Affiliations of authors: Y. S. Kuppumbatti, S. Waxman, R. Mira-y-Lopez (Department of Medicine), I. J. Bleiweiss (Department of Pathology), J. P. Mandeli (Department of Biomathematical Sciences), Mount Sinai School of Medicine, New York, NY.
Correspondence to: Rafael Mira-y-Lopez, M.D., Ph.D., Department of Medicine, Mount Sinai School of Medicine, Box 1178, One Gustave L. Levy Place, New York, NY 10029-6574 (e-mail: mira{at}msvax.mssm.edu).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The concept of aberrant RA signaling in cancer, first highlighted by the leukemogenic role of
the promyelocytic leukemia-RAR fusion protein (5), was extended
to solid human carcinomas by the demonstration of progressive RARß underexpression in
various cancers, including breast cancer (6). We reported earlier that
CRBP is not expressed in several breast cancer cell lines in routine culture (7). Because the human CRBP gene is CpG rich (8) and
tissue-specific genes harboring CpG islands have been shown to be susceptible to
hypermethylation in culture (9), the lack of CRBP expression that we
observed could be a culture artefact. To address this question, we analyzed CRBP expression in
normal and malignant breast tissues. We show that CRBP is underexpressed in about one in four
breast cancers in situ. This finding extends the concept of aberrant RA signaling in
breast cancer to prenuclear RA signaling events.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sections of formalin-fixed, paraffin-embedded breast cancer specimens mounted on charged slides were culled by a breast pathologist (I. J. Bleiweiss) over a period of several weeks. These slides represent an essentially unselected cross-section of cases of breast cancer (the only criterion for inclusion in the study was the availability of residual sections). Reduction mammoplasty specimens were used as a source of normal breast tissue; analysis of the histologic sections used for in situ hybridization confirmed normal morphology. Patient privacy was protected by the use of a code that was breakable only by the breast pathologist.
In Situ Hybridization
CRBP expression was evaluated by in situ hybridization in six reduction mammoplasty specimens and in 49 human breast carcinoma specimens. In situ hybridization analysis was carried out as described (10) with minor modifications. Briefly, tissue sections were deparaffinized in two changes of fresh xylene (each incubated for 10 minutes), hydrated by sequential transfers in a series of solutions containing decreasing concentrations of ethanol, washed twice in phosphate-buffered saline (PBS), deproteinized by an initial incubation in 0.2 N HCl for 10 minutes at 23 °C, and followed by a 15-minute incubation at 37 °C with proteinase K at 2 mg/mL in 10 mM Tris (pH 8.0) containing 2 mM CaCl2. Proteinase K was predigested for 20 minutes at 37 °C to destroy any contaminating ribonuclease (RNase). The slides were then washed three times with PBS, postfixed in PBS containing 4% paraformaldehyde for 5 minutes, washed two more times in PBS, and washed once in acetylation buffer (0.25% acetic anhydride and 0.1 M triethanolamine [pH 8.0]). The slides were next washed briefly in PBS, dehydrated in a graded ethanol series up to 100% ethanol, and air-dried. The slides were then prehybridized in humid boxes at 42 °C for at least 1 hour with hybridization solution (50% deionized formamide, 2x standard saline citrate [SSC], 2x Denhardt's solution, 10% dextran sulfate, yeast transfer RNA [400 µg/mL], salmon sperm DNA [250 µg/mL], and 20 mM dithiothreitol in diethyl pyrocarbonate-treated water). Then the slides were incubated with hybridization solution (containing 20 ng of freshly denatured digoxigenin-labeled probe in 50 µL of hybridization solution), covered with parafilm (American Can Co., Greenwich, CT) placed in humidified boxes, and incubated at 42 °C overnight. The slides were then allowed to cool to room temperature, the parafilm was removed in 2x SSC, and the sections were incubated at 37 °C for 30 minutes with RNase A (40 µg/mL) and RNase T1 (10 U/mL; Boehringer Mannheim Biochemicals, Indianapolis, IN) in 10 mM Tris (pH 8.0), 1 mM EDTA, and 0.5 M NaCl. The sections were then washed twice in 2x SSC and incubated in 2x SSC containing 2% normal sheep serum and 0.05% Triton X-100 for 2 hours at 23 °C with mild agitation.
