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Cancer Research | Clinical Cancer Research |
Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
Molecular Cancer Research | Cell Growth & Differentiation |
Max-Delbrück-Center for Molecular Medicine, D-13122 Berlin, Germany [N. P. K., J. M., S. K., G. B., B. A., A. O., M. Z., P. B.], and Institute of Molecular Genetics, 166 37 Prague 6, Czech Republic [P. B.]
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Abstract |
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Introduction |
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A number of studies demonstrated that NRs have a profound impact on development of hematopoietic cells, e.g., RXR and TR/c-erbA effectively regulate erythroid cell growth and differentiation (Refs. 7
and 8
and references therein). Also in this context, an oncogenic version of TR/c-erbA, the v-erbA oncoprotein, arrests differentiation of erythroblasts and cooperates with v-erbB in producing a fatal erythroleukemia in chickens (9
, 10)
. Additionally, activation of both glucocorticoid receptor and ER is required for an efficient induction of self renewal of erythroid progenitor cells (11, 12, 13, 14)
. In other cell systems, activation of RAR by retinoic acid directly mediates a growth arrest of human HL60 leukemia cells and promotes their differentiation into neutrophils (15)
. Furthermore, a chromosomal translocation that characterizes most of acute PMLs results in a fusion protein containing the NH2-terminal part of the PML gene and the DNA binding and ligand binding domains of RAR
(16
, 17)
. Interestingly, introduction of a dominant-negative RAR in bone marrow cells immortalized a lymphohematopoietic progenitor, which can be induced to differentiate into lymphoid, myeloid, and erythroid lineages (18)
.
In view of the central role of NRs as key factors in regulating molecular mechanisms of hematopoietic cell development, we focused on the identification of potential new members of the NR gene family by applying a reverse transcriptase-PCR approach. This strategy used degenerate primers encompassing the region encoding the DNA binding domain of most NR genes (19) . During the course of this study, we consistently found the TR4 gene as the most abundant cDNA in primary cultures of chicken bone marrow cells.
TR4 (TAK1 and NR2C2; Ref. 20
) is an orphan receptor that is closely related to TR2, and both TR2 and TR4 define a subclass within the steroid/thyroid receptor family (21, 22, 23)
. TR4 and TR2 form homodimers as well as TR4/TR2 heterodimers that synergize in repressing gene expression (24)
. However, TR4 and TR2 do not heterodimerize with RXR, which is an obligate partner for most NRs that are able to form heterodimers. Several lines of evidence suggest that TR4 is an important regulator of distinct NR signaling pathways, e.g., it has been shown that TR4 effectively represses RAR, RXR, peroxisome proliferator-activated receptor, vitamin D3 receptor, and TR-mediated gene expression (Refs. 22
, 24, 25, 26
, and references therein). TR4 is able to repress transcription by interaction with DR1-HREs and DR2 elements (27)
. TR4 also appears to repress ER-mediated transactivation specifically in bone cells but not in kidney or breast cancer cells (28)
. In contrast, some genes containing DR4-HRE motifs or nonclassical thyroid HREs were described to be activated by TR4 (27)
. Such a positive transcriptional activity of TR4 was observed for the ciliary neurotrophic factor receptor gene (29)
, and a cooperation of TR4 with COUP-TF and TR1 in activation of the HIV-long terminal repeat promoter was also reported (30)
. More recently, it was demonstrated that TR4 interacts with the androgen receptor, and as a consequence, both TR4 and androgen receptor mediated pathways were influenced (31)
. The current mode of TR4 action is still a subject of debate and may involve distinct mechanisms, such as: (a) occupation of different HREs by TR4 homodimers or TR4/TR2 heterodimers; (b) sequestration of (a) coactivator(s); (c) association of TR4 with (an) as yet undefined corepressor(s); (d) modulation of TR4 activity by thus far unknown ligand(s); or (e) other posttranslational events.
In this work, we demonstrate that the nuclear orphan receptor TR4 is highly expressed in hematopoietic cells. We additionally analyzed the impact of TR4 on primary bone marrow cells by ectopic expression of TR4 via retrovirus vector. Additionally, we show that a DNA binding-deficient TR4 protein exerts a similar effect as its natural counterpart and provide information of the potential role of such proteins on hematopoietic cell growth and differentiation.
