RAR (BMS188,
753 (BMS753)) (37), RAR
(BMS189, 453 (BMS453)) (38), RAR
(BMS188,
961 (BMS961)) (37), and all three RXRs (BMS188, 649 (BMS649)) (39) were a gift from Bristol-Myers Squibb, Pharmaceutical Research Institute (Buffalo, NY). Treatments of F9 cells with synthetic retinoids were for
24 h.
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RESULTS |
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Cloning of P450RAI-- A cDNA library derived from RA-treated P19 in Unizap XR (Stratagene) was screened with a full-length cDNA probe from human P450RAI according to procedures previously described. Out of 16 clones isolated, 1 cDNA encoding the full open reading frame was obtained and was found to be 93% identical to the human P450RAI and 68% identical to the zibrafish P450RAI at the amino acid level (Fig. 1). Regions previously identified as having a high degree of homology between human and zibrafish P450RAIs were almost identical between human and mouse cytochromes, with most differences clustered elsewhere in the cDNA. The genomic sequence of both the mouse and human P450RAIs has also been determined, and there is very close homology in location, size, and sequence of the introns and in the proximal promoter.3 The high degree of homology among these P450RAIs from different species, their catabolic activities, and their location on syntenic chromosomal regions suggests that these genes are homologous.4
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RA Metabolic Activity of Mouse P450RAI Expressed in COS-1 Cells-- To determine whether the mouse P450RAI had similar RA metabolizing activity to its human and zibrafish counterparts, we cloned the full-length mouse P450RAI into the expression vector pRC/CMV, in which transcription is driven by the CMV promoter. COS-1 cells were transfected with the expression vector containing mouse P450RAI or with the empty vector as a control, using methods previously described (28). RA metabolism in the transfected cells was measured by HPLC analysis of the lipid-soluble components of the media after incubation with either all-trans[11,12-3H]RA (575 pM) or unlabeled all-trans RA (1 µM). In addition, the aqueous-soluble fraction of the media from cells incubated with 3H-labeled RA was counted by liquid scintillation. The pattern of metabolism was the same using this cytochrome P450 as with the human and zibrafish P450RAIs (27, 28). Cells treated with all-trans-[11,12-3H]RA and transfected with pRC/CMV-P450RAI (Fig. 2A) showed a decrease in the amount of RA substrate compared with cells transfected with empty vector and production of metabolites that eluted at 9.5 and 11.5 min, corresponding to the elution positions of 4-OH-RA and 4-oxo-RA standards. In the case of the 3H-labeled substrate, products were measured by collecting and counting fractions, and the broad peaks observed may also contain other metabolites, such as 18-OH-RA, which has been identified as a metabolic product in microsomes from rat testis incubated with RA (23). The counts in the aqueous-soluble fraction, which may represent glucuronide conjugates and/or products representing further metabolism of RA, are significantly increased in the mouse P450RAI transfectants compared with control cells (Fig. 2B). With the unlabeled RA substrate (Fig. 2C), there was also a decrease in the amount of substrate in cells transfected with mouse P450RAI compared with controls. Three metabolites with spectral properties characteristic of retinoids were observed, co-migrating with 18-OH-, 4-OH-, and 4-oxo-RA standards at 9.3, 9.6, and 11.5 min, respectively. An additional uncharacterized peak, present in the culture medium and lacking the characteristic retinoid UV spectrum, was observed at 10.5 min with both P450RAI-transfected and control samples.
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Expression of P450RAI in Wild-type and Mutant F9 Cells-- F9 cells have previously been shown to exhibit RA-inducible RA metabolism (26, 41, 42). To determine whether any of the RA metabolic activity in F9 cells could be accounted for by P450RAI expression, we assessed P450RAI mRNA expression in F9 cells treated with RA for various lengths of time. In these experiments, we used a plating density (1.3 × 106 cells/100-mm culture dish) that was found to give optimal expression of P450RAI (data not shown). Levels of P450RAI mRNA were higher in RA-treated cells plated at lower density (0.6-1.3 × 106 cells/100-mm culture dish) at all time points tested than in cells plated at higher density (2.5-5 × 106 cells/100-mm culture dish).
