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[Cancer Research 61, 556-564, January 15, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Increased Retinoic Acid Responsiveness in Lung Carcinoma Cells that Are Nonresponsive Despite the Presence of Endogenous Retinoic Acid Receptor (RAR) ß by Expression of Exogenous Retinoid Receptors Retinoid X Receptor {alpha}, RAR{alpha}, and RAR{gamma}1

Haisu Wan, Waun K. Hong2 and Reuben Lotan3, 4

Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are thought to mediate most of the effects of retinoids on cell growth and differentiation. Despite expressing abundant levels of RARß mRNA, lung adenocarcinoma H1792 cells are resistant to the growth-inhibitory effects of all-trans-retinoic acid, suggesting that they have a defect in retinoid signaling. To determine whether transfection of exogenous receptors can restore retinoid responsiveness, we transiently transfected into H1792 cells coexpression vectors containing cDNAs of cell surface antigen CD7 and either RAR{alpha}, RARß, RAR{gamma}, or RXR{alpha}. The cells were then treated with retinoids and incubated with 5'-bromo-2'deoxyuridine. Cells that express exogenous receptor were identified using antibodies against CD7, and cells that synthesized DNA were identified with anti-5'-bromo-2'-deoxyuridine antibodies using secondary antibodies with red and green fluorescence, respectively. RXR{alpha} and RAR{alpha} enhanced growth inhibition by all-trans-retinoic acid or 9-cis-retinoic acid, whereas RAR{gamma} was less effective, and RARß was ineffective. The effects of the transfected receptors were associated with antagonism of activator protein 1 (AP-1) activity. Studies with RXR{alpha} deletion and point mutants indicated that growth suppression is: (a) dependent on intact DNA-binding and ligand-binding regions but not on the NH2-terminal region, which contains a ligand-independent transactivation function; (b) dependent on RXR homodimer formation and transactivation of RXR response element; and (c) associated with AP-1 antagonism. These results demonstrate that transfected receptors can restore responsiveness to retinoids by antagonizing AP-1 in H1792 cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoids, a group of natural and synthetic vitamin A analogues, include compounds that suppress carcinogenesis in experimental animals (1 , 2) and have shown promise as chemopreventive and therapeutic agents (2 , 3) presumably by modulating the growth, differentiation, and apoptosis of normal, premalignant, and malignant cells (2, 3, 4, 5) . Retinoids can regulate gene expression by activating nuclear retinoid receptors, which are members of the steroid hormone receptor gene superfamily and function as ligand-dependent DNA-binding transcription enhancing factors (6 , 7) . Each of the two subtypes of retinoid receptors, RARs5 and RXRs, includes three isotypes designated {alpha}, ß, and {gamma}. The RARs bind both ATRA and 9-cis-RA, whereas the RXRs bind only 9-cis-RA. Each RAR and RXR subtype can be expressed in several isoforms (e.g., RARß1, RARß2), which differ in their NH2-terminal domain as a result of alternative promoter usage and splicing (6) . RARs can form heterodimers with RXRs, and RXRs can also form homodimers. Such dimers can bind to RAREs in the regulatory regions of certain target genes (6 , 7) . The RAREs consist of DRs PuG(G/T)TCA(X)nPuG(G/T)TCA with one or five intervening nucleotides (X) or closely degenerate motifs (6) . Activation of transcription by RAR-RXR and RXR-RXR dimers is usually mediated via DR5 (RARE) and DR1 (RXRE), respectively (6 , 7) . Each receptor isotype and isoform can regulate a distinct subset of retinoid-responsive genes because deletion of individual receptors by homologous recombination resulted in loss of induction by RA of different genes (6, 7, 8, 9) . However, studies in mice by knockout of single or multiple receptors have demonstrated that receptors may possess both specific and redundant functions (6) . Transcription regulation by retinoid receptors is determined by interplay of cofactors with opposite effects. Corepressors bind to complexes formed between retinoid receptors and response element and suppress transcriptional activation. However, ligand binding causes corepressors to dissociate and coactivators to associate with the retinoid receptors and activate the transcriptional machinery (6 , 10) .

Retinoid receptors contain six domains designated A to F. The NH2 terminus (domains A and B) includes a ligand-independent activation function (AF-1). The following C domain contains a highly conserved DNA-binding domain, which may also participate in protein-protein interaction with cofactors. The D domain is involved in ligand-induced functional change and is critical for the binding of receptor to corepressors. The E and F domains, which are moderately conserved among receptors, are involved in ligand binding and include a ligand-dependent transactivation function (AF-2) and a dimerization surface (6) .

Endogenous and transfected retinoid receptors can antagonize the function (transrepression) of AP-1 (11, 12, 13) , a complex comprised of dimers of members of the Jun and Fos family of DNA-binding proto-oncogenes that mediate mitogenic signals from a variety of growth factors and tumor promoters (14) .

Abnormalities in the expression or function of retinoid receptors have been found in various cell types (15 , 16) . Decreased expression of RAR{alpha} in breast cancer (17) and of RARß in lung cancer cells in vitro (18, 19, 20, 21, 22, 23, 24) and in vivo (25, 26, 27) , in head and neck cancers (28 , 29) , and in breast cancer cells in vitro (17 , 30) and in vivo (31 , 32) has been proposed to result in resistance to the effects of retinoids on cell growth and differentiation and to enhance the development of certain malignancies (15 , 16) . Transfection of exogenous receptors into some of these cells restored responsiveness to certain effects of retinoids (16 , 17 , 19 , 33, 34, 35, 36, 37) .

Although RARß expression decreases in lung cancer cells and tissues (18, 19, 20, 21, 22, 23, 24, 25, 26, 27) , about 50% of lung tumors express this receptor (25) , suggesting that the latter cancers develop despite the presence of RARß. Likewise, certain lung cancer cell lines were found to express RARß, yet they resisted the growth-inhibitory effects of ATRA (20 , 24 , 38) . It is possible that the downstream steps in RARß signaling are defective in such tissues and cells (23 , 24) or that the expression or function of other retinoid receptors is abnormal (16) .