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Results |
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TR4 Effectively Induces Proliferation of Myeloid Progenitor Cells.
Whereas previous studies demonstrated the importance of TR4 in repressing several NR pathways, the impact of TR4 in primary cells and particularly in those of the hematopoietic system has thus far not been studied. Therefore, to get insight into its potential role in hematopoietic cells, TR4 was ectopically expressed in chicken bone marrow cells, and its effects on proliferation and/or development of such cells were investigated. To this end, a retrovirus vector harboring TR4 cDNA was constructed and transfected into CEF, and neomycin-resistant cells were selected. Cells effectively expressed TR4 protein (see below, Fig. 3
) and were used as a source of virus to infect bone marrow cells. Pilot experiments using different culture conditions with various combinations of growth factors, hormones, sera, and conditioned media allowed us to optimize the growth of TR4-infected precursor cells. Most significantly, TR4 caused an outgrowth of cells within 45 days, whereas this was not observed in control cultures infected with empty vector (Fig. 2A)
. Cells grew in large aggregates containing >100 cells (Fig. 2B
, left panel) and ceased proliferation after 1416 days.
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In conclusion, morphological features and the cell surface marker profile clearly indicate that TR4 cells are myeloid progenitors. This is in accordance with expression in these cells of myeloid proteins, such as C/EBPß (see below Fig. 7
).
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Bandshift assays demonstrated that HA-TR4 effectively bound to the TR4 binding site contained in DR1-HRE (AGGTCAAAGGTCA; Ref. 26
), whereas HA-TR4 did not (Fig. 4A)
. The band corresponding to HA-TR4 migrated slightly higher than untagged TR4. HA-TR4 binding was clearly sequence specific because it was efficiently competed by unlabeled DR1-HRE but not by a mutated oligonucleotide (Fig. 4A
and data not shown). Moreover, the anti-HA-specific monoclonal antibody was able to supershift the band of HA-TR4, whereas it did not affect the pattern of HA-
TR4. These data were additionally confirmed by Shift-Western experiments (38)
where the proteins in the bandshift gel were transferred to a membrane and probed with the anti-HA antibody; as expected, the antibody detected HA-TR4, which corresponds to the shifted band (data not shown).
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Having determined that HA-TR4 does not bind DNA nor represses transcription, we next sought to examine whether the DNA binding activity of TR4 was responsible for the TR4-induced phenotype observed. Therefore, HA-TR4 and HA-
TR4 retroviruses were used to infect bone marrow cells as above. HA-TR4 behaved identically to unmodified TR4, and growing myeloid cells were readily obtained (Fig. 5
and data not shown). Surprisingly, an outgrowth of myeloid cells was also observed for HA-
TR4, though the total cell numbers were somewhat lower than for HA-TR4 (Fig. 5)
. As expected, control cells infected with neo-virus exhibited a restricted growth potential and ceased proliferation after 46 days in culture. In addition, retrovirus-transduced cells efficiently expressed HA-tagged TR4 proteins as revealed by Western blot analysis using the anti-HA-specific antibody (Fig. 5)
. Finally, both by morphology and cell surface marker expression, TR4, HA-TR4, and HA-
TR4 cells were identical (data not shown).
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RXR and Its DNA Binding-deficient Variant RXR Do Not Fully Recapitulate TR4 Effects on Bone Marrow Cells.
TR4 functions as a strong repressor of RXR-mediated transcription as published elsewhere and also shown here. Therefore, to test whether a dominant-interfering RXR would display a similar phenotype as TR4, RXR lacking the DNA binding domain (7)
was introduced into bone marrow cells via retrovirus.