To study the role of retinoid receptors in RA induction of P450RAI, we compared expression in wild-type F9 cells with expression in several F9 derivatives in which various RARs, RXRs, or combinations of these receptor genes had been knocked out by homologous recombination. In wild-type F9 cells, a high level of P450RAI expression was seen in cells continuously treated with 10
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Analysis of RA Metabolism in Wild-type and Mutant F9
Cells--
Following a 48-h incubation period in RA, we analyzed the
abilities of these different F9 cell lines to metabolize
[3H]RA. Aqueous-soluble and lipid-soluble extracts were
prepared from the media of cultured cells exposed to
all-trans-[3H]RA for 4 h. Radioactivity
recovered from aqueous- and lipid-soluble fractions of the cells
pretreated with 1 µM RA or with DMSO vehicle alone
averaged 81% of total radioactivity added. The aqueous-soluble counts
shown in Fig. 3B demonstrate that RA treatment of wild-type and RAR/
cells resulted in a large increase in aqueous-soluble radioactivity compared with DMSO-treated control cells. RAR
/
and
RXR
/
cell lines also showed an increase in aqueous-soluble radioactivity relative to untreated controls, but the counts were significantly lower than in wild-type and RAR
/
cells. RA
treatment of RXR
/
/RAR
/
cells resulted in no increase in
aqueous-soluble counts. Although 4-OH-RA, 18-OH-RA, and 4-oxo-RA were
detectable with all of the F9 derivatives except for the
RXR
/
/RAR
/
cell line (data not shown), the levels were
much lower than those observed in P450RAI-transfected COS cells (Fig.
2, A and C). This may reflect more efficient
downstream processing of hydroxylated RA in F9 cells. The percentage of
total recovered counts in the RA substrate peak after HPLC analysis was
also determined for all cell lines. Fig. 3, A and
C, shows that the RA recovered is inversely proportional to
the level of P450RAI mRNA expression in all the cell lines tested.
Thus, in both wild-type and mutant F9 cells, metabolic activity as
measured both by generation of water-soluble products and by
disappearance of substrate parallels expression of P450RAI
mRNA.
RT-PCR Analysis of P450RAI Expression in Wild-type and Mutant F9
Cells--
RNA from F9 cells was reverse transcribed, and P450RAI mRNA
levels were measured by PCR analysis, to corroborate and extend the
results obtained by Northern blot analysis and to study the effects of
receptor-specific retinoids on P450RAI expression. Results
similar to those observed in Northern blot analysis were obtained. The
level of P450RAI message in RAR/
cell line was less than that
observed in wild-type F9 (Fig.
4A and B, lanes 1-4 and 21-24). In RAR
/
cells,
P450RAI was expressed at a higher level than in wild-type
cells at all time points measured (Fig. 4, A and B,
lanes 1-4 and 17-20); this was also seen, though not as clearly, with Northern blot analysis (Fig. 3, lanes
1-6). The RT-PCR analysis included an additional cell line,
RXR
/
/RAR
/
, which also clearly exhibited a compromised
response to RA as measured by the lower level of induction of
P450RAI compared with wild-type.
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DISCUSSION |
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We have isolated a cDNA clone that represents the mouse homolog of P450RAI, first described in zibrafish and subsequently in human (27, 28). The mouse P450RAI closely resembled the zibrafish P450RAI (68% homology at the amino acid level) and was almost identical to the human P450RAI (93% amino acid homology). P450RAI cDNA from all three species, when transfected into COS cells, catalyzes metabolism of RA into hydroxylated derivatives, including 4-OH- and 4-oxo-RA (this work and Refs. 27 and 28). After this report was submitted for publication, two reports appeared describing the cloning of mouse cDNA homologs of P450RAI (43, 44). At the amino acid level, these are identical to the mouse P450RAI described here. However, the metabolic products described by Fujii et al. (43) differ from those observed with our mouse P450RAI; in that study, 5,8-epoxy all-trans-RA (a compound not observed in previous metabolic studies) is described as an important metabolite, in contrast to our study, which indicated that 4-OH-, 18-OH-, and 4-oxo-RA are the most abundant lipid-soluble products. It is likely that these discrepancies have arisen from technical differences in the extraction and characterization of metabolites.
In agreement with previous studies using F9 and other embryonal
carcinoma cell lines (26, 41), our work demonstrates that RA is rapidly
metabolized to more polar metabolites in wild-type F9 cells. F9
derivatives lacking RAR or RXR
showed a reduced rate of RA
metabolism, whereas F9 cells lacking both RAR
and RXR
showed no
detectable RA metabolism. The rapid induction of P450RAI and
its expression in F9 wild-type and mutant cell lines correlate exactly
with the observed rates of RA metabolism, suggesting that P450RAI may
be responsible for most of the RA metabolism observed in F9 cells.
Although we did not observe a significant difference in RA metabolism
between the wild-type and RAR
/
cell lines, we note that it has
been previously reported that at earlier time points following RA
exposure, RA metabolic activity in RAR
/
cells is greater than
that observed in wild-type cells (19). In the same study, Boylan
et al. (19) also demonstrated that RAR
/
cells have a
decreased rate of RA metabolism, which is in agreement with our
results. The correlation that we observed between RA metabolism and
P450RAI expression does not preclude the possibility that
other enzymes are involved in the observed RA metabolism. RA-inducible
retinol metabolism has been reported in F9 cells (45, 46), and because
P450RAI does not metabolize retinol in transfected-COS cells (28), a
different enzyme may be involved in retinol metabolism. The enzyme
responsible for this activity has not been identified, but it may also
have the ability to metabolize RA.