In the present study, we attempted to restore responsiveness to the growth-inhibitory effects of retinoids in NSCLC H1792 adenocarcinoma cells, which express RARß but are resistant to the growth-inhibitory effects of ATRA, by transfecting expression vectors for different receptors. We demonstrated that overexpression of RAR{alpha}, RAR{gamma}, or RXR{alpha} but not RARß resulted in the suppression of the growth of H1792 cells to different degrees, in a ligand-dependent fashion. We further demonstrated that the growth-inhibitory effects of nuclear retinoid receptors were closely correlated with their ability to antagonize AP-1 transcriptional activity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
NSCLC Cell Culture.
The H1792 cell line was obtained from Dr. Adi Gazdar (University of Texas Southwestern, Dallas, TX). The Calu-1 NSCLC cell line was purchased from the American Type Culture Collection (Manassas, VA). The cells were maintained in DMEM containing 10% FBS. In some experiments, delipidized serum (free of endogenous retinoids) was used. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2:95% air.

Retinoids.
The retinoids ATRA, 9-cis-RA, and TTNN were obtained from Dr. Werner Bollag (Hoffmann La Roche, Ltd., Basle, Switzerland). AM80 was obtained from Dr. Koichi Shudo (University of Tokyo, Tokyo, Japan). SR11217 was obtained from Dr. Marcia Dawson (Stanford Research Institute International, Menlo Park, CA). CD2314, CD2325, and CD437 were obtained from Dr. Braham Shroot (Centre Internationale de Recherche Dermatologique/Galderma, Sophia Antipolis, France). LG69 was obtained from Dr. Richard Heyman (Ligand Pharmaceuticals, San Diego, CA). The receptor selectivity of these retinoids and their transactivation potencies have been reported elsewhere (24) . All of the retinoids were dissolved in DMSO at a concentration of 10 mM and stored briefly under N2 in the dark at -20°C. The stock solutions were diluted to the desired final concentrations in growth medium. Control cultures received the same amount of DMSO as retinoid-treated cultures.

Electrophoretic Mobility Shift and Supershift Assays.
The assay was performed exactly as described by us elsewhere (37) .

Construction of Plasmids.
Plasmids containing human cDNAs for RAR{alpha}1, RARß2, RAR{gamma}1, and RXR{alpha} were obtained from Dr. Magnus Pfahl (Sidney Kimmel Cancer Center, San Diego, CA). To increase translation efficiency, we deleted the 5' region and introduced a new Kozak sequence into the RARß cDNA to form the RARk12ß construct (37) . cDNA fragments containing the entire open reading frames of the different receptors were inserted into plasmid pMARKCD7D5 (36) obtained from Dr. Jonathan Kurie (The University of Texas M. D. Anderson Cancer Center).

RXR{alpha} deletion mutants (shown schematically in Fig. 5ACitation ) were inserted into pMARKCD7D5. Specifically, the mutant RXR{alpha}DA was prepared by deleting part of the 5' end of wt RXR{alpha} cDNA with HindIII and SmaI. Mutant RXR{alpha}DD was prepared by deleting a fragment between nucleotides 29 and 197 in wt RXR{alpha} cDNA. Mutant RXR{alpha}DF was prepared by deleting the 3' end of RXR{alpha} from nucleotide 402. The RXR{alpha} point mutant pMARKRXR{alpha}F313A was constructed using the QuickChange site-directed mutagenesis kit (Stratagene, San Diego, CA). The point mutant constructs pMARKRXR{alpha}L430F and pMARKRXR{alpha}K431Q were prepared from pBSRXR{alpha}L430F and pBSRXR{alpha} K431Q (39) , respectively (provided by Dr. Xiao-kun Zhang; Burnham Cancer Institute, La Jolla, CA).



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  		Fig. 5. RXR{alpha} structure requirements for mediating retinoid-induced inhibition of DNA synthesis and AP-1 antagonism. A, schematic representation of the RXR{alpha} protein and the predicted deletion mutants. The line at the bottom of the figure and the letters A–F represent the domains of the receptor, and the numbers between 1 and 462 represent the amino acids from the NH2 to the COOH terminus, respectively. B, effects of transiently transfected RXR{alpha} and its mutants on the BrdUrd labeling index in H1792 cells treated with DMSO (control) or with 1 µM ATRA or 9-cis-RA. C, anti-AP-1 activity of RXR{alpha} deletion mutants determined in cells cotransfected with RXR{alpha} or its mutants and the AP-1 reporter construct. The experiments were performed as describeds in the legends to Figs. 2Citation and 4Citation .

 
Transient Transfection and Luciferase Assays.
Cells were seeded at a concentration of 2 x 105 cells/well in 6-well plates. After an overnight incubation, cells in each well were transfected with 1.2 µg of DNA (0.7 µg of luciferase reporter plasmid described below, 0.1 µg of the ß-galactosidase expression vector pCH110, and 0.4 µg of pMARKCD7D5 or pMARKCD7D5-derived expression vectors) in 6 µl of LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) following the manufacturer’s procedure. Two reporter plasmids were used: (a) (RARE)3-tk-LUC, which contains three tandem repeats of DR5 RARE from the P2 promoter region (-59 to -33 bp) of the human RARß2 gene (40) connected to the minimal herpes simplex virus tk promoter and a luciferase cDNA; and (b) RXRE-tk-LUC, which contains five tandem repeats of DR1 RXRE, a 35-bp sequence (-605 to -639) from the promoter of the mouse CRBP-II gene (41) inserted immediately upstream of tk-luciferase in a reporter plasmid (both provided by Dr. Richard A. Heyman). The reporter plasmid for analysis of AP-1 transcriptional activity (Col-AP-1-LUC) was obtained from Dr. Jonathan Kurie. The promoter of this reporter construct was derived from the matrix metalloproteinase 1 promoter (-73 to +63), which contains the AP-1 binding site TGAGTCA (42) . The pCH110 ß-galactosidase expression vector (Pharmacia Biotech, Piscataway, NJ) was used at 0.1 µg for transfection as the internal control for transfection efficiency. After a 6-h exposure to the transfection mixture, cells were treated in medium containing 10% FBS and various concentrations of retinoid or DMSO alone for 20 h and then harvested for measurement of ß-galactosidase activity and luciferase activity using a luciferase assay system and protocol from Promega (Technical Manual TM033; Promega, Madison, WI) and a Lumat luminometer. Triplicate wells were used for each experimental group. Relative luciferase activity was related to the ß-galactosidase activity to normalize for transfection efficiency.