RXR was demonstrated before to act as a dominant-interfering RXR allele in transient transactivation experiments (7)
. Full-length RXR was used as a control. While there was an initial outgrowth of
RXR and RXR cells, at day 9 of culture, cells ceased proliferation, and cell numbers remained constant or started to decrease (Fig. 5)
. All these data indicate that while RXR promotes growth of myeloid cells, extended proliferation is specifically accomplished only by TR4 and TR4 variants. This difference in their proliferative potential is likely not to be due to different expression levels of TR4 and RXR, because both are expressed from the very same retroviral vector. Additionally,
RXR had a similar effect on bone marrow cells as full-length RXR, indicating that as seen for TR4, the DNA binding of RXR is dispensable for this activity.
To examine the differentiation potential of TR4 cells, inducers of myeloid differentiation, such as 9cRA or TPA, were added, and cells were investigated for morphological changes and adhesive properties (Fig. 6, A and B)
. As a control, cells were treated with chicken myelomonocytic growth factor, which in previous experiments showed growth-promoting but no differentiation-inducing activity (data not shown). On 9cRA treatment, only 510% of the HA-TR4 and HA-
TR4 cells became adherent (Fig. 6B)
. However, the majority of RXR and
RXR cells adhered to the dish after the addition of 9cRA and differentiated into macrophage-like cells (Fig. 6, A and B
and data not shown). This might indicate that TR4 and RXR transformants might represent different cell types, at least in response to 9cRA, and this might account for the differences observed. TPA rapidly induced adhesion of the entire cell population, and no obvious differences were observed for TR4,
TR4 or RXR,
RXR cells.
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To further extend the analysis of TR4 cells, the presence of C/EBPß, another protein that is highly expressed in myeloid progenitors and required for efficient Mim-1 expression (42
, 43)
, was tested by immunoblotting. It was found that TR4- and HA-TR4-containing cells displayed high levels of C/EBPß (Fig. 7C)
. C/EBPß was also expressed in RXR and
RXR cells, although its level was lower.
Taken together, all these data support a role of TR4 in inducing outgrowth of committed granulocyte progenitors and/or in preventing terminal differentiation of such cells.
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Discussion |
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As shown here, TR4 is expressed in several hematopoietic lineages, but after ectopic expression in bone marrow cells, an effective outgrowth was only observed in the myeloid compartment. Interestingly, whereas erythroid cells showed a prominent expression of TR4 in all species analyzed, an outgrowth of erythroid progenitor cells was not observed. In addition, if introduced into established erythroid progenitors (STED progenitors; Ref. 7 ), TR4 had only a marginal growth promoting activity (data not shown). This might be because ectopic expression represents one approach to assess gene function but does not necessarily recapitulate all activities of the respective protein in its physiological environment.
TR4 is believed to exert its transcriptional activity as a repressor by several mechanisms, including target site competition (24
, 26 , 27
, and references therein). Accordingly, TR4-mediated repression on certain DNA elements requires an intact DNA binding domain as also demonstrated here. Alternatively, transcriptional repression might be mediated by sequestration of cofactors (transrepression) that, e.g., bind to AF-2, and, therefore, both TR4 and TR4 might exert transrepression. Interestingly, in vivo TR4 and
TR4 exhibit similar biological activities in promoting promyelocyte proliferation. It is therefore tempting to speculate that most, if not all, of the activity of ectopically expressed TR4 in myeloid cells results from the sequestration of (a) cofactor(s) that is/are involved in proper myeloid development. Evidence coming from experiments in other hematopoietic cell types would also be in line with this idea. For example, in erythroid cells, the ER can arrest differentiation via a mechanism that requires an intact AF-2 domain but is independent of AF-1 and DNA binding domains (45)
.
Recently, it has become evident that NRs are capable of modulating target gene expression not only as DNA-bound factors but also by mechanisms that do not require direct DNA interactions (6
, 12) . Such DNA independent activities of NRs are in most situations executed by protein-protein interactions. The most likely candidates for such function are corepressors and coactivators that are known to interact with the AF-2 domain of NRs (reviewed in Ref. 6
). The observations reported here demonstrating a similar activity for TR4 and TR4 in promyelocyte proliferation would be in accordance with such a DNA-independent mechanism of TR4 action. A candidate for such a function is RIP-140, a coactivator that has been shown to interact with TR4 (25
, 26)
. Additionally, transrepression by TR4 attributable to sequestration of RIP-140 is not without precedence and was observed, e.g., in peroxisome proliferator-activated receptor/RXR-controlled gene expression (26)
.