For immunodetection of the in situ hybridization signal, the slides were briefly washed with buffer 1 (100 mM maleic acid and 150 mM NaCl [pH 7.5]), incubated in buffer 1 containing 2% normal sheep serum and 0.3% Triton X-100 for 30 minutes at 23 °C, and then incubated overnight at 4 °C with sheep antidigoxigenin antibody conjugated with alkaline phosphatase (Boehringer Mannheim Biochemicals) diluted to 0.375 U/mL in buffer 1 containing 1% normal sheep serum and 0.3% Triton X-100. On the next day, the slides were washed in buffer 1 for two 10-minute periods at 23 °C and equilibrated with buffer 3 (100 mM Tris-HCl [pH 9.5], 100 mM NaCl, and 50 mM MgCl2). The color reaction was developed by incubating the slides in a chromogen solution (45 µL of a nitroblue tetrazolium solution at 100 mg/mL and 35 µL of a 5-bromo-4-chloro-3-indolyl phosphate solution at 50 mg/mL in 10 mL of buffer 3; both reagents were from Boehringer Mannheim Biochemicals) up to 8 hours at 23 °C in humidified light-tight boxes, with occasional examination of color development. The color reaction was stopped by washing slides with TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA), and the slides were mounted with a coverglass in aqua mounting medium (Baxter, Houston, TX).
The CRBP RNA probes used were generated by amplifying a 320-base-pair (bp) fragment
(exons 1, 2, and part of 3) from the human CRBP (hCRBP) complementary DNA (cDNA; from
Dr. Winnie Eskild, University of Oslo, Norway) with forward primer
5'-TTGTGGCCAAACTGGCTCCA-3' and reverse primer
5'-ACACTGGAGCTTGTCTCCGT-3' and subcloning of the polymerase chain
reaction (PCR) product into the pCRII transcription vector (Invitrogen Corp., Carlsbad, CA).
Antisense and sense digoxigenin-labeled CRBP RNA probes, respectively (insert orientation was
confirmed by DNA sequencing), were transcribed from the T7 and SP6 promoters with DIG
RNA labeling kit (Boehringer Mannheim Biochemicals). Because the transcription from the T7
promoter proved to be more efficient, a clone in reverse orientation was used for the
T7-promoted synthesis of sense CRBP RNA. The sense CRBP RNA probe did not react with
normal breast tissue, as expected, but it did react with breast carcinoma tissue, suggesting that it
hybridized to a messenger RNA (mRNA) preferentially expressed in cancer. A BLAST search
revealed that the sense hCRBP RNA probe contained a stretch of 21 nucleotides complementary
to the GLUT-1 gene, which is overexpressed in breast cancer (11).
Deletion of this region (nucleotides 39-60 of the CRBP sequence) by digestion with EcoRV (polycloning site) and HincII (nucleotide 68), followed by religation and T7
transcription, generated a modified sense CRBP RNA probe that did not react with breast
carcinoma tissue, confirming the specificity of the method. RARß2 and RXR antisense
RNA probes were provided by Dr. Xiao-Chun Xu (The University of Texas M. D. Anderson
Cancer Center, Houston). RNA probes were quantitated by a spot assay as described in the DIG
RNA labeling kit instructions.
The breast pathologist evaluated the in situ hybridization sections by use of hematoxylin-eosin sections from the same blocks for orientation. Multiple low-power fields were inspected in every section. Where adjacent normal tissue and/or carcinoma in situ and/or invasive carcinoma tissue coexisted in a section, their staining was evaluated separately. For any given sample, the staining within a given tissue type was uniformly positive or negative, although in some samples, there was variation in the intensity of the stain. There was wide variation in staining intensity among sections from different patients, particularly (but not exclusively) in the tumor compartment, but rather than assigning a subjective intensity score, a binary evaluation (negative versus positive) was made.