Single Cell DNA Synthesis Assay.
The single cell proliferation assay was performed by a modification of the method described by Frangioni et al. (36) . H1792 cells were seeded at a concentration of 105 cells/well in 6-well plates. After 18–24 h, cells were transfected with various pMARKCD7D5 vectors using LipofectAMINE. Each well received 1 µg of plasmid DNA and 6 µl of LipofectAMINE. After 6 h, the transfection solution was removed by aspiration, and the cells were refed with medium containing 10% delipidized serum and the indicated concentration of retinoids or DMSO control. After 36 h, the cells were incubated for 7 h with a labeling reagent containing 10 µM BrdUrd and 1 µM 5'-fluoro-2'-deoxyuridine (Amersham, Arlington Heights, IL) to incorporate BrdUrd into DNA in cells engaged in DNA synthesis. Cells were then washed three times with PBS and fixed with absolute methanol (prechilled to -20°C) for 10 min. Cells were then rehydrated with PBS and washed once in water. Chromosomal DNA was depurinated by treatment with 2 M HCl for 15 min at room temperature. The acid was neutralized by one wash with 0.1 M Na2B4O7 (pH 8.5), followed by a 2-min incubation in the same solution. Cells were then washed twice with 0.1% NP40 in PBS (0.1% NP40/PBS) and incubated for 1–2 h at room temperature with anti-BrdUrd mAb (IgG1; Becton Dickinson, San Jose, CA) and anti-CD7 mAb (IgG2b clone 3A1E-12H7; Sera-Lab, Sussex, United Kingdom), both of which were diluted 1:6 in 0.3% BSA and 0.1% NP40/PBS. The cells were washed five times with 0.1% NP40/PBS and incubated for 45 min at room temperature with Texas Red-conjugated goat antimouse IgG2b and FITC-conjugated goat antimouse IgG1 (Southern Biotechnology, Birmingham, AL) diluted 1:200 in 0.1% NP40/PBS. Cells were washed four times with 0.1% NP40/PBS and twice with PBS. The cells were then observed using an immunofluorescence microscope with filters for the red fluorescence of Texas Red in the cytoplasm and on the cell surface and the green fluorescence of FITC in the cell nuclei. Cells that have taken up the plasmid and synthesized DNA are stained both red and green (RG), and their nuclei appear yellow because of the overlap of the two colors. Cells that have taken up the plasmid but failed to synthesize DNA stained only red (R). The BrdUrd labeling index (the percentage of cells synthesizing DNA among the cells that have taken up the plasmid) was determined using the following formula: [(RG/(R + RG)]100. Usually, >500 cells were analyzed in several arbitrarily chosen microscopic fields.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Nuclear Retinoid Receptors in H1792 Cells and Their Ability to Form Complexes with RARE.
Previously, we found that NSCLC H1792 cells express abundant amounts of RARß mRNA compared with other NSCLC cells, in which no RARß mRNA could be detected (24) . We found no mutations in RARß transcripts from H1792 cells after amplification by reverse transcription-PCR and sequencing (data not shown). High levels of RAR{gamma}, RXRß, and RXR{gamma} mRNAs were detected in H1792 cells, whereas RXR{alpha} and RAR{alpha} transcripts were about 80% and 60% less abundant, respectively, in H1792 cells than in other NSCLC cells (24) .

Electrophoretic mobility shift and supershift assays shown in Fig. 1Citation revealed two major shifted bands (Lane 1), which appeared to be specific for RARE because they could be competed by excess unlabeled DR5 RARE oligonucleotide (Fig. 1Citation , compare Lanes 1 and 13), but not by a mutated oligonucleotide (Fig. 1Citation , Lane 14). Complexes containing RARß and RXR{alpha} represented the major RARE binding heterodimer, as indicated by supershift with anti-RARß (Fig. 1Citation , Lane 3) and anti-RXR{alpha} (Fig. 1Citation , Lane 5) antibodies as well as by double shift using a mixture of antibodies against both RARß and RXR{alpha} in the same reaction (Fig. 1Citation , Lane 8). The antibodies against all RXRs produced a shifted complex that migrated differently from the complex shifted with RXR{alpha} antibody (compare Fig. 1Citation , Lanes 5 and 6), suggesting that RXRß and/or RXR{gamma} may also be associated with RARß on the RARE.



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  		Fig. 1. Characterization of the binding of retinoid receptors present in H1792 nuclear extracts to RARß-RARE by electrophoretic mobility shift assay. Nuclear extracts from H1792 cells containing 10 µg of protein were incubated with 32P-labeled RARß-RARE alone (Lane 1) or in the presence of 100-fold excess of unlabeled wt (wt) or mutated (mu) RAREß2 (Lanes 13 and 14, respectively). Supershifted bands were analyzed after the addition of anti-receptor antibodies (anti-RAR{alpha}, anti-RARß, and anti-RAR{gamma} in Lanes 2–4; anti-RXR{alpha} and anti-all RXRs in Lanes 5 and 6, respectively; and mixtures of RAR- and RXR-specific antibodies in Lanes 7–12) to the incubation mixture with nuclear extracts and 32P-labeled wt RARß-RARE before electrophoresis. Additional details are described in Reference 37.