Investigation of the subcellular localization of the TR4 proteins showed that TR4 was nuclear, whereas TR4 was localized mostly in the nucleus but also present in the cytoplasm of TR4-transduced CEF and myeloid cells. This might be because the putative nuclear localization signal of TR4 (corresponding to the respective nuclear localization signal in the DNA binding region of TR2; Ref. 37
) is deleted in
TR4. Thus, the reduced nuclear expression of
TR4 might well explain the somewhat diminished proliferative potential of
TR4 cells as compared with TR4 cells.
There is a large body of evidence demonstrating that different RAR alleles, such as PML-RAR or full-length and truncated RARs, effectively block myeloid cell development at the promyelocyte stage (Refs. 17
, 18
, 46, 47, 48
, and references therein) and thus exhibit a phenotype similar to the TR4 promyelocytes as reported here. However, RAR, RAR variants, and TR4 might mediate their effects on myeloid cells by different mechanisms that do or do not require DNA binding, respectively. In addition, thus far no ligand has been identified that regulates TR4 activity. In this study, the experiments with TR4 cells involved serum-containing media, and, therefore, we cannot exclude the presence of a serum-borne ligand (or a metabolite) that might influence TR4 activity. In this respect, we note though that both TR4 and TR4 proteins would be affected similarly by ligand because the putative ligand-binding domain is present in both constructs.
Finally, TR4 as a regulator of myeloid development might represent a pharmacological target and thus might open new possibilities for treatment of myeloid leukemia. In particular, synthetic agonists and/or antagonists that would modulate TR4 activity might provide valuable agents in combination with established treatments for differentiation therapy.
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Materials and Methods |
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CEFs were grown in standard growth medium. Production of retrovirus-producing CEFs and infection of bone marrow cells were as described before (7) . Infections were performed in the presence of 1 µg/ml recombinant human insulin (Actrapid HM40; Novo Nordisk Pharma) and 100 ng/ml SCF.
Construction of Retroviral Vectors.
A retroviral vector containing the full-length TR4 cDNA downstream of the cytomegalovirus promoter was constructed by subcloning wild-type human TR4 (21)
, kindly provided by J. P. H. Burbach (Utrecht, the Netherlands), into the EcoRI site of pSFCV-LE (50)
. The HA-tagged versions of TR4 were first engineered in a shuttle vector containing coding sequences for three HA epitopes upstream of the cloning sites.5
In HA-TR4, amino acid positions 64232, comprising the entire DNA binding domain of human TR4, are deleted. The HA-TR4 and HA-
TR4 were then subcloned into the EcoRV-EcoRI sites of pSFCV-LE. Recombinant retrovirus vectors containing RXR and
RXR (that is devoid of its DNA binding domain;
DBD RXR) were described (7)
.
cDNA Synthesis and PCR.
For RNA preparation, cDNA synthesis, and PCR amplification, see Ref. 32
. Briefly, first-strand cDNA was synthesized from 25 µg of total RNA using 100 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and random hexamers in a total volume of 2050 µl. For PCR amplification, a set of degenerate primers for the region encoding the DNA binding domain of NRs was designed as described previously (19)
. The sequences of these primers were as follows:
NHR1: 5'-GAYRARDCNWSNGGNTDYCAYT-3';
NHR2: 5'-GAYMGNDSNWSNGGNAARCAYT-3';
NHR3: 5'-CAYTTNYKNWRNCKRCANKMNKGRCA-3';
NHR4: 5'-TGYAARNNNTTYTTYMRNMG-3'.
First-strand cDNA was subjected to 40 cycles of PCR amplification using Taq DNA polymerase (Boehringer Mannheim) and applying the following cycle profile: 94°C for 30 s, 37°C for 90 s, and 72°C for 20 s. The primer combinations used were as follows: in the first reaction, NHR1 plus NHR3 or NHR2 plus NHR3. The product of this reaction was diluted 1:300 and used as a template for a hemi-nested reaction with NHR3 and NHR4. PCR products were purified from agarose gels and cloned into pCRII vector (Invitrogen, San Diego, CA), and clones were subjected to DNA sequencing using an Applied Biosystems DNA sequencing device. Sequences were then analyzed using the BLAST alignment tool. GenBank accession no. of chicken TR4: AF133054.
RNA Analysis.
RNA was isolated by standard procedures from tissues and the following cells. Human: K562 (erythroid); U937 and HL60 (myeloid); Namalwa and Raji (B-lymphoid); and CEM, Jurkat (T-lymphoid), and HepG2 (hepatocytes). Mouse: MEL and E31 (erythroid); WEHI-3 and RAW (myeloid); A20 (B-lymphoid); and NIH3T3 (fibroblast). Chicken: DT40 (B-lymphoid); MSB-1 (T-lymphoid); and v-relER-transformed progenitors and dendritic cells (51)
. Primary cells were as follows. Human: erythroid progenitors (52)
, peripheral blood T cells, and monocytes (53)
. Mouse: dendritic cells (53)
. Chicken: SCF and STED erythroblasts (7
, 32)
. A total of 15 µg of RNA/lane was analyzed by Northern blotting (GeneScreen membranes; DuPont) as described (7)
. Filters were hybridized with 32P-labeled human (21)
or chicken TR4-specific probes, washed at moderate stringency, and exposed to film.
Flow Cytometry.
For flow cytometry, cells were recovered, washed with 1% BSA (fraction V; Sigma Chemical Co.) in PBS, and incubated with primary antibody (1 h). The following monoclonal antibodies were used: MC4783, MC512, and MC223 (54)
; K1 (55)
and MHC class II ß chain [2G11, specific for the nonpolymorphic region of chicken BL (56)
; JS4, JS8 (57
, 58)
, CD3, CD4, and CD8 (all Southern Biotechnology Associates); MEP17, MEP21, MEP26, and EOS47 (34)
; DM-GRASP (BEN1; Ref. 59
); HEM-CAM (60)
; and CD44 (AV6, kindly provided by T. F. Davison, Compton, United Kingdom). Cells were then reacted with FITC-conjugated antimouse IgG (1 h; Sigma Chemical Co.), washed, and resuspended in PBS containing 1% BSA and propidium iodide (2 µg/ml; Sigma Chemical Co.) for gating on viable cells. Cells were analyzed by flow cytometry using a FACS Calibur device with CellQuest software (Becton Dickinson).
Western Blotting.
Total cell extracts were separated on a 10% SDS-PAGE gel and blotted onto nitrocellulose membranes (BA85; Schleicher & Schuell) using a semidry blotting system (Pharmacia). Antibody reactions were performed as described previously (49
, 51)
. The following antibodies were used: anti-HA 12CA5, anti-C/EBPß (kindly provided by K. H. Klempnauer, Münster, Germany), and anti-Mim-1 (Ref. 40
; kindly provided by A. Leutz and E. Kowenz-Leutz, Berlin, Germany). Subsequently, blots were washed and incubated with the respective secondary antibody (enhanced chemiluminescence kit; Amersham) in Tris-buffered saline supplemented with 5% nonfat milk powder for 45 min (49)
. All incubations were done at room temperature. Blots were developed with enhanced chemiluminescence reagents and exposed to film.
Immunofluorescence.
CEF containing TR4 versions were cultured as above, trypsinized, and allowed to adhere to glass slides (1 h, 37°C). Cells were washed and fixed with 3.7% paraformaldehyde in PBS (15 min). After fixation, cells were permeabilized with 0.5% NP-40 in PBS (15 min), incubated with 0.5% heat-inactivated FCS in PBS (30 min) to block unspecific binding, and reacted with anti-HA monoclonal antibody (12CA5) for 1 h, followed by reaction with FITC-conjugated antimouse IgG (1 h; Sigma Chemical Co.). Samples were also stained for actin with tetramethylrhodamine isothiocyanate-labeled phalloidin (Sigma Chemical Co.). DAPI (0.5 µg/ml; Sigma Chemical Co.) was used to stain nuclei. Samples were washed with PBS and mounted in Mowiol 4.88 (Hoechst) containing 50 mg/ml DABCO (Sigma Chemical Co.) as antibleaching agent. All incubations were done at room temperature. Photographs were taken with an Axioplan 2 fluorescence microscope (Zeiss) equipped with a Photometrics Quantix CCD camera. Images were processed with IPLab Spectrum and Adobe PhotoShop software.