Northern Blot Analysis
Total RNA was isolated with the PureScript RNA isolation kit (Gentra Systems,
Minneapolis, MN). Twenty micrograms of total RNA was size fractionated on a 1.2%
agarose-2.2 M formaldehyde gel, transferred to a Hybond-N+ nylon membrane
(Amersham Life Science Inc., Arlington Heights, IL) in 20x SSC, and UV-crosslinked
(UV Stratalinker 2400; Stratagene, La Jolla, CA). Hybridization probes were the 320-bp hCRBP
cDNA described above, a 236-bp human Mat-8 cDNA derived by reverse
transcription-coupled-PCR (RT-PCR) from MDA-MB-468 RNA by use of primers as described (12), a c-myc cDNA probe (from Dr. Nicole Schreiber-Agus, Albert
Einstein College of Medicine, New York, NY), and a 316-bp human glyceraldehyde-3-phosphate
dehydrogenase cDNA fragment (Ambion, Austin, TX). The probes were labeled with
[-32P]deoxycytidine 5'-triphosphate by random priming
to a specific activity of 109 cpm/µg. The membrane was prehybridized for 4
hours at 42 °C in 50% formamide, 6x standard saline phosphate-EDTA
(SSPE), 5x Denhardt's solution, and salmon sperm DNA (0.2 mg/mL), hybridized
overnight with one of the radiolabeled probes (1-5 x 106 cpm/mL), and
washed twice at room temperature in 6x SSPE-0.1% sodium dodecyl sulfate (SDS)
and once at 65 °C in 0.1x SSPE-0.1% SDS before autoradiography.
Subsequent rounds of hybridization were done as above after stripping the blot for 15 minutes at
90 °C-100 °C in 0.2x SSPE-0.1% SDS (stripping was ascertained by
overnight autoradiography).
Statistical Methods
Statistical significance was evaluated with the 2 test or Fisher's
exact test if sample sizes were small. McNemar's test for paired-sample testing was also
used. All P values are from two-sided tests.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All six reduction mammoplasty specimens analyzed for CRBP expression
by in situ hybridization showed specific CRBP staining of the
ductal epithelium, as shown in Fig. 1, A, for one
specimen. Primary human breast epithelial cell cultures generated from
nine additional reduction mammoplasty specimens as described
(7,13) were probed for CRBP expression by northern (three
specimens) or western (six specimens) blot analysis. In all cases,
abundant CRBP expression was detected (7) (Mira-y-Lopez R,
Zheng WL, Kuppumbatti YS, Rexer B, Jing Y, Ong DE: unpublished
results). Thus, we have documented CRBP expression in all 15 reduction
mammoplasty specimens examined.
|
Table 1 summarizes the results of the CRBP in
situ hybridization analysis of 49 human breast carcinomas, 35 of
which contained adjacent normal breast tissue in the same section. CRBP
was expressed in 33 of the 35 specimens of adjacent normal breast
tissue but was not expressed in 12 of the 49 breast carcinoma
specimens. Thus, the incidence of CRBP-negative specimens, which was
0% for reduction mammoplasty tissue, increased to 6% for adjacent
normal tissue (not statistically significant) and to 24% for breast
carcinoma tissue (P = .023 compared with adjacent normal
tissue). The 95% confidence interval for the frequency of CRBP loss in
human breast carcinoma is 12.5%-36.5%. The frequency of CRBP loss
was similar in ductal carcinoma in situ (DCIS) (six [27%]
of 22) compared with invasive carcinoma (six [22%] of 27) and in
low-grade tumors compared with high-grade tumors (Table
2
). These findings and the two cases of adjacent
normal tissue that were CRBP negative (Table 1
) suggest that the loss
of CRBP occurs relatively early in the multistep process of
carcinogenesis. For instance, the loss of CRBP appears to be an earlier
event than the loss of RARß, which was reported to be more pronounced
in advanced disease (6). However, we did encounter one
specimen in which the DCIS component expressed CRBP and the invasive
carcinoma component did not (data not shown).
|
|
The loss of CRBP was not associated with patient age or with steroid receptor status,
although there was a statistically nonsignificant association with progesterone receptor-negative
disease (Table 2). No association was apparent between CRBP expression
and lymph node status
(32 informative cases) or Her2/neu overexpression (26 informative cases) (data not shown).