 
Complexes containing RAR{gamma} were also detected (Fig. 1Citation , Lanes 4, 9, and 12), but the low intensity of the RAR{gamma} band indicates that it is a minor component of the DNA-binding heterodimer. Although RXR{alpha} did not show abundant mRNA expression in Northern blotting, we detected high-level expression of RXR{alpha} protein as indicated by the intensity of the bands supershifted and double supershifted by the RXR{alpha}-specific antibody (Fig. 1Citation , Lane 5). In contrast, antibodies specific for RAR{alpha} failed to produce any supershifted band (Fig. 1Citation , Lanes 2, 7, and 10), although we had previously found that the same antibodies could supershift RAR{alpha} when incubated with nuclear extracts from head and neck squamous cell carcinoma cells and labeled RARE (37) .

Use of pMARKCD7D5 Vector and BrdUrd Incorporation to Evaluate the Growth-inhibitory Effects of Exogenous Nuclear Retinoid Receptors in H1792 Cells.
To determine whether exogenous retinoid receptors can restore retinoid responsiveness, we transfected H1792 cells transiently using coexpression vector pMARKCD7D5 (Ref. 36 ; Fig. 2ACitation ), into which cDNAs of the different retinoid receptors were inserted. The cells were then grown without or with 1 µM ATRA and labeled with BrdUrd. After fixation and double immunostaining with anti-CD7 antibodies and anti-BrdUrd antibodies (detected with different secondary antibodies tagged with red and green fluorescent fluorophores, respectively), cells were observed under a fluorescence microscope (Fig. 2B)Citation . The proportion of red cells with labeled nuclei within a population of about 500 cells exhibiting red fluorescence was considered to represent the mitotic index (Fig. 2C)Citation . The mitotic indices in cell populations transfected with pMARKCD7D5 vector harboring no retinoid receptor was about 32% and was reduced only slightly by ATRA treatment. Transient expression of RAR{alpha} or RXR{alpha} decreased DNA synthesis in the absence of ATRA to 24% (about 25% inhibition relative to pMARKCD7D5 vector only), whereas transfection of the other receptors did not decrease BrdUrd incorporation in the absence of ATRA. Overexpression of RAR{alpha} and RXR{alpha} caused about 60% inhibition of DNA synthesis, and overexpression of RAR{gamma} caused 45% inhibition of DNA synthesis, whereas RARß failed to enhance the response of the cells to inhibition of DNA synthesis by ATRA (Fig. 2C)Citation .



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  		Fig. 2. Schema of the pMARKCD7D5 vector (A) and its use for the visualization (B) and quantitation (C) of transfected cells and their ability to synthesize DNA after transfection of pMARKCD7D5 constructs with RARs or RXR{alpha}. The pMARKCD7D5 vector (36) contains AmpR, ampicillin resistance gene; two SV40 origins of replication and enhancer elements (ori/enh) for replication and transcriptional activation; an adenovirus major late promoter for transcription initiation (Ad MLP); a cDNA copy of the majority of the adenovirus tripartite leader for efficient translation (TPL); an intervening sequence composed of the 5' splice site from the adenovirus first leader of late mRNA and a 3' splice site from an immunoglobulin gene for mRNA processing (IVS); SV40 polyadenylation signal for transcription termination and mRNA processing [SV40 Poly(A)]; adenovirus VA I gene product to enhance translation; bacterial origin of replication from pBR322 (ori); and two polylinkers into which can be inserted cDNAs of the cell surface antigen CD7 truncated in its cytoplasmic domain (CD7Dc5) to prevent signaling by binding of anti-CD7 antibody and one of the retinoid receptors. B, detection of H1792 cells that have been transfected with pMARKCD7D5 vector and cells that have incorporated BrdUrd by the immunofluorescence antibody technique. H1792 cells were transiently transfected with pMARKCD7D5 plasmids encoding various nuclear retinoid receptors, labeled with BrdUrd, and stained as described in "Materials and Methods." The nuclei of cells in S phase, which have incorporated BrdUrd during the period of labeling, were detected with an anti-BrdUrd mAb and a FITC-conjugated secondary antibody (green). Cells expressing CD7Dc5, which represent the transiently transfected cells, were detected with an anti-CD7 mAb and Texas Red-conjugated secondary antibody (red). Nuclei of S-phase cells with expression of CD7Dc5 appear yellow due to the overlap of the red and green colors. C, effects of various nuclear retinoid receptors on the growth of H1792 cells. Cells were transfected with pMARKCD7D5 vector harboring various nuclear retinoid receptors, cultured in delipidized FBS supplemented with either 1 µM ATRA or DMSO, fixed, and processed for the single cell proliferation assay as described in "Materials and Methods." The data represent the mean values from three independent experiments. Bars, SD.

 
Similar experiments with the RA-resistant NSCLC cell line Calu-1, which expresses no detectable RARß but does express the other RARs and RXRs (24) , have demonstrated that overexpression of RAR{alpha}, RARß, RAR{gamma}, and RXR{alpha}, followed by treatment with 1 µM ATRA, caused inhibition of DNA synthesis of 35.5%, 40%, 10.1%, and 70%, respectively.