We have observed (Fig. 5) that following removal of RA from the medium, F9 cells are capable of RA-independent expression of P450RAI. This sustained level of P450RAI expression in F9 cells following withdrawal of RA could explain the high level of RA metabolism observed when cells pretreated with RA are exposed to a second dose of RA. Such observations have been made in F9 cells by Williams and Napoli (26), who found that the half-life (3.5 h) of RA in F9 cells is reduced 1.5-fold when cells pretreated with RA are re-exposed to RA. Similarly, it has been observed that in vitamin A deficient hamsters, orally administered RA is slowly metabolized, whereas in animals pre-fed RA, metabolism of subsequently administered RA was much more extensive (47).
Our results from Northern analysis and RT-PCR analysis of knockout
lines suggest that RAR and RXR
may be important in mediating the
induction of P450RAI. These observations were substantiated using several receptor subtype-specific synthetic retinoids. The RAR
-specific ligand strongly induced P450RAI, whereas the
RAR
and RAR
-specific ligands did not induce detectable P450RAI
mRNA expression. The activity of the RAR
agonist was further
enhanced by the addition of a RXR panagonist, almost to the level of
P450RAI-induced expression in wild-type by RA. This is
consistent with the view that in most instances, RAR-RXR heterodimers
are the functional units, transducing the effects of retinoids both
in vivo and in vitro (14-17,
48).2
A clear understanding of RA metabolic activity is critical when considering the activities of retinoids in different cell types or tissues. For example, it may be very important when considering the use of retinoids in the treatment of disease. RA has been found to induce a complete but temporary remission in acute promyelocytic leukemia (10). Further treatment with RA at relapse is ineffective. This resistance to RA has been attributed to induced RA metabolism by a cytochrome P450 (49), which could be P450RAI. Because our studies with synthetic retinoids show that P450RAI induction is mediated in a receptor subtype-specific manner, the specificity of synthetic retinoids may be crucial in determining whether or not the RA metabolic pathway is induced. It is possible that some retinoids that either fail to induce RA metabolism or are not substrates for the metabolic pathway may be more effective than all-trans-RA as therapeutic agents in cancer and other diseases (34, 40, 50-52). In addition, RA is known to play a crucial role in development, and P450RAI, which is known to be expressed in a precise spatiotemporal pattern during development (43),5 may provide a mechanism for tight control of RA at the tissue level. An important test of both this notion and our hypothesis on the involvement of P450RAI in RA metabolism will be the ablation of the P450RAI gene from F9 cell lines and the mouse genome.
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ACKNOWLEDGEMENTS |
---|
We thank Tudor Moldoveanu for performing the mouse P450RAI transfection studies reported here and Jay White for comments and discussion. We are grateful to P. R. Reczek (Bristol-Myers Squibb, Pharmaceutical Research Institute, Buffalo, NY) for the gift of synthetic retinoids.
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FOOTNOTES |
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* This work was supported by grants from by the Medical Research Council of Canada (to M. P. and G. J.).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 contribution of these authors to the work was equal.
Supported by fellowships from the Center National de la
Recherche Scientifique and the Fondation pour la Recherche
Médicale.
¶¶ Supported by funds from CNRS, INSERM, the Collège de France, the Center Hospitalier Universitaire Régional, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Human Frontier Science Program, and Bristol-Myers Squibb.
|| To whom correspondence should be addressed: Cancer Research Laboratories, Rm. 355, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-545-6791; Fax: 613-545-6830; E-mail: petkovic{at}post.queensu.ca.
1 The abbreviations used are: RA, retinoic acid; 4-OH-RA, 4-hydroxy-RA; 18-OH-RA, 18-hydroxy-RA; RAR, RA receptor; RXR, retinoid X receptor; HPLC, high performance liquid chromatography; RT-PCR, reverse transcriptase polymerase chain reaction.
2 Chiba, H., Clifford, J., Metzger, D., and Chambon, P. (1997) J. Cell Biol. 139, 735-747.
3 B. Beckett, J. White, and M. Petkovich, unpublished observations.
4 White, J. A., Beckett, B., Scherer, S. W., Herbrick, J.-A., and Petkovich, M. (1998) Genomics, in press.
5 A. Iulianella, D. Lohnes, L. Luu, K. Hsu, B. R. Beckett, and M. Petkovich, unpublished results.
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REFERENCES |
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