Protein Purification and Sequencing.
Coomassie blue-stained protein bands were excised and digested in the gel with sequencing-grade modified trypsin (Promega; Ref. 61
). Tryptic peptide maps were obtained by reversed-phase high-performance liquid chromatography on a µRPC C2/C18 SC 2.1/10 column using the Smart system (Pharmacia Biotech). The flow rate was 100 µl/min at 25°C using a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Peptides of interest were loaded onto a Biobrene-coated glass filter fiber of a Procise sequencer (Perkin-Elmer/Applied Biosystems), and sequencing was carried out using standard protocols.
Bandshift Assays.
CEFs were lysed in a buffer containing 20 mM Tris (pH 7.5), 2 mM DTT, 20% glycerol, and 400 mM KCl (1 h on ice). Whole cell extracts (510 µg) were incubated for 15 min on ice with a 32P-labeled oligonucleotide containing a DR1-HRE binding site: (5'-GGGGATCCGGCAGAGGTCAAAGGTCAAACGT-3'; Ref. 26
). Poly(dI-dC) (1 µg/reaction) was added as a nonspecific competitor. For competition experiments, a mutated version of the DR1-HRE site was used: (5'-GGGGATCCGGCAGTGGACAATGGACAAACGT-3'). Binding reactions were separated on 5% polyacrylamide gels in 0.25 x Tris-borate EDTA (44)
.
Transient Transfections.
Calcium phosphate coprecipitation was used to transiently transfect CEFs with retroviral expression vectors containing TR4, HA-TR4, and HA-TR4 in combination with eukaryotic expression vectors carrying RXR in pSG5 vector and a luciferase reporter with the rat CRBPII-RXR responsive element (7)
. Rous sarcoma virus-ß-gal or pCH110 plasmids were cotransfected to normalize for transfection efficiencies. Transfected cells were grown in standard growth medium containing FCS and ChS that were extensively deprived of retinoids (7)
. 9cRA (10-6 M final concentration) was added, and 48 h later, cell extracts were prepared and analyzed for luciferase and ß-galactosidase activity as described (44)
. Luciferase values were normalized for ß-galactosidase activity.
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Acknowledgments |
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Footnotes |
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1 Supported in part by Deutsche Forschungsgemeinschaft Grant Ze432/1 to M. Z. N. P. K. was a recipient of a Max-Delbrück postdoctoral fellowship and is an Investigator of CONICET (Argentina).
2 Present address: Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina.
3 To whom requests for reprints should be addressed, at Max-Delbrück-Center for Molecular Medicine, Robert-Rössle Str. 10, D-13122 Berlin, Germany. Phone: 49-30-9406 3343; Fax: 49-30-9406 3329; E-mail: zenke{at}mdc-berlin.de
4 The abbreviations used are: NR, nuclear receptor; 9cRA, 9-cis retinoic acid; CEF, chicken embryo fibroblast; DR, direct repeat; HA, hemagglutinin; HRE, hormone responsive element; RAR, retinoic acid receptor; RXR, retinoid X receptor; TR, thyroid hormone receptor; ER, estrogen receptor; PML, promyelocytic leukemia; C/EBP, CAAT/enhancer binding protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; ChS, chicken serum; SCF, stem cell factor; DAPI, 4',6-diamidino-2-phenylindole.
5 N. P. Koritschoner, P. Bartunek, and M. Zenke, unpublished data.
Received for publication 7/25/01. Revision received 9/20/01. Accepted for publication 9/20/01.
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References |
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HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cancer Research | Clinical Cancer Research |
Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
Molecular Cancer Research | Cell Growth & Differentiation |