Effects of Overexpressed Nuclear Retinoid Receptors on Activation of Transcription of Reporter Constructs Containing DR1 and DR5 Response Elements in H1792 Cells.
To determine whether the transfected receptors alter retinoid-regulated transcription in intact H1792 cells, the cells were cotransfected with pMARKCD7D5 vector bearing no receptor or individual retinoid receptors and a luciferase reporter construct containing DR5 RARE (Fig. 3A)Citation . ATRA or 9-cis-RA treatment of cells transfected with pMARKCD7D5 vector alone activated luciferase transcription by 11- and 15-fold, respectively, via endogenous retinoid receptors (Fig. 3ACitation , top left panel). The RAR{alpha}-selective retinoid AM80 and the RAR{gamma}-selective retinoid CD437 activated transcription by 4-fold, whereas the RARß-selective retinoid CD2314 increased transcription by only 1.6-fold (Fig. 3ACitation , top left panel). In cells transfected with pMARKCD7D5 containing RAR{alpha} (Fig. 3ACitation , top right panel), the activation of the DR5 reporter by ATRA, 9-cis-RA, and AM80 was 25-, 32-, and 13-fold, respectively; more than 2-fold higher than that in cells transfected with pMARKCD7D5 vector alone. There was no increase in the response of the cells to RARß- and RAR{gamma}-selective retinoids. This indicated that the overexpressed RAR{alpha} increased the response of the cells to retinoid signaling. Cells transfected with pMARKCD7D5 vector containing RARß (Fig. 3ACitation , bottom left panel) showed a 35% increase in transcriptional activation of DR5 RARE by ATRA and 9-cis-RA compared with cells transfected with pMARKCD7D5 only but showed no increase in response to AM80, CD2314, or CD437. Treatment of these cells with several other RARß-selective agonists such as TTNN and LG030369 also failed to show any stimulation of transcription (data not shown). These results indicate that exogenous RARß contributes much less than RAR{alpha} to the activation of transcription via RARE. Cells overexpressing RAR{gamma} (Fig. 3ACitation , bottom right panel) exhibited a higher transcriptional activation of DR5-driven reporter in the absence of retinoids than cells transfected with the other receptors. The activation of transcription by ATRA, 9-cis-RA, AM80, CD2314, and CD437 was 4.4-, 6.3-, 1.5-, 1.0-, and 2.1-fold, respectively. Overexpression of RXR{alpha} in H1792 cells failed to increase activation of DR5 RARE by any of the RAR-selective retinoids (data not shown).



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  		Fig. 3. Transcriptional activities of nuclear retinoid receptors on DR5 RARE (A) and DR1 RXRE (B). A, cells were cotransfected with pMARKCD7D5 only or with pMARKCD7D5 containing each of the indicated RARs and the reporter DR5 RARß-RARE-tk-LUC. ß-Galactosidase was also transfected to normalize for transfection efficiency. The cells were then treated with DMSO as a control or with the indicated retinoids (all at 1 µM). The activation of reporter gene transcription was analyzed by the luciferase assay as described in "Materials and Methods." B, the cells were transfected and treated with retinoids as described in A, except that pMARKCD7D5 containing RXR{alpha} was used, and more RXR-selective retinoids were used. Transfection efficiency was normalized using ß-galactosidase activity. Bars, SD.

 
H1792 cells transfected with pMARKCD7D5 alone showed no increase in transcriptional activation of DR1 RXRE (38) on treatment with a variety of retinoids including some that bind to RXRs (e.g., 9-cis-RA, LG69, and SR11217; Fig. 3Citation B, top panel). This finding is surprising because the cells express high levels of RXR protein as detected by the electrophoretic mobility shift assay. Nonetheless, overexpression of exogenous RXR{alpha} via the pMARKCD7D5 vector enhanced transcriptional activation of DR1 RXRE by ATRA, 9-cis-RA, LG69, and SR11217 by 5.0-, 6.6-, 8.5-, and 5.6-fold, respectively. However, AM80, TTNN, and CD2325, which are selective for RAR{alpha}, RARß, and RAR{gamma}, respectively, showed no increase or up to a 1.6-fold increase in transcription (Fig. 3BCitation , bottom panel).

The Growth-inhibitory Effects of Various Nuclear Retinoid Receptors Are Associated with Their Anti-AP-1 Activity.
Nuclear retinoid receptors can antagonize AP-1 activity in a ligand-dependent fashion, and this antagonism often leads to growth inhibition (12 , 14 , 35 , 43, 44, 45, 46, 47, 48) . Therefore, we examined whether an association exists between the growth-inhibitory effects mediated by the receptors and their anti-AP-1 activity in the H1792 cells. Overexpression of each of the three RARs or RXR{alpha} had only minor effects on AP-1 activity in the absence of exogenous retinoids (Fig. 4Citation ; cells were treated with DMSO as a control). Treatment of cells transfected with pMARKCD7D5 alone using ATRA or 9-cis-RA not only failed to suppress AP-1 activity but rather increased it by 30–40% (Fig. 4Citation , {square}), suggesting that the anti-AP-1 activity of retinoids via constitutively expressed retinoid receptors is aberrant in the H1792 cells. However, overexpression of RAR{alpha} and RXR{alpha} and, to a lesser extent, RAR{gamma} resulted in marked suppression of AP-1 activity in H1792 cells after ATRA or 9-cis-RA treatment compared with AP-1 activity in cells transfected with pMARKCD7D5 alone (Fig. 4)Citation . 9-cis-RA was more effective than ATRA in suppressing AP-1 activity in cells overexpressing RXR{alpha}. Overexpression of RARß resulted in a smaller decrease in AP-1 activity after retinoid treatment compared with the other transfected receptors. These results suggest a positive association between retinoid-induced growth-inhibitory effects mediated by the different retinoid receptors and their respective anti-AP-1 activity.



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  		Fig. 4. Analysis of the ability of various nuclear receptors to mediate ligand-dependent anti-AP-1 activity in H1792 cells. Cells were cotransfected with pMARKCD7D5 vector only or with pMARKCD7D5 vector containing the indicated retinoid receptors and the reporter construct Col-AP-1-LUC and a ß-galactosidase expression vector. The cells were then treated with DMSO as a control or with 1 µM ATRA or 9-cis-RA. Luciferase activity was analyzed as described in "Materials and Methods." Transfection efficiency was normalized using ß-galactosidase activity. Columns, mean of triplicate determinations; bars, SD.