Concomitant Loss of CRBP and RARß
To assess whether the loss of CRBP and the loss of RARß are
associated events, available duplicate sections from CRBP-negative and
CRBP-positive breast cancers were evaluated for RARß expression by
in situ hybridization. We found that four of five
CRBP-negative and four of seven CRBP-positive specimens were
negative for RARß expression, suggesting that the losses of CRBP and
RARß are not associated and occur in partially overlapping tumor
subsets. However, these preliminary findings suggest that the
concomitant loss of CRBP and RARß is quite frequent, with about one
in five breast cancers estimated to have lost both CRBP and RARß.
(The frequency of CRBP loss, or 0.24, times the frequency of RARß
loss in CRBP-negative tumors, or 0.80, yields 0.19 as the estimated
frequency of CRBP and RARß loss.) Thus, the biologic activity of
vitamin A may be doubly compromised in a substantial fraction of breast
cancers. Fig. 1, C, shows the lack of RARß staining in the tumor
tissue of specimen 47, which also lacked CRBP (Fig. 1,
B). Adjacent
normal tissue in the same specimen was positive for RARß.
Comparison of CRBP, c-myc, and Mat-8 Expression
CRBP has been identified as one of six genes that are expressed in
mouse mammary tumors induced by neu or ras but that are usually not
expressed in tumors induced by c-myc or int-2 (12).
Overexpression of neu did not induce expression of this set of genes in
c-myc-induced tumors; conversely, overexpression of c-myc did not
decrease their expression in neu-induced tumors. This led to the
hypothesis that there may be two types of mouse mammary epithelial
cells at risk for malignant transformation, one (CRBP negative) that is
targeted by c-myc/int-2 and the other (CRBP positive) that is targeted
by neu/ras (12). Therefore, we asked whether the loss of CRBP
in human breast cancer is associated with c-myc overexpression or is
accompanied by the loss of Mat-8, one of the genes whose expression
pattern matches that of CRBP in mouse mammary tumors. To answer this
question, we measured the content of CRBP, Mat-8, and c-myc mRNAs in
nine human breast carcinoma cell lines. Only MDA-MB-231 cells, which
overexpressed c-myc, showed loss of both CRBP and Mat-8; five cell
lines expressed Mat-8 only, and one cell line expressed CRBP only
(Fig. 2). Also, the expression of c-myc and CRBP
were not associated. These results and the lack of association
between CRBP and Her2/neu expression indicate that the above
observations made on mouse mammary tumors do not translate to human
breast cancer.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study provides important in situ validation of our earlier study showing CRBP
underexpression in human breast cancer cell lines in vitro [(7); Fig. 2]. The frequency of CRBP underexpression that
we observed in
situ (24%) is lower than the frequency that we observed in vitro (six
[60%] of 10 cell lines were CRBP negative by northern blot analysis). This
difference may be simply because of a sampling error or the different sensitivities of the
techniques used. Alternatively, culture conditions are more conducive to the loss of CRBP.
An RT-PCR study (15) that used bulk tumor RNA concluded that CRBP was expressed in all 18 human breast carcinomas examined. This apparent discrepancy can be readily explained by the amplification of CRBP mRNA from adjacent normal breast tissue or from other normal CRBP-expressing cells, such as fibroblasts and infiltrating lymphocytes. However, we cannot exclude the possibility that tumors identified as CRBP negative by in situ hybridization express low levels of CRBP mRNA that can be detected by RT-PCR.
What, if any, is the biologic relevance of CRBP underexpression in breast cancer? CRBP is postulated to regulate the uptake and intracellular fate of retinol. For instance, retinol uptake in vivo is thought to proceed via a facilitated transport system in which retinol bound to plasma retinol-binding protein on the outer surface of the cells is transported to CRBP on the inner cell surface (16). If this is the case in normal breast epithelial cells, then loss of CRBP would be expected to compromise retinol uptake and thus RA synthesis, providing a growth advantage to cancer cells. Loss of CRBP may also compromise RA synthesis via mechanisms independent of retinol uptake. For instance, CRBP is postulated to function in a chaperone-like capacity to regulate the formation of retinyl esters and the synthesis of RA (3,4). Recent studies (17) of CRBP null mice demonstrated that CRBP is essential for efficient retinol storage but is not essential for RA synthesis. Nevertheless, it remains intriguing that CRBP co-localizes with retinal dehydrogenase-2 in regions of the central nervous system that are active in RA synthesis (18). An alternative view is that CRBP prevents the partitioning of retinol into cell membranes, where it may have deleterious effects on normal cell function. Finally, the possibility that CRBP exerts a function that is independent of its retinol-binding ability cannot be ruled out. We are currently performing gain-of-function experiments to elucidate the role of CRBP in breast cells.
