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[Cancer Research 59, 3518-3526, July 1, 1999]
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[Cancer Research 59, 3518-3526, July 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Overexpression of Retinoic Acid Receptor ß in Head and Neck Squamous Cell Carcinoma Cells Increases Their Sensitivity to Retinoid-induced Suppression of Squamous Differentiation by Retinoids1

Haisu Wan, Nobuhiko Oridate2, Dafna Lotan, Waun K. Hong and Reuben Lotan3

Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 7703


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear retinoic acid receptor ß (RARß) expression is suppressed in many head and neck squamous cell carcinomas (HNSCCs), and an inverse relationship was found between squamous differentiation and RARß expression in such cells. To investigate the role of RARß in HNSCC growth and differentiation, we transfected a retroviral RARß2 expression vector (LNSß) into HNSCC SqCC/Y1 cells, which do not express endogenous RARß but do express RAR{alpha}, RAR{gamma}, and retinoid X receptors. Transfected clones expressing RARß2 mRNA and protein exhibited enhanced sensitivity to the suppressive effects of all-trans-retinoic acid (ATRA) on squamous differentiation compared with cells transfected with the LNSX vector only; transglutaminase type I level was suppressed after a 3-day treatment with 10-10 M ATRA in four of five LNSß clones, whereas it was not suppressed in LNSX cells even by 10-6 M ATRA. Similarly, cytokeratin 1 mRNA level was more suppressed in ATRA-treated LNSß clones than it was in LNSX cells. This effect was independent of transrepression of activator protein-1. None of the LNSß-transfected clones showed an increased growth inhibition by ATRA, 9-cis-retinoic acid, or the synthetic retinoid 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthalenecarboxylic acid. These findings suggest that, in SqCC/Y1 cells, RARß mediates suppression of squamous differentiation by ATRA without enhancing its growth-inhibitory effects.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been proposed that the clinical efficacy of retinoids in cancer prevention and therapy depends on their ability to modulate cell growth, differentiation, and apoptosis in premalignant and malignant cells by regulating gene expression (1, 2, 3, 4, 5) . This ability, in turn, is thought to be mediated by nuclear retinoid receptors, which are members of the steroid hormone receptor gene superfamily and function as ligand-dependent, DNA-binding, transcription-enhancing factors (1 , 6, 7, 8, 9) .

Two subfamilies of nuclear retinoid receptors have been identified: RARs 4 and RXRs. These subfamilies both include three isotypes, designated {alpha}, ß, and {gamma}, which are encoded by distinct genes. Like other members of the superfamily, nuclear retinoid receptors consist of a conserved modular structure, which contains six domains, designated A–F, from the NH2 terminus to COOH terminus of the molecule. The C domain binds DNA, and the E/F domain serves for ligand binding, transactivation, and dimerization (6, 7, 8, 9) . The RARs bind both ATRA and 9-cis-RA, whereas the RXRs bind only 9-cis-RA. These receptors also bind a variety of synthetic retinoids, some of which exhibit preferential binding to specific subtypes. For each RAR and RXR subtype, there are two to four isoforms (e.g., RARs ß1, ß2, and ß4) generated by differential usage of alternative promoters and alternative splicing, which, consequently, results in differences in their A domains (6, 7, 8, 9) . RARs can form heterodimers with RXRs and recognize RAREs that consist of DRs with intervening nucleotides (X) numbering 1 or 5 [PuG(G/T)TCA(X)nPuG(G/T)TCA or closely degenerate motifs, although more complex motifs have also been identified in certain gene promoters; Refs. 6, 7, 8, 9 ]. The binding of retinoid receptors, generally in the form of hetero- or homodimers, can regulate the expression of the target genes in either a positive or negative way. Corepressors and other nuclear factors recognize the complexes formed by nuclear retinoid receptors with DNA response element. Ligand binding can alter receptor conformation such that the corepressors dissociate and coactivators associate with the holoreceptors and activate the transcriptional machinery (6, 7, 8, 9, 10) . It is thought that each receptor isotype and even each isoform regulate a distinct subset of retinoid-responsive genes because their expression is regulated spatiotemporally during embryonal development and their expression patterns in certain adult tissues are distinct (9) . Moreover, these RARs exhibit higher nucleotide sequence homologies within a single isotype across different species (e.g., rat and human RARß) than between isotypes within a single species (e.g., human RAR{alpha} and human RARß), implying that RARs may have distinct functions that have been conserved during evolution (6, 7, 8, 9) .

In addition, retinoid receptors can transrepress the function of AP-1, 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 (11) . It has been proposed that retinoid receptors antagonize AP-1 activity by either directly binding the AP-1 complex (11) or competing for the limited amount of CBP/p300, which is required for the transcriptional activity of both nuclear retinoid receptors and AP-1 (12) .

One of the physiological functions of vitamin A is to maintain the proper differentiation of many epithelial tissues, as indicated by aberrations in epithelial cell differentiation, such as squamous metaplasia in vitamin A deficiency (1, 2, 3) . In addition, vitamin A deficiency has been associated with enhanced carcinogenesis (1 , 4) . Because these receptors are the proximate mediators of many of the effects of retinoids, it is plausible to assume that changes in their expression and function may cause aberrations in the response of cells to ATRA and, thereby, alter the regulation of cell growth and differentiation. Indeed, normal nonkeratinizing oral mucosa epithelial cells express RAR{alpha}, RARß, RAR{gamma}, and RXR{alpha} (13, 14, 15) , whereas many premalignant oral lesions (15) and HNSCCs (14 , 16 , 17) exhibit a selective suppression of RARß expression. These findings have led to the proposal that loss of RARß expression is associated with the development of oral cancer (14 , 17) and suggest that restoration of RARß expression to HNSCCs may enhance response to RA and confer a more normal pheno-type (15) .