 
The DNA-binding Domain and Carboxyl-terminal Domain F Are Required for Both the Growth-inhibitory Effect and Anti-AP-1 Activity of RXR{alpha}.
Because RXR{alpha} appeared to be the most potent of the four receptors in mediating both inhibition of DNA synthesis and antagonism of AP-1, we generated and used the deletion mutants shown in Fig. 5ACitation to determine which of the different domains of this receptor are required for the two activities. The construct RXR{alpha}DA, which was predicted to express a mutant protein with a deletion of the 71 NH2-terminal amino acids of wt RXR{alpha}, was as effective as intact RXR{alpha} in mediating both inhibition of DNA synthesis (Fig. 5B)Citation and antagonism of AP-1 (Fig. 5C)Citation . In contrast, mutant RXR{alpha}DD, which was predicted to express a protein with a deletion between amino acids 29 and 197, and mutant RXR{alpha}DF, which was predicted to express a truncated protein lacking the 61 COOH-terminal amino acids of RXR{alpha}, completely lost the growth-inhibitory effect (Fig. 5B)Citation and anti-AP-1 (Fig. 5C)Citation effect of RXR{alpha}. These findings indicated that both the DNA-binding domain and the ligand-binding/heterodimerization domain are required for the two RXR{alpha} activities.

To assess additional aspects of the expression and function of these RXR{alpha} mutants in H1792 cells, their ability to activate the transcription of DR1- and DR5-containing luciferase reporter constructs was examined. RXR{alpha}- and RXR{alpha}DA-transfected cells exhibited similar potency for activation of DR5 RARE (Fig. 6Citation , compare B and C). However, compared with wt RXR{alpha} (Fig. 6B)Citation , RXR{alpha}DA was able to partially (50–60% of the effect of wt RXR{alpha}t) activate transcription via DR1 after treatment of the cells with ATRA, 9-cis-RA, and the RXR-selective retinoids LG69 and SR11217 (Fig. 6C)Citation . Mutants RXR{alpha}DD and RXR{alpha}DF showed no ability to activate DR1 (data not shown).



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  		Fig. 6. Effect of deletion of domain A of RXR{alpha} on ligand-induced transcriptional activation of DR1 and DR5 retinoid response elements in H1792 cells. Cells were transfected with pMARKCD7D5 vector only (A), pMARKCD7D5 containing wt RXR{alpha} (B), or with RXR{alpha} mutant in which domain A was deleted (C). The cells were then transfected with DR1 or DR5 reporter constructs and treated with either DMSO (control) or one of the indicated retinoids (each at 1 µM). Transcription activation was determined from the luciferase activity as described in "Materials and Methods."

 
Although they were unable to activate DR1, RXR{alpha}DD and RXR{alpha}DF interfered with the ligand-induced transcriptional activation of DR1 by wt RXR{alpha} in cotransfection experiments. RXR{alpha}DD (Fig. 7A)Citation was more effective than RXR{alpha}DF (Fig. 7B)Citation in antagonizing RXR{alpha}. It is possible that these mutants formed abortive heterodimers with wt RXR{alpha} or competed for the same coactivators.



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  		Fig. 7. Interference by the deletion mutants RXR{alpha}{Delta}D and RXR{alpha}{Delta}F in the activation of DR1 RXRE by wt RXR{alpha}. Cells were cotransfected with the indicated amounts of wt RXR{alpha} and RXR{alpha}{Delta}D (A) or RXR{alpha}{Delta}F (B). The total amount of DNA in each transfection reaction was brought up to 1.2 µg using pMARKCD7D5 vector. The cells were then cotransfected with DR1 luciferase reporter construct and treated with DMSO or 1 µM 9-cis-RA. Luciferase activity was determined as described in "Materials and Methods." Columns, mean of triplicate assays; bars, SD.

 
Modification of Anti-AP-1 Activity of RXR{alpha} by Point Mutation also Results in Alteration of Its Growth-inhibitory Effect.
Several RXR{alpha} point mutations in the F domain were found to interfere with RXR-RXR and RXR-RAR dimerization (39) . One such mutant, RXR{alpha}L430F, had lost the ability to act as homodimer, whereas a mutation in the adjacent codon (RXR{alpha}K431Q) retained this ability. We cloned these two point-mutated RXR{alpha}s into pMARKCD7D5 vector and analyzed their activities after transient transfection into H1792 cells and treatment with 9-cis-RA. Fig. 8Citation shows that the point mutation L430F, which abrogated DR1 activation as expected but retained some ability to activate DR5, resulted in a loss of ability to suppress DNA synthesis and to antagonize AP-1. In contrast, the point mutation RXR{alpha}K431Q, which retained the ability to activate DR5 as well as DR1, like wt RXR{alpha}, also exhibited the ability to both suppress DNA synthesis and antagonize AP-1 in the presence of 9-cis-RA (Fig. 8)Citation .



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  		Fig. 8. Analysis of the effects of RXR{alpha} point mutations that affect its homodimerization on activation of retinoid response elements, anti-AP-1 activity, and suppression of DNA synthesis. Cells were cotransfected with pMARKCD7D5 vector without receptor or with pMARKCD7D5 vector constructs containing wt RXR{alpha} or the indicated point mutants of RXR{alpha} and either luciferase reporter constructs containing DR5 RARE (top panel), DR1 RXRE (second panel from the top), or AP-1 consensus sequence (third panel from the top). The cells were then incubated with medium with DMSO or 1 µM 9-cis-RA. Luciferase activity was analyzed as described in "Materials and Methods." Transfection efficiency was normalized using ß-galactosidase activity. Columns, mean of triplicate determinations; bars, SD. Bottom panel, cells were transfected with the indicated pMARKCD7D5 vectors, incubated in medium with DMSO or 1 µM 9-cis-RA, and then analyzed for BrdUrd incorporation as described in "Materials and Methods."

 
Mutation F318A in the ligand-binding pocket of mouse RXR{alpha} was found to cause the receptor to exhibit constitutive activation of DR1 (as a homodimer) and, to a lesser extent, a DR5 reporter (as a heterodimer with RARs) in the absence of a RXR ligand (49) ; namely, to act like a ligand-bound receptor. We generated a homologous mutant in human RXR{alpha} (RXR{alpha}F313A) and cloned it into pMARKCD7D5 vector. After transient transfection into H1792 cells, we found that this mutant has a constitutive transcriptional activity on DR1 but not on DR5 in the absence of 9-cis-RA (Fig. 8Citation , top two panels, respectively). Furthermore, this mutant exhibited ligand-independent antagonism of AP-1 and inhibition of DNA synthesis (Fig. 8Citation , bottom two panels, respectively). These results indicate that DR1 activation, antagonism of AP-1, and inhibition of DNA synthesis are strongly associated and may be causally related.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Most NSCLC cell lines that we (24) and others (22 , 22) have examined previously were found to be resistant to the growth-inhibitory effects of ATRA and other retinoids. Many of these cell lines failed to express RARß (18, 19, 20, 21, 22, 23, 24) , and this aberration has been proposed to be the reason for their retinoid resistance (22) . In another lung carcinoma cell line, transfection of RARß restored sensitivity to growth inhibition by ATRA when the cells were grown in low serum concentration (50) . Furthermore, knockout by homologous recombination of the RARß gene in F9 embryonal carcinoma cells resulted in loss of growth inhibition by ATRA (51) . Despite this evidence that RARß can mediate growth inhibition, the H1792 cells were resistant to ATRA (24) , although they express high levels of RARß mRNA as confirmed and extended in the present study. The H1792 cells were found to express mRNAs for all of the RARs and for RXR{alpha} (24) . Furthermore, RARß and, to a much lesser degree, RAR{gamma} proteins were detected in complexes with DR5 RARE, presumably as heterodimers with RXR{alpha} (Fig. 1)Citation . These endogenous receptors were able to mediate transactivation of DR5 RARE (Fig. 3A)Citation . However, there was no tight association between the ability to transactivate DR5 RARE and the ability to inhibit DNA synthesis, as seen in cells transfected with RARß (Fig. 2CCitation and Fig. 3ACitation , bottom left panel). The endogenous RXRs failed to mediate activation of DR1 RXRE (Fig. 3B)Citation in H1792 cells exposed to ATRA or 9-cis-RA, and endogenous RARs and RXRs failed to mediate AP-1 antagonistic activity in H1792 cells exposed to ATRA or 9-cis-RA (Fig. 4Citation , {square}). Based on these findings, we suggest that the resistance of H1792 cells to the growth-inhibitory effects of retinoids is due to the inability of the constitutively expressed receptors to mediate DR1 activation and AP-1 antagonism.

Several previous studies have demonstrated that stable transfection of different retinoid receptors can restore response to the effects of retinoids on cell growth, differentiation, or apoptosis. RARs were able to restore responsiveness in leukemia cells (52) ; RAR{alpha} and RARß were able to restore responsiveness breast carcinoma cells (17) ; RARß was able to restore responsiveness in breast (33) , lung (50) , and head and neck (37) carcinoma cells; RAR{gamma} was able to restore responsiveness in head and neck squamous carcinoma (53) and teratocarcinoma cells (54) ; and RARs plus RXR{alpha} were able to restore responsiveness in ovarian carcinoma cells (35) .

In the present study, we have chosen the H1792 cells to determine whether transient transfection of different receptors can overcome the apparent block in response to growth inhibition by retinoids. We found that exogenous retinoid receptors can mediate growth inhibition by ATRA in the following rank order: RAR{alpha} = RXR{alpha} > RAR{gamma} (Fig. 2C)Citation . RARß overexpression was without an effect. A similar ranking of activity of the exogenous receptors was noted when their effects on AP-1 antagonism were determined. Combined, these results suggest that the growth inhibition mediated by the transfected receptors may be associated with antagonism of AP-1.

In another RA-resistant NSCLC cell line, Calu-1, which does not express RARß, transfection of pMARKCD7D5 vectors containing RAR{alpha}, RARß, or RXR{alpha} but not RAR{gamma} restored growth inhibition by ATRA. These results show that some receptors (e.g., RAR{alpha} and RXR{alpha}) can restore ATRA responsiveness in more than one NSCLC cell line, whereas other receptors (e.g., RARß and RAR{gamma}) may do so in some cells, but not in others. Thus, cell context may determine the ability of a transfected receptor to mediate growth inhibition.

Another interesting result was that although H1792 cells express endogenous RXR{alpha}, this receptor does not activate DR1 RXRE after the cells are treated with 9-cis-RA and other RXR-selective retinoids (Fig. 3BCitation , top panel). However, after transfection of exogenous RXR{alpha}, the same retinoids activated DR1 RXRE (Fig. 3Citation B, bottom panel). Thus, the putative increase in the level of RXR{alpha} in the transfected cells causes a qualitative change in ability to activate RXRE. The lack of RXRE activation may be due to low endogenous RXR{alpha}-RXR{alpha} levels and transrepression by more abundant RXR{alpha}-RARß heterodimers that can bind to the DR1 with a higher affinity than RXR{alpha}-RXR{alpha} homodimers but fail to activate this response element (55 , 56) . One explanation for the effect of overexpression of RXR{alpha} is that the increased RXR{alpha} level leads to the formation of RXR homotetramers, which, having a higher affinity for DR1 than RXR-RAR, may displace RXR-RAR heterodimers from the DR1 RXRE (57) . In the presence of RXR ligands, the DNA-bound tetramers dissociate to dimers (57) that activate DR1-mediated transcription of the reporter.

Because of the interesting effects of RXR{alpha}, we wished to gain some additional understanding of its mechanism of action. Therefore, we generated several deletion and point mutants of this receptor and compared their effects with that of wt RXR{alpha}. Studies with deletion mutants indicated that the DNA-binding and ligand-binding domains were important for growth inhibition (Fig. 5B)Citation , DR1 RXRE activation, and AP-1 antagonism (Fig. 5C)Citation . Some deletion mutants of RARs have been shown to possess dominant negative activity (58 , 59) . Our results indicate that mutants with deletions in the DNA-binding domain (RXR{alpha}DD) or ligand-binding domain (RXR{alpha}DF) can exert a dominant negative effect on RXR{alpha}, as indicated by the partial suppression of the effect of wt RXR{alpha} on the activation of DR1 RXRE (Fig. 7)Citation . The RXR{alpha} mutant with a deletion in the NH2-terminal domain was almost as active as wt RXR{alpha} when examined for all of the activities, suggesting that the ligand-independent transactivation function residing in this domain is dispensable for all activities. The finding that point mutation L430F, which inhibits the ability of RXR{alpha} to transactivate transcription as a homodimer, but not mutation K431Q, which has intact ability to act as a homodimer (39) , has lost the ability to transactivate DR1, suppress DNA synthesis, and transrepress AP-1 (Fig. 8)Citation provides further support for the conclusion that the mechanisms underlying the growth-inhibitory effects mediated by RXR{alpha} in H1792 cells involve homodimerization, transactivation of RXRE (DR1), and transrepression of AP-1. The data point to the possibility that RXR{alpha} homodimers can mediate AP-1 transrepression. Interestingly, this conclusion is also supported by the ability of the point mutant RXR{alpha}F313A to activate DR1, inhibit DNA synthesis, and antagonize AP-1 in the absence of 9-cis-RA (Fig. 8)Citation . The effects of F313A are important because they exclude pleiotropic effects resulting from different signaling pathways that can be provoked by 9-cis-RA and point to the central role of the RXR receptor in the above-mentioned effects.

AP-1 is a complex comprised of dimers of members of the Jun and Fos family of DNA-binding proto-oncogenes that mediate mitogenic signals from a variety of growth factors and tumor promoters (14) . AP-1 activity is particularly important for the progression of lung cancer cells because altered expression of members of AP-1 family was found to be an early event in human lung carcinogenesis (60) . The transrepression of AP-1 by liganded retinoid receptors has been demonstrated previously both in vitro (11, 12, 13 , 35 , 43, 44, 45, 46, 47, 48) and in vivo (61) . The retinoids that induced the repression of AP-1 activity were also found to be able to inhibit the growth of many types of cancer cells (45, 46, 47, 48) . Normal HBE cells are sensitive to both the anti-AP-1 and growth-inhibitory effects of RA. However, tumorigenic HBE cells and many lung cancer cells are resistant to both effects of RA (48) . Our study has extended these reports by demonstrating that several overexpressed retinoid receptors can mediate anti-AP-1 effects of retinoids. The mechanism of AP-1 transrepression is not entirely clear. No binding of liganded retinoid receptors to the AP-1 consensus DNA sequence was observed, thus excluding competition for DNA binding as a mechanism of antagonism. Several mechanisms have been suggested including binding of liganded retinoid receptors to c-Jun or c-Fos, which would thus interfere with c-Jun/c-Jun homodimerization and c-Jun/c-Fos heterodimerization and prevent the formation of AP-1 complexes capable of DNA binding (12 , 13) . A variation on this mechanism was suggested by the demonstration that RARs, RXRs, and c-Jun form a complex at the AP-1 site of the collagenase promoter in which c-Jun binds directly to the DNA and apparently links the retinoid receptors to the complex (62) . Competition for the limited amount of the coactivator CBP/p300, which is required for the transcriptional activity of both nuclear retinoid receptors and AP-1, has also been proposed as a mechanism of mutual retinoid and AP-1 antagonism (13) . Interference with the Jun NH2-terminal kinase signaling pathway represents another mechanism by which nuclear hormone receptors can antagonize AP-1. This mechanism is based on the blockade by hormone-activated nuclear receptors of c-Jun phosphorylation on Ser63/Ser73, which is required to recruit the transcriptional coactivator CBP (63) . A similar mechanism was observed in HBE cells, in which ATRA decreased the amount and activation of AP-1 components. ATRA inhibited Jun NH2-terminal kinase and, to a lesser extent, extracellular signal-regulated kinase activity and also reduced c-fos mRNA (48) . Recently, pretreatment of human skin with ATRA was found to inhibit UV induction of c-Jun protein and, consequently, AP-1 via a posttranscriptional mechanism because ATRA did not inhibit UV induction of c-Jun mRNA (61) .

Taken together, our data suggest that the sensitivity to the growth-inhibitory effect of RA can be restored by overexpression of several exogenous nuclear retinoid receptors in lung cancer cells. One potential clinical implication is that one could combine receptor gene transfer and retinoid treatment as a strategy for therapy or prevention of lung cancer. With respect to the mechanism of growth inhibition, the data indicate that antagonism of AP-1 by the transfected receptors may be important for inhibition of DNA synthesis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by USPHS Grant U19 CA68437 from the National Cancer Institute (to W. K. H. and R. L.) and a University of Texas M. D. Anderson Cancer Center Faculty Achievement Award (to R. L.). DNA sequencing was supported in part by M. D. Anderson Cancer Center Core Grant P30 CA16672 from the National Cancer Institute. Back

2 American Cancer Society Clinical Research Professor. Back

3 Incumbent of the Irving and Nadine Mansfield and Robert David Levitt Cancer Research Chair. Back

4 To whom requests for reprints should be addressed, at Department of Thoracic/Head and Neck Medical Oncology, Box 80, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-7480; Fax: (713) 794-0209; E-mail: rlotan{at}mdanderson.org Back

5 The abbreviations used are: RAR, retinoic acid receptor; ATRA, all-trans-retinoic acid; BrdUrd, 5'-bromo-2'-deoxyuridine; 9-cis-RA, 9-cis-retinoic acid; FBS, fetal bovine serum; RARE, retinoic acid response element; RXR, retinoid X receptor; RXRE, RXR response element; AP-1, activator protein 1; DR, direct repeat; RA, retinoic acid; NSCLC, non-small cell lung cancer; tk, thymidine kinase; mAb, monoclonal antibody; HBE, human bronchial epithelial; wt, wild-type. Back

Received 7/19/00. Accepted 11/ 8/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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