CRBP belongs to a large family of fatty acid-binding protein genes, whose members encode cytosolic proteins of about 15 kd involved in the intracellular binding and targeting of hydrophobic substrates (19). Of interest, another member of this family, mammary-derived growth inhibitor (MDGI), regulates mammary differentiation (20) and is underexpressed in breast cancer (21), and other fatty acid-binding proteins are underexpressed in experimental carcinomas of the small intestine and colon (22). Thus, decreased fatty acid-binding protein expression may constitute a recurring theme in cancer.
Our finding that at least two genes encoding retinoid signaling proteins, CRBP and RARß, are underexpressed in about one in five breast cancers is of interest because the retinoid signaling pathway is safeguarded by the expression of functionally redundant proteins. Thus, compound gene deletions are required for its inactivation (1). We hypothesize that compound loss of CRBP and RARß hinders the bioactivity of endogenous vitamin A more extensively than the single loss of either gene. For instance, CRBP underexpression may compromise ligand availability and thus the activation of multiple RAR and RXR isoforms, leading to a more extensive phenotype than loss of RARß expression alone. Conversely, RARß underexpression may ensure silencing of this growth-suppressive receptor, leading to a stronger phenotype than loss of CRBP expression alone. The phenotype of mice with compound CRBP and RARß gene deletions remains to be learned. It also remains to be seen whether CRBP underexpression in cancer is as widespread as underexpression of RARß, which has been demonstrated for multiple types of cancers.
What is the mechanism of CRBP underexpression in breast cancer? Like RARß, CRBP has been shown to be transcriptionally induced by RA (23). Although the responsible regulatory cis site in the hCRBP gene has not been characterized, RA induction of hCRBP has been demonstrated (24). In oral leukoplakia, the loss of RARß has been associated with low RA steady-state levels (25). By extension, it is possible that the loss of CRBP in breast cancer is secondary to altered RA metabolism. (In this scenario, CRBP loss would represent a consequence rather than a cause of low RA levels.) However, the lack of association between the loss of CRBP and RARß in breast cancer, the apparently different time courses of these events (early versus progressive, respectively), and the fact that RA treatment of breast cancer cells in vitro fails to restore the expression of either CRBP or RARß (7) suggest that there are alternative explanations, which we are currently seeking.
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1
Chambon P. A decade of molecular biology of retinoic acid
receptors. FASEB J 1996;10:940-54.
2
Kurlandsky SB, Gamble MV, Ramakrishnan R, Blaner WS.
Plasma delivery of retinoic acid to tissues in the rat. J Biol Chem 1995;270:17850-7.
3 Ong DE, Newcomer ME, Chytil F. Cellular retinoid-binding proteins. In: Sporn MB, Roberts AB, Goodman DS, editors. The retinoids: biology, chemistry and medicine. New York (NY): Raven Press; 1994. p. 283-317.
4
Napoli J. Retinoic acid biosynthesis and metabolism. FASEB J 1996;10:993-1001.
5 Zelent A. Translocation of the RAR alpha locus to the PML or PLZF gene in acute promyelocytic leukaemia. Br J Haematol 1994;86:451-60.[Medline]
6 Xu XC, Sneige N, Liu X, Nandagiri R, Lee JJ, Lukmanji F, et al. Progressive decrease in nuclear retinoic acid receptor beta messenger RNA level during breast carcinogenesis. Cancer Res 1997;57:4992-6.[Abstract]
7
Jing Y, Zhang J, Bleiweiss I, Waxman S, Zelent A, Mira-y-Lopez
R. Defective expression of cellular retinol binding protein type I and retinoic acid receptors
2, ß2, and
2 in human breast cancer cells. FASEB J 1996;10:1064-70.