In previous studies, we found that retinoids can inhibit the growth of HNSCC cells in vitro (16 , 18) and suppress the expression of squamous differentiation markers, including CK1, TGase I, and involucrin, as well as inhibit the formation of cornified envelopes (16 , 18 , 19) . Similar results of suppression of squamous differentiation by retinoids were reported in HNSCC cells growing as xenotransplants in nude mice (20) . Recently, we found an inverse relationship between the induction of endogenous RARß expression by retinoids in HNSCC 1483 and SqCC/Y1 cells and the suppression of squamous cell differentiation (21) .

To determine more directly whether RARß plays a role in retinoid-mediated suppression of squamous cell differentiation, we transfected a human HNSCC cell line SqCC/Y1 with RARß using a retroviral vector, isolated stable transfectants, and examined the effect of RARß expression on the phenotype of the cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Retinoid Treatment.
Cell lines PE501 and PA317 (provided by Dr. S. Collins, Fred Hutchinson Cancer Center, Seattle, WA) and thymidine kinase-negative NIH3T3 cells (purchased from American Type Culture Collection, Manassas, VA) were cultured in DMEM containing 10% FBS. Parental SqCC/Y1 cell line, derived from a well-differentiated SCC of the buccal mucosa (22) , was provided by Dr. M. Reiss (Yale University, New Haven, CT). This cell line and all transfectants derived from it in this study were cultured in DMEM containing 5% FBS that was delipidized to minimize the effect of endogenous retinoids found in regular serum (18 , 23) . FBS was delipidized by vigorously mixing 1.5 volumes of serum with 1 volume of Seroclear (Calbiochem-Novabiochem Corp., San Diego, CA) for 30 min, followed by a 10-min centrifugation at 1000 x g to separate the phases and collecting the serum fraction. Retinol was present in the FBS at a concentration of {approx}0.7 µM but could not be detected in delipidized FBS by high-performance liquid chromatography analysis. Likewise, ATRA, 13-cis-RA, and 9-cis-RA were not detected by high-performance liquid chromatography in delipidized FBS. All cells were incubated at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air.

Retinoids.
ATRA, 9-cis-RA, and TTNN were provided by Dr. W. Bollag (F. Hoffmann-La Roche, Basel, Switzerland). These retinoids were dissolved in DMSO at a concentration of 10-2 M and stored in the dark at -20°C under N2. Stock solutions were diluted to the appropriate final concentrations in growth medium immediately prior to each experiment. Control cultures received the same amount of DMSO as retinoid-treated cultures.

Retroviral RARß Vector Construction.
The transfection efficiency of SqCC/Y1 cells by standard methods was very low. Therefore, we decided to use a retroviral vector (24 , 25) . To generate a retroviral vector expressing RARß with a high translation efficiency, we deleted the 5' untranslated region of wild-type human RARß2 cDNA from the plasmid pGEMI-hRARß2 and replaced the original sequence with one that contains a presumably better Kozak sequence. Specifically, the A2 region and part of B region of RARß2 (26) were amplified by the PCR using the following oligonucleotide primers: 5'-AAGCT TGTCG ACGCC ACCAT GTTTG ACTGT ATGGA TG-3', corresponding to the upstream sequence, and 5'-AGCCC TTACA TCCTC ACAG-3', corresponding to the downstream sequence using pGEMI-hRARß2 as a template. A 229-bp fragment was obtained and cloned into pCRII vector using TA cloning kit (Invitrogen, San Diego, CA). A clone with the required insert direction was sequenced to confirm that no mutations had occurred during the PCR amplification. The vector harboring the PCR product was then digested with XhoI, and the small fragment released from it was subcloned back into the same site of pGEMI-hRARß2. Then the modified cDNA of hRARß2 was inserted into the retroviral vector LNSX (Ref. 24 ; provided by Drs. S. Collins and D. Miller, Fred Hutchinson Cancer Center, Seattle, WA). The resulting construct with the desired cloning direction was designated LNSß (Fig. 1)Citation .



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  		Fig. 1. Schematic structure of the LNSß retroviral vector. This vector was constructed by inserting the cDNA of a modified hRARß2 into the HindIII site (arrow) of the retroviral vector LNSX. The 5' untranslated region of wild-type hRARß2 cDNA was deleted, and the original Kozak sequence was replaced with a modified optimal one (underlined) to enhance the translation efficiency.

 
Retroviral Transduction.
LNSß and LNSX (as vector control) were first transfected into the ecotropic retrovirus packaging cell line PE501 using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) following the manufacturer’s protocol. After a 6-h exposure to the DNA/Lipofectamine suspension, cells were fed with 1 ml of the medium containing 20% serum. After 10 h, medium was replaced with fresh medium containing 10% FBS. Thirty-six h after the start of transfection, the supernatant-containing virus particles were collected, filtered through a 0.4-µm filter, and then used to infect PA317 amphotropic packaging cell line. Supernatants from selected retroviral producers were titered on thymidine kinase-negative NIH3T3 target cells, as reported by Robertson et al. (27) , and used to infect the HNSCC SqCC/Y1 cells. These cells were first cultured for 24 h in medium supplemented with 5% delipidized FBS and polybrene (6 µg/ml), together with various amounts of supernatant from retroviral vector producer cells. To obtain a population of transfected cells (pool), we used high virus titer (>105 colony-forming units per 100-mm dish) to transfect SqCC/Y1 cells. Subsequently, all of the cells in the transfected dish were trypsinized, suspended, and split at various ratios for subculturing in growth medium supplemented with G418 (800 µg/ml) for 14–21 days. All of the G418-resistant cells from these dishes were harvested together and designated LNSX and LNSß pool populations, respectively. In addition, individual clones of transfected cells were isolated after SqCC/Y1 cells were infected with low titer of LNSß virus (200 colony-forming units for each 100-mm dish) using the same protocol as above. Individual clones (clones 3, 4, 6, 8, and 10) were expanded. All transfectants were maintained in a medium containing G418 (300 µg/ml). Samples of the transfected cell populations and individual clones were frozen in liquid nitrogen for storage.

cDNA Probes and Their Use for Southern and Northern Analyses.
For Southern analysis, genomic DNA was extracted from cell lines by the method of Maniatis et al. (28) . The DNA was then digested with restriction enzymes, electrophoresed, and blotted onto Hybond N+ membranes (Amersham, Arlington Heights, IL). For Northern analysis, total RNA was isolated from cells by the method of Chirgwin et al. (29) and subjected to electrophoresis on 1.2% formaldehyde agarose gel. The RNA was stained with ethidium bromide and transferred to Hybond N+ membranes by capillary transfer.

The following probes were used for hybridization in this study: pSG5 expression vector harboring RAR{alpha} (30) , obtained from Dr. P. Chambon (Institute of Genetics and Molecular and Cellular Biology, Illkirch, France); the RARß2 probe spanning the entire ORF of human RARß2 cDNA (26) , prepared from a 1.5-kb fragment released from LNSß vector; the GAPDH probe, prepared from a 1.3-kb fragment spanning the whole ORF of cDNA cloned from chicken muscle (31) ; the cDNA probe for TGase type I>, a 1-kb fragment obtained by digesting plasmid pTG-7 with XhoI, provided by Dr. A. Jetten (National Institute of Environmental Health Sciences, Research Triangle Park, NC; Ref. 32 ); and the cDNA probe for human CK1, a 0.5-kb BamHI-PstI fragment released from HK1, a pGEM3 plasmid in which human CK1 cDNA has been cloned, provided by Dr. D. Roop (Baylor College of Medicine, Houston, TX; Ref. 33 ). Probes were labeled with 32P using random hexanucleotides (Stratagene, La Jolla, CA) as primers. Hybridization was performed in a solution containing the following: 50% formamide, 5x SSC (0.15 M NaCl-0.015 M sodium citrate), 1x Denhardt’s solution (0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, and 0.02% BSA), 0.5% SDS, and 0.1 mg/ml salmon sperm DNA. Blots were washed in 0.1x SSC and 0.1% SDS for 1 h at 60°C and then placed against a Kodak X-OMAT film between two intensifying screens for 1–4 days of autoradiography at -80°C.

Detection of RARß by Immunoblotting of Nuclear Proteins.
Nuclear extracts were prepared by a modification of the method of Dignam et al. (34) . All procedures were carried out on ice. Briefly, cells from each 100-mm dish were washed twice with ice-cold PBS and scraped off in 2–3 ml of PBS. After low-speed centrifugation, cell pellets were solubilized by mixing in 200 µl of buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM MgCl2, 1 mM DTT, and 0.3 mM PMSF] containing 0.1% NP40 for 10 min. The crude nuclear fraction was recovered by centrifugation in a microcentrifuge for 30 s, resuspended in 100 µl of buffer C [20 mM HEPES (pH 7.9), 25% glycerol, 400 mM NaCl, 0.2 mM EDTA, 1 mM DTT, and 0.5 mM PMSF], and nuclear proteins were extracted by vigorous mixing for 1 h. Following removal of nuclei by a 5-min centrifugation, the supernatant was further concentrated by using a Centricon-30 (Amicon, Beverly, MA) or directly stored at -70°C in small aliquots. The concentration of proteins was determined by a Coomassie blue dye-binding assay (Pierce, Rockford, IL).

For Western immunoblotting, nuclear proteins (20–60 µg) were subjected to electrophoresis in 10% polyacrylamide slab gels in the presence of SDS, transferred electrophoretically onto nitrocellulose membranes, and stained with Ponceau S to determine uniformity of protein loading and transfer. The blots were blocked by incubation for 1 h in PBS containing 15% powdered nonfat milk and 0.2% Tween 20, washed in PBS containing 0.2% Tween 20, and then incubated for 2 h with a 1:1000 dilution of rabbit polyclonal antiserum RPß(F). This antibody, which recognizes the human RARß F domain (35) , was provided by Dr. P. Chambon. The blot was washed again as above and incubated for 1 h at room temperature with 125I-labeled protein A (1 x 106 cpm/blot; 38.9 µCi/µg, ICN Radiochemicals, Irvine, CA). After extensive washes in PBS/Tween 20, the membrane was dried and subjected to autoradiography.

Electrophoretic Mobility Shift and Supershift Assays.
Nuclear and cytosolic extracts were prepared by the method of Dignam et al. (34) with some modifications. Cells were trypsinized, harvested in PBS, and collected by centrifugation. The cells were then suspended and washed with ice-cold PBS twice and then resuspended in 5x the cell pellet volume in buffer A [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, 10 mM KCl, 10 mM monothioglycerol, 1 mM PMSF, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin]. The pellet was homogenized at 4°C with a Dounce homogenizer in 2x the cell pellet volume in buffer A. The nuclei were collected by centrifugation (6000 x g, 5 min at 4°C). The supernatant fraction representing the cytosolic extract was frozen at -80°C until further use. Nuclear pellet was solubilized in 3x the pellet volume of buffer B [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, 600 mM KCl, 1 mM DTT, 10 mM monothioglycerol, 1 mM PMSF, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin] on ice for 60 min. After ultracentrifugation (100,000 x g, 20 min at 4°C), the supernatant was dialyzed for 6 h against buffer C [10 mM Tris-HCl (pH 7.5), 10 mM KCl, 1 mM EDTA, 20% glycerol, 1 mM DTT, 10 mM monothioglycerol, 1 mM PMSF, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin].

The synthetic oligonucleotide representing the native RARß2 RARE promoter sequence from nucleotide -63 to -33 (5'-TCGAGGGTAGGGTTCACCGAAAGTTCACTCG-3') and a mutated RARE (5'-TCGAGGGTAGGcTTacCCGAAAGTTCACTCG-3'), which was used as a control for specificity of the binding to DNA, were labeled with [{gamma}-32P]ATP (4000 Ci/mol) using T4 polynucleotide kinase (Ref. 36 ; the underlined sequences indicate the DRs that constitute the DR5 RARE).

The DNA-binding reactions were performed as described by Wagner and Green (37) with some modifications as follows: nuclear and cytosolic extracts were preincubated with 2 µg of poly(dI·dC) for 15 min at 4°C and then incubated with 32P-labeled oligonucleotides (6000 cpm) for 15 min at 4°C in the presence of 10 mM Tris-HCl (pH 7.5), 10 mM KCl, 1 mM EDTA, 1 mM DTT, 5 mM MgCl2, and 20% glycerol. For supershift assays, monoclonal antibodies specific for RAR{alpha}, RARß, or RAR{gamma} (Refs. 35 and 38 ; provided by Dr. P. Chambon) were added (0.5 µl) to the above incubation mixture. The reaction mixture (20 µl) was subjected to electrophoresis in a 5% polyacrylamide gel containing 25 mM Tris-HCl (pH 8.5), 192 mM glycine, and 1 mM EDTA, and the gels were processed for autoradiography. The specificity of DNA binding was analyzed by adding 100-fold molar excess of nonradioactive wild-type RARE or mutated RARE.

Determination of Growth Rate and Calcein-AM Assay.
For determination of doubling time of parental SqCC/Y1 cells and the transfectants, cells were plated in medium containing 5% delipidized FBS at a density of 5 x 104 cells in each well of six-well plates. After 24 h, medium were replaced with serum-free medium supplemented with DMSO or 10-7 M ATRA. Cells were refed with fresh medium on day 3. At 24-h intervals, cells in triplicate wells were detached, suspended, and counted using an electronic particle counter (Coulter Electronics, Hialeah, FL). Doubling times were calculated from the linear areas of slopes of growth curves constructed with cell number on a logarithmic scale on the Y axis versus time on a linear scale in the X axis.

To determine the effects of different retinoids on the growth of SqCC/Y1 cells and the transfected derivatives, we used serum-free medium to minimize contribution of serum retinoids. Cells were seeded in 96-well cluster well plates (Corning Costar Corp. Cambridge, MA) at 2 x 103 cells per well. After a 48-h culture in medium containing 5% delipidized serum, medium was replaced with serum-free one supplemented with various concentrations of retinoids or DMSO for control. The cells were also refed with fresh medium on day 3. Six replicates wells were set for each group. Relative cell density was determined on day 6 using the Calcein-AM assay (Molecular Probes, Eugene, OR). Medium in each well was aspirated, and the cells were washed three times with calcium- and magnesium-free PBS; subsequently, cells were covered with 100 µl of 2 µM Calcein-AM, which was diluted in calcium- and magnesium-free PBS from a 0.2 mM stock solution dissolved in DMSO and incubated for 30 min at room temperature. The same amount of DMSO solution diluted in PBS was used as control. Following the incubation, relative fluorescence intensity was measured using Millipore’s Cytofluor 2300 Fluorescence Plate Reader connected to a NEC Power Mate 386/33i computer and an NEC Pinwriter printer. The excitation wavelength was set at 480 nm, and the emission was measured at 530 nm. The fluorescence of cells in control cultures (Fc) and in treated cultures (Ft) was used to calculate growth inhibition according to the equation: % growth inhibition = (1 - Ft/Fc) x 100.

Transient Transfections and Reporter Gene Assay.
The COL-AP1-LUC reporter plasmid, which contains the luciferase gene controlled by a promoter fragment of the collagenase gene (nucleotides -74 to +63) was obtained from Dr. Jonathan Kurie (University of Texas M. D. Anderson Cancer Center, Houston, TX). This fragment contains a consensus AP-1-binding site (TGAGTCA). Cells were seeded at a concentration of 1 x 105 cells per well in 6-well cluster plates and cultured in medium containing 10% delipidized serum. After 24 h, the cells were transfected with 1.3 µg of luciferase reporter construct, 0.5 µg of expression vector (for parental SqCC/Y1 cells), or pBluescript(-) (for LNSX and LNSß transfectants) and 0.2 µg of pRL-SV40 (Promega), which contains the Renilla luciferase gene driven by the SV40 early promoter and was used as internal control for transfection efficiency. Transfection was performed using Lipofectace reagent (Life Technologies, Inc., Bethesda, MD) following the manufacturer’s protocol, with a minor modification. Briefly, after 12 h of exposure to the above mixture of 2 µg of DNA and 8 µl of Lipofectace reagent (for each well) in 1 ml of serum-free medium, cells were refed with 2 ml of fresh serum-free medium supplemented with DMSO or different ATRA concentrations. After 20 h, cell lysates were prepared for dual-luciferase reporter assay (Promega, Madison, WI). Both firefly and Renilla luciferase activities were determined using a luminometer (Lumat model LB9501; EG&G Berthold, Bad Wildbad, Germany). Triplicate wells were used for each experimental group. The relative firefly luciferase activity generated from the AP-1 reporter construct was normalized using the Renilla luciferase activity generated from the pRL-SV40 construct to account for transfection efficiency.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Introduction of Exogenous RARß2 Gene into SqCC/Y1 Cells by Retroviral Transfection.
The transfection of SqCC/Y1 cells by standard DNA transfection methods occurs at a very low efficiency (39 , 40) ; therefore, we decided to use transfection with a retrovirus to introduce RARß2 cDNA into the SqCC/Y1 cells. A retroviral vector harboring human RARß2 cDNA was constructed using the previously described LNSX vector (24) . In this vector, the Moloney murine leukemia virus long terminal repeat (L) drives the neomycin resistance gene (N), and the SV40 (SV) 40 promoter (S) drives the desired cDNA (X; in our case, RARß2) inserted into a multiple cloning site. To ensure optimal translation efficiency, we deleted all of the 5' untranslated region of wild-type hRARß2 cDNA because it contains several short ORFs, which may decrease the translation efficiency of RARß mRNA (41) , and introduced a new Kozak sequence (42) into the RARß cDNA. The resulting vector was designated LNSß (Fig. 1)Citation .

The LNSß vector and the retroviral vector LNSX were packaged into a replication defective virus in the amphotropic cell line PA317, and then the virus was used to transfect SqCC/Y1 cells. Five stable (G418-resistant) transfected clones and a stably transfected population of LNSß and LNSX were used for the further analysis. The presence of the exogenous RARß gene in the stable LNSß transfectants was confirmed by Southern blotting (Fig. 2A)Citation . Similar level of exogenous gene were found in the individual clones and the population. The other bands observed in all of the cells analyzed represent endogenous RARß gene, and they were similar in parental and LNSX- and LNSß-transfected cells, suggesting that no gross rearrangements had occurred during transfection. No exogenous RARß was detected in the parental SqCC/Y1 cells or in the LNSX-transfected population (Fig. 2A)Citation .



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  		Fig. 2. Characterization of RARß- and LNSX-transfected cells. A, identification of the presence of exogenous RARß gene in stable transfectants by Southern blotting. Genomic DNA (10 µg) isolated from the indicated cells and transfected clones (3, 4, 6, 8, and 10) was digested with the restriction enzyme HindIII and subjected to agarose gel electrophoresis. The expected 1.5-kb band of integrated RARß gene was detected with a 32P-labeled RARß cDNA probe (released from LNSß vector by HindIII) in all LNSß transfectants but not in parental or LNSX control cells. The numbers on the right represent the size of standard commercial markers of DNA size. B, detection of exogenous and endogenous RARß mRNA and endogenous RAR{alpha} and RARß mRNAs in SqCC/Y1 cells and transfectants by Northern blotting. B, total RNA (12.5 µg) fractions isolated from the indicated cell lines and clones were analyzed by Northern blotting as described in "Materials and Methods." Total RNA samples from SqCC/Y1 parental cells treated for 5 days with DMSO or 10-6 M ATRA were used as a control of the size of endogenous

 
Expression of Exogenous RARß2 Gene in SqCC/Y1 Cells.
The expression of the exogenous RARß2 gene was analyzed at the mRNA and protein levels by Northern and Western blotting, respectively. As shown in Fig. 2BCitation , parental SqCC/Y1 cells and the LNSX-transfected cells have no detectable endogenous or exogenous RARß mRNA. In contrast, the LNSß transfected population and clones expressed detectable levels of exogenous RARß mRNA. The size of the exogenous RARß2 transcript was larger than the endogenous one that can be induced by ATRA in parental SqCC/Y1 cells (Fig. 2BCitation , Lanes -RA and +RA).

The levels of RAR{alpha} transcripts in all of the cell lines and clones were comparable to the levels in parental SqCC/Y1 cells, indicating that RAR{alpha} expression was not altered by the transfection and expression of exogenous RARß2 (Fig. 2C)Citation . Similar results were observed for RAR{gamma} (data not shown).

To determine whether the LNSß transfectants express the RARß protein, we performed Western blotting using polyclonal antibodies against a peptide located in the F region of human RARß protein (36) . Cells were grown in medium containing delipidized serum in which no endogenous RARß mRNA or protein can be detected in parental SqCC/Y1 cells. As shown in Fig. 2DCitation , the RARß protein was present in all of the LNSß transfectants but was undetectable in LNSX control cells. An extract of the HNSCC 1483 cells treated with 10-6 M ATRA for 3 days was used as a positive control for the endogenous RARß protein because ATRA induces RARß in these cells (21) The LNSß transfectants produced from the exogenous RARß cDNA the same size protein (Mr {approx}51,000) and in comparable amounts to those that 1483 cells did after ATRA induction of the endogenous gene (Fig. 2D)Citation . A few other protein bands were detected in all extracts including the LNSX control and, therefore, were presumed to be nonspecific. One of them (Mr 120,000), shown in Fig. 2DCitation (NS), was used as an internal control for loading. We also performed Western blotting with commercial anti-RARß antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and obtained comparable results for the RARß protein detected with the RPß(F) antibodies, although the nonspecific bands were distinct for the two antibodies (data not shown).

RARE Binding Activity of RARß Protein Expressed from Transduced RARß2 cDNA.
In previous studies we have established that of the different retinoid receptors only RAR{gamma} and RXRs participate in binding RARß2 RARE in parental SqCC/Y1 cells, probably as a RAR{gamma}-RXR heterodimers (39 , 40) . To determine whether the RARß protein expressed in the LNSß transfectants is capable of binding this RARE, we performed gel shift analysis. Fig. 3Citation demonstrates that, in the parental SqCC/Y1 cells, a single shifted band is formed between nuclear proteins and RARE, but no such complex is formed with a cytosolic extract. The complex is specific because it is competed by excess unlabeled RARE but not by unlabeled mutated RARE. A similar complex is also formed by nuclear proteins from LNSß cells. Gel supershift analysis revealed that, of the RARs, only RAR{gamma} was present in the complex formed in the parental cells (Fig. 3Citation , Lane 7), whereas RAR{alpha}, RARß, and RAR{gamma} were supershifted from the complex formed in LNSß-transfected cells (Fig. 3Citation , Lanes 12, 13, and 14). These results demonstrated that the RARß protein expressed from the exogenous RARß2 cDNA is functional because it can bind RARE. In addition, RAR{alpha} was also identified in the complex formed in LNSß cells, whereas it could not be supershifted from complex formed in the parental SqCC/Y1 cells (Fig. 3)Citation .



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  		Fig. 3. Identification of nuclear proteins with specific RARE-binding activity in parental SqCC/Y1 cells and in LNSß transfectants. Nuclear extracts (Lanes 2–7 and 9–14) from SqCC/Y1 or LNSß cells were incubated with 32P-labeled wild-type (Wt) or mutated (Mu) RARß2 RARE and then analyzed by gel shift assay. Specificity of the binding was determined by competition with 100-fold molar excess of unlabeled Wt or mutated RARE. Cytosolic extracts (Lanes 1–8) were also analyzed for binding RARE as a control. The appearance of supershifted bands was analyzed by the addition of subtype-specific monoclonal antibodies against each RAR (as indicated above the relevant lanes) to the incubation mixture containing nuclear extracts and wild-type RAREß2 before electrophoresis.

 
To determine whether ATRA treatment affects RARß protein levels in transfected cells, we compared the supershifted RARß protein levels using a gel shift analysis of nuclear extracts from clones grown in the absence or presence of 0.1 and 1 µM ATRA for 24 h and found that no RARß could be detected by supershift in parental SqCC/Y1 or vector only transfected cells (LNSX), whereas similar amounts of RARß were supershifted in extracts from untreated or ATRA-treated, RARß-transfected cells (Fig. 4)Citation . Thus, the expression of the transfected RARß is not suppressed in ATRA-treated cells by posttranscriptional mechanisms.



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  		Fig. 4. An analysis of the effect of ATRA treatment on the levels of RARß protein as detected by gel supershift assay. Cells indicated at the top of the figure were grown in the absence or presence of 0.1 or 1 µM ATRA for 24 h, and then nuclear extracts were prepared and incubated with 32P-labeled wild-type RAREß2 and with antibodies against RARß. The mixture was then analyzed by gel electrophoresis as described in "Materials and Methods." n.s., nonspecific band.

 
The results of Southern, Northern, and Western blotting and supershift analysis demonstrate that a functional RARß was expressed in SqCC/Y1 cells infected by the LNSß retroviral vector.

Comparison of the Responses of Parental SqCC/Y1 Cells and LNSß-transfected Cells to the Growth-inhibitory Effects of Retinoids.
To determine whether the expression of RARß alters the response of the SqCC/Y1 cells to the growth inhibitory effects of retinoids, we compared the doubling times of cells grown in serum-free medium in the absence or presence of 10-7 M ATRA. The results presented in Table 1Citation show that the increase in doubling times in ATRA-treated cultures relative to their DMSO controls were 15–20% in parental and LNSX-transfected cells and 23.5–28% in the LNSß population and the transfected clones (Table 1)Citation . The difference between the groups was not significant. Two LNSß transfectant clones (clones 3 and 4) exhibited longer doubling times than the rest of the clones and cell lines. Because the LNSß population and several of the clones had doubling times similar to those parental and LNSX cells (Table 1)Citation , it is unlikely that the longer doubling times in the two clones were due to RARß expression. However, the slower growth could be an inherent property of the cells from which the clones were derived or an interaction of inherent properties and RARß expression. In any event, these clones were neither more nor less sensitive to ATRA than the other clones.


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  		Table 1 Comparison of the response of RARß transfectants (LNSß), control vector transfectants (LNSX), and parental SqCC/Y1 cells to the growth-inhibitory effects of RAa

 
In addition to the above experiments, we also examined the response of the cells to the growth-inhibitory effects of 10-7 M or 10-9 M ATRA, 9-cis-RA, or TTNN after a 6-day treatment. 9-cis-RA was used because it binds to both RARs and RXRs, and TTNN was used because it exhibits a preferential binding to RARß, although it also binds RAR{gamma}. Fig. 5Citation shows that all of the cells were sensitive to all three retinoids at 10-7 M, that the potencies of ATRA and 9-cis-RA were similar in most of the cell lines, and that TTNN was less potent than these retinoids in most of the cell lines. There was no consistent difference between the responses of cells transfected with RARß and the parental and vector control cells (Fig. 5)Citation . These results indicate that expression of RARß did not enhance the response of the cells to the growth-inhibitory effects of any of the retinoids tested.



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  		Fig. 5. An analysis of the inhibitory effects of different retinoids on the growth of parental SqCC/Y1 cells, LNSX-transfected cells, and LNSß-transfected cells and clones. Cells were treated with the retinoids ATRA, 9-cis-RA, or TTNN at either 10-9 or 10-7 M for 6 days, and then their numbers were estimated. Columns, mean percentages of growth inhibition in six replicate wells, calculated as described in "Materials and Methods"; bars, SD.

 
Comparison of the Responses of LNSX Cells and LNSß-transfected Clones to the Suppressive Effects of ATRA on the Expression of Squamous Differentiation Markers.
Previously, we reported that ATRA can suppress the expression of squamous cell differentiation markers in SqCC/Y1 cells treated for 7 days with 10-6 M ATRA (16) . To determine whether RARß expression enhances the ability of ATRA to suppress squamous cell differentiation, we analyzed the levels of transcripts for the squamous differentiation markers TGase I and CK1 under suboptimal (short 3-day) treatment conditions where 10-6 M ATRA was insufficient for suppression of TGase I in the LNSX cells (Fig. 6Citation , Lanes LNSX). Fig. 6Citation shows that, in the absence of ATRA, TGase expression was similar in the LNSß clones and in LNSX cells, and the expression of CK1 was even higher in some clones (e.g., clone 8) than in the LNSX cells. However, whereas 10-6 M ATRA was not effective in LNSX cells, 10-7 M ATRA was very effective in suppressing TGase I in all five LNSß clones. Furthermore, even 10-10 M ATRA was effective in suppression of this marker in four of the five clones. These results indicate that most of the clones were 10,000 times more sensitive than LNSX cells for suppression of TGase I expression.



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  		Fig. 6. An analysis of the effects of ATRA on the expression of the squamous differentiation markers TGase I and CK1 in LNSX-transfected cells and LNSß-transfected clones. Cells were treated for 3 days with control serum-free medium supplemented with DMSO (Lanes C), or the same medium supplemented with 10-10, 10-7, or 10-6 M ATRA (Lanes 10, 7, and 6, respectively). Total RNA (20 µg/lane) extracted from the indicated cells and clones was subjected to Northern blotting and the same blot was hybridized consecutively with 32P-labeled probes for TGase, CK1, and GAPDH. After each hybridization, the membrane was analyzed by autoradiography and then stripped and reprobed with the next probe.

 
A similar analysis of CK1 transcript level and modulation by ATRA has shown that CK1 was suppressed somewhat by 10-10 M and completely by 10-7 M ATRA in LNSX cells. However, it was suppressed more effectively by 10-10 M ATRA in all of the LNSß clones than in the LNSX cells. The results indicate that RARß expression enhanced the ability of ATRA to suppress the expression of both squamous markers, especially TGase I.

RARß Expression Does Not Enhance the Anti-AP-1 Effects of ATRA in SqCC/Y1 Cells.
AP-1 activity has been implicated in the regulation of the differentiation-specific expression of the CK1 gene through an AP-1 site in the 3' flanking sequence (43) . Several retinoids, including ATRA, can transrepress AP-1 (11) . Therefore, we investigated whether the enhanced inhibition of K1 expression in RARß-transfectants was due to increased anti-AP-1 activity. Fig. 7ACitation shows that AP-1 activity was suppressed by ATRA in a dose-dependent fashion in stable transfectants of SqCC/Y1, irrespective of whether they were transfected with vector only (LNSX) or with RARß (LNSß). A similar result was obtained when the LNSX and LNSß retroviral vectors were transiently cotransfected into SqCC/Y1 cells with the COL-AP1-LUC reporter construct, as shown in Fig. 7BCitation . Thus, the enhanced suppression of CK1 expression by ATRA in cells expressing exogenous RARß does not appear to be mediated by increased antagonism of AP-1.



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  		Fig. 7. An analysis of the effects of ATRA on AP-1 transrepression in SqCC/Y1 cells expressing RARß, either stably (A) or transiently (B). A, SqCC/Y1 cells transfected with the LNSX vector, LNSß vector (a population of stable transfectants), or clone LNSß3 (a clone of stably transfected cells) were transfected transiently with the AP-1-LUC reporter construct and treated with either control medium containing DMSO or medium supplemented with the indicated ATRA concentrations for 20 h and then analyzed for luciferase activity as described in "Materials and Methods." B, SqCC/Y1 cells were cotransfected with the AP-1-LUC reporter construct and either the control retroviral vector LNSX or LNSß and analyzed for AP-1 transrepression by ATRA as in A.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
More than 90% of the malignancies that occur in the head and neck region are SCCs, and therefore, not surprisingly, abnormal keratinization occurs frequently in nonkeratinizing epithelium during head and neck carcinogenesis and is detected in certain premalignant lesions (e.g., leukoplakia) and in moderately to well-differentiated HNSCCs (44, 45, 46) . Interestingly, squamous metaplasia is also a characteristic of vitamin A deficiency because vitamin A is important for the maintenance of proper differentiation of many epithelial tissues (47) .

Previous studies with human HNSCCs have demonstrated that some of them, including the SqCC/Y1 cell line used in this study, exhibit characteristic markers of squamous differentiation including TGase I, involucrin, and CK1, especially when cultured in delipidized serum or in serum-free medium. Furthermore, retinoids suppress squamous differentiation (15 , 17 , 18 , 21 , 22 , 46 , 48) . The formation of cross-linked envelopes was suppressed by retinoids as a result of inhibition of TGase activity and expression (15 , 17 , 18 , 32 , 49 , 50) and decreasing the level of envelope precursors, including involucrin (15 , 17 , 18 , 22) .

Several studies have demonstrated that some HNSCC cell lines (13 , 15) and oral premalignant and malignant lesions (14 , 16) express little or no RARß, whereas they do express RAR{alpha} and RAR{gamma} mRNAs. Because the normal counterparts of these cancer cells in the nonkeratinizing oral cavity mucosa express RARß mRNA (12, 13, 14, 15) , it was suggested that the selective suppression of RARß expression may enhance malignant transformation in oral epithelial cells. In addition, the expression of RARß in normal cell strains derived from oral cavity and from oral leukoplakia lesions was related inversely to the degree of keratinization (12) , and RARß expression in HNSCCs was related inversely to the expression of CK1 and TGase I (15 , 21) . These findings suggested that RARß may mediate suppression of aberrant keratinization in normal oral mucosa epithelial cells and that the loss of its expression during carcinogenesis may result in abnormal squamous differentiation.

To examine this hypothesis more directly, we studied the effect of restoration of RARß expression by a transfection of an RARß2 cDNA in a retroviral vector (LNSß) in HNSCC SqCC/Y1 cells, which do not express endogenous RARß when grown in serum-free medium. We found that LNSß-transfected SqCC/Y1 cells that expressed exogenous RARß became much more sensitive than vector-transfected cells to suppression of the squamous differentiation markers TGase I and, to a lesser extent, CK1. These results indicate that RARß may enhance the suppression of squamous differentiation in SqCC/Y1 cells beyond what the constitutively expressed RAR{alpha} and RAR{gamma} can do. In a previous study, we overexpressed RAR{gamma} in the same parental SqCC/Y1 cells and found that the cells exhibited enhanced expression of CK1, TGase I, and involucrin in the absence of ligand but the expression was still suppressed effectively in the presence of ligand (39) . The effect of introduction of RARß in this study was different from the introduction of RAR{gamma} because RARß did not increase TGase I expression above the level found in the vector transfectants, whereas the level of CK1 increased only in one of five clones. Thus, RARß and RAR{gamma} may exert different effects in the SqCC/Y1 cells in support of the observation that retinoid receptors may exhibit specificity for distinct promoters in some cell context (51) . The appearance of supershifted RAR{alpha} band in the RARß-transfected cells but not in the parental cells is not clear. It is not simply due to an increase in RAR{alpha} expression because no increase in RAR{alpha} mRNA was noted in transfectants compared to parental cells or vector transfectants. A possible explanation is the formation of RAR{alpha}-RARß dimers that bind the RARE in the gel shift experiment by analogy with the report that RAR{alpha}-RAR{alpha} homodimers bind RARE (52) .

It is not clear how retinoids regulate the expression of TGase I and CK1 in HNSCC cells. Studies with tracheal epithelial cells (32) and epidermal keratinocytes (49 , 50) demonstrated that the suppression of TGase I by retinoids occurred at least in part at the transcriptional level. Because there is no evidence for the presence of RARE in the human TGase I gene promoter, whereas there is an AP-1 site, it is possible that retinoids function by antagonizing AP-1 activity (11) and thereby suppress TGase I expression (50) . Likewise, the CK1 gene promoter contains an AP-1 site (43) , and retinoids could suppress CK1 expression by antagonizing AP-1. However, our results demonstrate that stable or transient overexpression of RARß was not accompanied by an increased anti-AP-1 activity. Another possibility, that the suppression is mediated by retinoid receptors, was raised by the finding that some genes contain negative RAREs (53 , 54) . Of potential relevance to this report are the findings that the promoters of the keratins K5, K10, and K14 contain a functional RARE that consists of a cluster of half-sites in various orientations that can be suppressed by ATRA (55) . Interestingly, the K14 promoter does not contain an AP-1 site, yet it is suppressed by ATRA by direct binding of RARs to the K14 RARE (56) . All three RARs were able to suppress the activation of K5, K14, and K17 promoter-reporter constructs upon cotransfection into HeLa cells and treatment with ATRA (55) . However, in our study, it appears that the constitutive RAR{alpha} and RAR{gamma} and RXR{alpha} were much less effective in suppressing CK1 than the overexpressed RARß.

Although they inhibit squamous differentiation, retinoids enhance differentiation of a variety of other cell types (2 , 3) .

The expression of RARß in the SqCC/Y1 cells did not increase their sensitivity to the growth-inhibitory effects of ATRA, 9-cis-RA, or TTNN. This finding is distinct from our observation that overexpression of RAR{gamma} in SqCC/Y1 cells enhanced their growth inhibition by ATRA (40) . Our findings are also different from reports by others that expression of RARß increased growth inhibition by ATRA of cervical carcinoma (57 , 58) , renal carcinoma cells (59) , and breast carcinoma cells (60, 61, 62) and suppressed the tumorigenicity of lung carcinoma cells (63) . The function of retinoid receptors depends on a variety of factors, including accessibility to the promoter, the nature of the flanking sequences in RAREs, and the presence and level of corepressors or coactivators. Thus, because the expression of both receptors and cofactors may be cell type specific (9 , 64, 65, 66) , it is plausible to assume that the overexpression of RARß may exert different effects in different cell types. In previous studies, we did not find a relationship between the expression of endogenous RARß and response to growth-inhibitory effects of retinoids in HNSCCs (16) and in lung cancer cell lines (67) . Recently, it has been reported that transfection of RARß into two HNSCC cell lines (SCC9 and SCC15) using the Lipofectamine method increased terminal differentiation (evidenced by increased involucrin and keratin K10) that caused cell death (68) . Our results differ from the latter findings because we failed to observe cell death, and only two of six clones of stable transfectants exhibited a prolonged doubling time. Furthermore, the constitutive expression of squamous differentiation marker TGase I was not elevated in our transfectants, and K1 level was higher in only one of five clones relative to vector control. The reason for the discordance between our results with SqCC/Y1 HNSCC cells and the results of Crowe (68) with SCC9 and SCC15 is not clear. However, it should be noted that our results are consistent with our recent findings of an inverse relationship between RARß expression and squamous differentiation (21) . More importantly, our findings are consistent with expression patterns in vivo because, in oral mucosa, normal epithelial cells express RARß (15) but do not express the keratinizing epithelial differentiation markers involucrin and K10 (or K1), which are expressed aberrantly in some HNSCC tumors and cell lines derived from them (45 , 46) . Thus, our transfectants represent cells in which RARß expression was restored, and the aberrant expression of squamous differentiation markers TGase I and K1 has become more suppressible by physiological levels of retinoids, presumably as it is in vivo.

Our findings that the RARß-transfected SqCC/Y1 cells are much more sensitive to suppression of squamous differentiation but not more sensitive to growth inhibition by retinoids than vector control cells are similar to what was found recently in an in vivo study of the effects of retinoids on the growth and differentiation of another HNSCC cell line, 1483. The growth of xenotransplanted 1483 tumors in nude mice was not inhibited by RA; however, RARß was induced and squamous differentiation was suppressed (19 , 69) . Thus, the effects of retinoids and RARß on squamous differentiation and on cell growth appear to be uncoupled in these two HNSCC cell lines in vitro and in vivo.

In conclusion, the results support the suggestion that decreased expression of RARß may be associated with increased keratinizing squamous differentiation in HNSCC cells and that pharmacological doses of ATRA can induce RARß in HNSCC cells, which may lead to restoration of a more normal differentiation.


   ACKNOWLEDGMENTS
 
We thank the following colleagues for the kind gifts of HNSCC cells, packaging cells, retroviral vectors, antireceptor antibodies, cDNA probes for squamous differentiation markers and nuclear receptors, and AP-1-reporter constructs: W. Bollag, P. Chambon, S. Collins, R. Heyman, A. Jetten, J. Kurie, D. Miller, D. Roop, and M. Reiss.


   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 This work was supported by USPHS/National Cancer Institute Grant PO1 CA52051, by National Institute of Dental and Craniofacial Research Grant P50 DE11906, and by the Irving and Nadine Mansfield and Robert David Levitt Cancer Research Chair (to R. L.). W. K. H. is an American Cancer Society Clinical Research Professor. Back

2 Present address: Department of Otolaryngology, Hokkaido University School of Medicine, Sapporo 060, Japan. Back

3 To whom requests for reprints should be addressed, at Department of Thoracic/Head and Neck Medical Oncology, Box 108, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. Phone: (713) 792-7480; Fax: (713) 794-0209. Back

4 The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; ATRA, all-trans-retinoic acid; RA, retinoic acid; RARE, RA response element; DR, direct repeat; AP-1, activator protein-1; HNSCC, head and neck squamous cell carcinoma; CK1, cytokeratin 1; TGase, transglutaminase; FBS, fetal bovine serum; SCC, squamous cell carcinoma; TTNN, 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthalenecarboxylic acid; ORF, open reading frame; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMSF, phenylmethylsulfonyl fluoride. Back

Received 11/19/98. Accepted 5/17/99.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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