8 Nilsson MH, Spurr NK, Lundvall J, Rask L, Peterson PA. Human cellular retinol-binding protein gene organization and chromosomal location. Eur J Biochem 1988;173:35-44.[Abstract]
9 Antequera F, Boyes J, Bord A. High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 1990;62:503-14.[Medline]
10 Xu XC, Clifford JL, Hong WK, Lotan R. Detection of nuclear retinoic acid receptor mRNA in histological tissue sections using nonradioactive in situ hybridization histochemistry. Diagn Mol Pathol 1994;3:122-31.[Medline]
11 Brown RS, Wahl RL. Overexpression of Glut-1 glucose transporter in human breast cancer. Cancer 1993;72:2979-85.[Medline]
12 Morrison BW, Leder P. neu and ras initiate murine mammary tumors that share genetic markers generally absent in c-myc and int-2-initiated tumors. Oncogene 1994;9:3417-26.[Medline]
13 Stampfer M, Hallowes RC, Hackett AJ. Growth of normal human mammary cells in culture. In Vitro 1980;16:415-25.[Medline]
14 Jing Y, Waxman S, Mira-y-Lopez R. The cellular retinoic acid binding protein II is a positive regulator of retinoic acid signaling in breast cancer cells. Cancer Res 1997;57:1668-72.[Abstract]
15 Pasquali D, Bellastella A, Valente A, Botti G, Capasso I, del Vecchio S, et al. Retinoic acid receptors alpha, beta and gamma, and cellular retinol binding protein-I expression in breast fibrocystic disease and cancer. Eur J Endocrinol 1997;137:410-4.[Medline]
16 Bavik C, Ward SJ, Ong DE. Identification of a mechanism to localize generation of retinoic acid in rat embryos. Mech Dev 1997;69:155-67.[Medline]
17
Ghyselinck NB, Bavik C, Sapin V, Mark M, Bonnier D,
Hindelang C, et al. Cellular retinol-binding protein I is essential for vitamin A homeostasis. EMBO J 1999;18:4903-14.
18 Yamamoto M, Drager UC, Ong DE, McCaffery D. Retinoid-binding proteins in the cerebellum and choroid plexus and their relationship to regionalized retinoic acid synthesis and degradation. Eur J Biochem 1998;257:344-50.[Abstract]
19 Glatz JF, van der Vusse GJ. Cellular fatty acid-binding proteins: their function and physiological significance. Prog Lipid Res 1996;35:243-82.[Medline]
20 Yang Y, Spitzer E, Kenney N, Zschiesche W, Li M, Kromminga A, et al. Members of the fatty acid binding protein family are differentiation factors for the mammary gland. J Cell Biol 1994;127:1097-109.[Abstract]
21 Huynh HT, Larsson C, Narod S, Pollak M. Tumor suppressor activity of the gene encoding mammary-derived growth inhibitor. Cancer Res 1995;55:2225-31.[Abstract]
22 Davidson NO, Ifkovits CA, Skarosi SF, Hausman AM, Llor X, Sitrin MD, et al. Tissue and cell-specific patterns of expression of rat liver and intestinal fatty acid binding protein during development and in experimental colonic and small intestinal adenocarcinomas. Lab Invest 1993;68:663-75.[Medline]
23 Smith WC, Nakshatri H, Leroy P, Rees J, Chambon P. A retinoic acid response element is present in the mouse cellular retinol binding protein I (mCRBPI) promoter. EMBO J 1991;10:2223-30.[Abstract]
24 Fisher GJ, Reddy AP, Datta SC, Kang S, Yi JY, Chambon P, et al. All-trans retinoic acid induces cellular retinol-binding protein in human skin in vivo. J Invest Dermatol 1995;105:80-6.[Abstract]
25 Xu XC, Zile M, Lippman SM, Lee JS, Lee JJ, Hong WK, et al. Anti-retinoic acid (RA) antibody binding to human premalignant oral lesions, which occurs less frequently than binding to normal tissue, increases after 13-cis-RA treatment in vivo and is related to RA receptor ß expression. Cancer Res 1995;55:5507-11.[Abstract]
Manuscript received August 5, 1999; revised December 8, 1999; accepted December 21, 1999.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |