Evidence That Retinoic Acid Receptor β Induction by Retinoids Is Important for Tumor Cell Growth Inhibition
2000; Elsevier BV; Volume: 275; Issue: 22 Linguagem: Inglês
10.1074/jbc.m000527200
ISSN1083-351X
AutoresShi‐Yong Sun, Haisu Wan, Ping Yue, Waun Ki Hong, Reuben Lotan,
Tópico(s)interferon and immune responses
ResumoRetinoic acid receptor β (RARβ) is thought to be involved in suppressing cell growth and tumorigenicity. Many premalignant and malignant cells exhibit a reduced RARβ expression. However, in some of these cells (e.g. H157 human squamous cell carcinoma cells), RARβ can be induced by retinoids (e.g. all-trans-retinoic acid, ATRA) because its promoter contains a retinoic acid response element. To examine the hypothesis that RARβ induction is important for inhibition of cell proliferation by retinoids, we blocked ATRA-induced RARβ expression in H157 cells using a retroviral vector harboring multiple copies of antisense RARβ2 sequences. Antisense RARβ-transfected cells showed not only decreased expression of ATRA-induced RARβ protein but also reduced ATRA-induced RARE binding activity and transactivation. Importantly, all antisense RARβ transfectants of H157 cells were less responsive than vector-transfected cells to the growth inhibitory effects of the retinoids ATRA and Ch55 in vitro. These results demonstrate that RARβ induction may play an important role in mediating growth inhibitory effects of retinoids in cancer cells. Retinoic acid receptor β (RARβ) is thought to be involved in suppressing cell growth and tumorigenicity. Many premalignant and malignant cells exhibit a reduced RARβ expression. However, in some of these cells (e.g. H157 human squamous cell carcinoma cells), RARβ can be induced by retinoids (e.g. all-trans-retinoic acid, ATRA) because its promoter contains a retinoic acid response element. To examine the hypothesis that RARβ induction is important for inhibition of cell proliferation by retinoids, we blocked ATRA-induced RARβ expression in H157 cells using a retroviral vector harboring multiple copies of antisense RARβ2 sequences. Antisense RARβ-transfected cells showed not only decreased expression of ATRA-induced RARβ protein but also reduced ATRA-induced RARE binding activity and transactivation. Importantly, all antisense RARβ transfectants of H157 cells were less responsive than vector-transfected cells to the growth inhibitory effects of the retinoids ATRA and Ch55 in vitro. These results demonstrate that RARβ induction may play an important role in mediating growth inhibitory effects of retinoids in cancer cells. retinoic acid receptor retinoid X receptor all-trans-retinoic acid retinoic acid responsive element thymidine kinase (E)-4-[3-(3,5-di-tert-butylphenyl)]-3-oxo-1-propenylbenzoic acid Retinoids, a group of natural and synthetic vitamin A (retinol) derivatives, exert fundamental effects on the regulation of cell growth, differentiation, and development (1.De Luca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (815) Google Scholar). Some retinoids are being evaluated as chemopreventive and chemotherapeutic agents for a variety of human cancers (2.Lotan R. FASEB J. 1996; 10: 1031-1039Crossref PubMed Scopus (398) Google Scholar). Effective use of retinoids requires an understanding of their mechanism of action. Retinoids are thought to exert most of their effects by regulating gene expression primarily through two classes of nuclear receptors, RARs1 and RXRs (3.Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 319-349Google Scholar, 4.Chambon P.A. FASEB J. 1996; 10: 940-945Crossref PubMed Scopus (2590) Google Scholar). Both types of receptor are members of the steroid hormone receptor gene superfamily of sequence-specific, ligand-activated transcriptional factors. The receptors are encoded by six distinct genes, RARα, RARβ, and RARγ, and RXRα, RXRβ, and RXRγ. Each of these receptors includes several isotypes (e.g. β1, β2, and β4) formed by different splicing and usage of alternative promoters (3.Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 319-349Google Scholar, 4.Chambon P.A. FASEB J. 1996; 10: 940-945Crossref PubMed Scopus (2590) Google Scholar). ATRA binds to and activates RARs, and 9-cis-retinoic acid binds to and activates both RARs and RXRs. RARs and RXRs modulate the expression of target genes by interacting as either homodimers or heterodimers with RAREs located in the promoter regions of target genes (3.Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. Raven Press, New York1994: 319-349Google Scholar, 4.Chambon P.A. FASEB J. 1996; 10: 940-945Crossref PubMed Scopus (2590) Google Scholar). Some RAR genes, in particular the RARβ gene, contain RAREs in their promoters and can be induced by retinoids (5.De The H. Vivanco-Ruiz M.M. Tiollais P. Stunnenberg H. Dejean A. Nature. 1990; 343: 177-180Crossref PubMed Scopus (843) Google Scholar, 6.Sucov H.M. Murakami K.K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5392-5396Crossref PubMed Scopus (413) Google Scholar, 7.Hoffman B. Lehmann J.M. Zhang X-k. Hermann T. Graupner G. Pfahl M. Mol. Endocrinol. 1990; 4: 1734-1743Crossref Scopus (197) Google Scholar, 8.De The Marchio A. Tiollais P. Dejean A. EMBO J. 1989; 8: 429-433Crossref PubMed Scopus (344) Google Scholar). The RARβ RARE consists of two direct repeats of the core motif sequence (G/A)GTTCA separated by five nucleotides (5.De The H. Vivanco-Ruiz M.M. Tiollais P. Stunnenberg H. Dejean A. Nature. 1990; 343: 177-180Crossref PubMed Scopus (843) Google Scholar, 6.Sucov H.M. Murakami K.K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5392-5396Crossref PubMed Scopus (413) Google Scholar, 7.Hoffman B. Lehmann J.M. Zhang X-k. Hermann T. Graupner G. Pfahl M. Mol. Endocrinol. 1990; 4: 1734-1743Crossref Scopus (197) Google Scholar). The level of RARβ transcript increases in many cell types in response to ATRA due to the presence of this RARE (5.De The H. Vivanco-Ruiz M.M. Tiollais P. Stunnenberg H. Dejean A. Nature. 1990; 343: 177-180Crossref PubMed Scopus (843) Google Scholar, 6.Sucov H.M. Murakami K.K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5392-5396Crossref PubMed Scopus (413) Google Scholar, 7.Hoffman B. Lehmann J.M. Zhang X-k. Hermann T. Graupner G. Pfahl M. Mol. Endocrinol. 1990; 4: 1734-1743Crossref Scopus (197) Google Scholar, 8.De The Marchio A. Tiollais P. Dejean A. EMBO J. 1989; 8: 429-433Crossref PubMed Scopus (344) Google Scholar). RARβ expression is suppressed in various types of malignant tumors including lung carcinoma (9.Gebert J.F. Moghal N. Frangioni J.V. Sugarbaker D.J. Neel B.G. Oncogene. 1991; 6: 1859-1868PubMed Google Scholar, 10.Houle B. Ledue F. Bradley W.E.C. Choromosomes Cancer. 1991; 3: 358-366Crossref PubMed Scopus (86) Google Scholar, 11.Hu L. Crowe D.L. Rheinwald J.G. Chambon P. Gudas L. Cancer Res. 1991; 51: 3972-3981PubMed Google Scholar, 12.Nervi C. Volleberg T.M. Gerore M.D. Zelent A. Chambon P. Jetten A.M. Exp. Cell. Res. 1991; 195: 163-170Crossref PubMed Scopus (101) Google Scholar, 13.Swisshelm, K., Ryan, K., Lee, X., Tsou, H. C., Beacocke, M., and Sager, R. Cell Growth Differ. 5, 133–141Google Scholar, 14.Xu X-C. Ro J.Y. Lee J.S. Shin D.M. Hong W.K. Lotan R. Cancer Res. 1994; 54: 3580-3587PubMed Google Scholar, 15.Xu X.C. Sozzi G. Lee J.S. Lee J.J. Pastorino U. Pilotti S. Kurie J.M. Hong W.K. Lotan R. J. Natl. Cancer Inst. 1997; 89: 624-629Crossref PubMed Scopus (199) Google Scholar). It has been suggested that the decrease in RARβ expression may lead to resistance to retinoids (16.Berg W.J. Nanus D.M. Leung A. Brown K.T. Hutchinson B. Mazumdar M. Xu X.C. Lotan R. Reuter V.E. Motzer R.J. Clin. Cancer Res. 1999; 5: 1671-1675PubMed Google Scholar, 17.Xu X.C. Liu X. Tahara E. Lippman S.M. Lotan R. Cancer Res. 1999; 59: 2477-2483PubMed Google Scholar, 18.Pergolizzi R. Appierto V. Crosti M. Cavadini E. Cleris L. Guffanti A. Formelli F. Int. J. Cancer. 1999; 81: 829-834Crossref PubMed Scopus (43) Google Scholar, 19.Hoffman A.D. Engelstein D. Bogenrieder T. Papandreou C.N. Steckelman E. Dave A. Motzer R.J. Dmitrovsky E. Albino A.P. Nanus D.M. Clin. Cancer Res. 1996; 2: 1077-1082PubMed Google Scholar, 20.Sabichi A.L. Hendricks D.T. Bober M.A. Birrer M.J. J. Natl. Cancer Inst. 1998; 90: 597-605Crossref PubMed Scopus (107) Google Scholar, 21.Li Y. Dawson M.I. Agadir A. Lee M.O. Jong L. Hobbs P.D. Zhang X.K. Int. J. Cancer. 1998; 75: 88-95Crossref PubMed Scopus (79) Google Scholar). Indeed, transfection of RARβ into RARβ-negative cervical, breast, and lung cancer cells increased cell responsiveness to growth inhibition and induction of apoptosis by retinoids (21.Li Y. Dawson M.I. Agadir A. Lee M.O. Jong L. Hobbs P.D. Zhang X.K. Int. J. Cancer. 1998; 75: 88-95Crossref PubMed Scopus (79) Google Scholar, 22.Si S.P. Lee X. Tsou H.C. Buchsbaum R. Tibaduiza E. Peacocke M. Cell Exp. Res. 1996; 223: 102-111Crossref PubMed Scopus (50) Google Scholar, 23.Seewaldt V. Johnson B.S. Parker M.B. Collins S.J. Swisshelm K. Cell Growth Differ. 1995; 6: 1077-1088PubMed Google Scholar, 24.Liu Y. Lee M-O. Wang H-G. Li Y. Hashimoto Y. Klaus M. Reed J.C. Zhang X-K. Mol. Cell. Biol. 1996; 16: 1138-1149Crossref PubMed Scopus (330) Google Scholar, 25.Weber E. Ravi R.K. Knudsen E.S. Williams J.R. Dillehay L.E. Nelkin B.D. Kalemkerian G.P. Feramisco J.R. Mabry M. Int. J. Cancer. 1999; 80: 935-943Crossref PubMed Scopus (38) Google Scholar), and restoration of RARβ expression in RARβ-negative lung cancer cell lines inhibited tumorigenicity in nude mice (26.Houle B. Rochette-Egly C. Bradley W.E.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 985-989Crossref PubMed Scopus (283) Google Scholar). RARβ induction by retinoids has been demonstrated in vivo(16.Berg W.J. Nanus D.M. Leung A. Brown K.T. Hutchinson B. Mazumdar M. Xu X.C. Lotan R. Reuter V.E. Motzer R.J. Clin. Cancer Res. 1999; 5: 1671-1675PubMed Google Scholar, 27.Lotan R. Xu X.C. Lippman S.M. Ro J.Y. Lee J.S. Lee J.J. Hong W.K. N. Engl. J. Med. 1995; 332: 1405-1410Crossref PubMed Scopus (378) Google Scholar, 28.Xu X.C. Lee J.S. Lee J.J. Morice R.C. Liu X. Lippman S.M. Hong W.K. Lotan R. J. Natl. Cancer Inst. 1999; 91: 1317-1321Crossref PubMed Scopus (71) Google Scholar). However, it is not clear to what extent if any the induced RARβ contributes to the response to growth-inhibitory effects of retinoids or whether it plays no role in the overall response to retinoids. To further understand the importance of RARβ induction, we constructed and transfected a retroviral expression vector harboring antisense RARβ2 into the H157 human lung squamous cell carcinoma cell line, which expresses RARβ only after ATRA treatment, and found that blocking RARβ induction decreases cell sensitivity to retinoids. ATRA and Ch55 were kindly provided by Dr. Werner Bollag (F. Hoffmann-La Roche, Basel, Switzerland) and Dr. Koichi Shudo (Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan), respectively. They were dissolved in dimethylsulfoxide at a concentration of 10 mm under N2 and stored in the dark at −80 °C. Stock solutions were diluted to the desired concentrations with growth medium just prior to use. The H157 lung squamous cell carcinoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in monolayer culture in a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium containing 5% fetal calf serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) at 37 °C. The retroviral vector LNSX, obtained from Dr. D. Miller (Fred Hutchinson Cancer Research Center, Seattle, WA) (29.Miller A.D. Rosman G. BioTechniques. 1989; 7: 980-990PubMed Google Scholar), was used in this study. We introduced into the LNSX vector multiple copies of hRARβ2 cDNA fragments corresponding to the initial site of RARβ2 translation in an antisense orientation by ligating 385-base pair BamHI/SphI cDNA fragments released from pSG5-RARβ plasmid (30.Brand N. Petkovitch M. Krust A. Chambon P. De The H. Marchio M. Tiollais P. Dejean A. Nature. 1988; 332: 850-853Crossref PubMed Scopus (812) Google Scholar) into unique HindIII,ClaI, and BamHI sites through blunt end ligation with T4 DNA ligase, respectively. We obtained a series of vectors harboring different number of copies of RARβ2 cDNA inserts in an antisense orientation as identified by sequencing and enzymatic digestions. Vectors harboring one, two, three, four, five, and six antisense RARβ2 inserts were designated as LNASβ, LNASβII, LNASβIII, LNASβIV, LNASβV, and LNASβVI, respectively. The LNASβVI shown in Fig. 1 was used in all subsequent experiments. LNASβVI or LNSX as a vector control was transfected directly into PA317 amphotropic packaging cell line by the calcium phosphate precipitation method (31.Shambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.30-16.36Google Scholar). The transfected cells were cultured in G418 (500 μg/ml), and individual clones of resistant cells were picked up using cloning cylinders after 14 days and expanded. The supernatants from these retroviral producers were titered on thymidine kinase-negative NIH3T3 target cells as described previously (31.Shambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.30-16.36Google Scholar). Supernatant from the highest titer producer clone for either LNASβVI or LNSX (up to 5 × 105/ml) was used in all subsequent experiments. H157 cells were plated at 1:10 split in 6-cm diameter tissue culture dishes (Corning). On the second day, the medium was replaced with 2 ml of fresh medium containing different amounts of viral supernatant and 8 μg/ml of polybrene, and 2 h later, 2 ml of fresh medium was added. Cells were split at 1:10 into 10-cm diameter dishes and selected after 24 h using G418 (1000 μg/ml)-containing medium. Surviving clones were isolated after 12–15 days using cloning cylinders. The rest of the clones from different dishes were pooled as pool transfectants. Transfectants were expanded and maintained under continuous G418 selection at 500 μg/ml. Total cellular RNA purification and Northern blotting were performed as described previously (32.Sun S-Y. Yue P. Dawson M.I. Shroot B. Michel S. Lamph W.W. Heyman R.A. Teng M. Chandraratna R.A.S. Shudo K. Hong W.K. Lotan R. Cancer Res. 1997; 57: 4931-4939PubMed Google Scholar). Twenty micrograms of RNA was loaded per lane. The 385-base pair BamHI/SphI fragment of RARβ from pSG5 expression vector harboring human RARβ2 cDNA (30.Brand N. Petkovitch M. Krust A. Chambon P. De The H. Marchio M. Tiollais P. Dejean A. Nature. 1988; 332: 850-853Crossref PubMed Scopus (812) Google Scholar), obtained from Dr. Pierre Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, C.U. de Strasbourg, France), was used as a probe for Northern blotting. The 340-base pairEcoRI/XbaI cDNA for glyceraldehyde-3-phosphate dehydrogenase, purchased from Ambion (Austin, TX), was used as a control of RNA loading. Nuclear extracts were prepared from H157 transfectants by a method described by Pollocket al. (33.Pollock R. Treisman R. Nucleic Acids Res. 1990; 18: 6197-6204Crossref PubMed Scopus (287) Google Scholar). Eighty micrograms of protein was electrophoresed through a 10% polyacrylamide slab gel and transferred to nitrocellulose membranes (Bio-Rad) by electroblotting. The blot was immersed in blocking solution (15% nonfat milk in phosphate-buffered saline) at room temperature for 1 h and then incubated with a 1:50 dilution of rabbit anti-RARβ polyclonal antiserum (generously provided by Dr. P. Chambon) in blocking solution overnight at 4 °C with agitation. The blot was washed four times with blocking solution and incubated with 125I-labeled goat anti-rabbit IgG at a 1:2500 dilution in blocking solution at room temperature for 1 h. The blot was then washed four more times with blocking solution containing 0.1% Tween 20 and exposed to x-ray film at −80 °C for 2–5 days. Nuclear extracts from H157 transfectants used in this assay were the same as those prepared for Western blotting. The following synthetic oligonucleotides were labeled with [γ-32P]ATP (4000 Ci/mol; ICN Rediochemicals, Irvine, CA) using T4 polynucleotide kinase: wild-type RARE-β2, 5′-TCGAGGGTAGGGTTCACCGAAAGTTCACTCG-3′; mutant RARE, 5′-TCGAGGGTAGGcTTacCCGAAAGTTCA-3′. Nuclear extracts were preincubated with 2 μg of poly(dI-dC)·poly(dI-dC) for 15 min on ice and then incubated with labeled RARE-β2 (approximately 10,000 cpm) for 15 min on ice in the presence of 10 mm Tris-HCl, pH 7.5, 10 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 5 mm MgCl2, and 20% glycerol. For the gel supershift assay, receptor-specific monoclonal antibodies of RARs and RXRs (provided by Dr. P. Chambon) (0.3 μl) were added to the reaction mixture. The reaction mixtures (10 μl) were electrophoresed on a 5% polyacrylamide gel containing 25 mm Tris-HCl, pH 8.5, 192 mm glycine, and 1 mm EDTA, dried, and exposed to x-ray film. The (RARE)3-TK-Luc reporter plasmid, which contains three direct repeats of RARE from the P2 promoter region of the human RARβ2 gene (from −59 to −33) connected to herpes simplex virus thymidine kinase promoter, and TK-LUC control reporter plasmids were provided by Dr. R. A. Heyman (Ligand Pharmaceuticals, San Diego, CA). The AP1-TK-Luc reporter plasmid, which contains the luciferase gene controlled by a promoter fragment of the collagenase gene (−74 to −63) harboring a consensus AP-1 binding site (TGAGTCA) connected to the thymidine kinase promoter, was obtained from Dr. J. Kurie (University of Texas M. D. Anderson Cancer Center, Houston, TX). pCH110 plasmid encoding β-galactosidase was purchased from Amersham Pharmacia Biotech. The plasmid purification, transfection, and luciferase activity assay procedures were the same as described previously (34.Sun S-Y. Yue P. Shroot B. Hong W.K. Lotan R. J. Cell. Physiol. 1997; 173: 279-284Crossref PubMed Scopus (84) Google Scholar). The effects of retinoids on the growth of different transfectants were evaluated by the sulforhodamine B assay as described previously (32.Sun S-Y. Yue P. Dawson M.I. Shroot B. Michel S. Lamph W.W. Heyman R.A. Teng M. Chandraratna R.A.S. Shudo K. Hong W.K. Lotan R. Cancer Res. 1997; 57: 4931-4939PubMed Google Scholar). Colony formation assay was performed as follows. Cells were plated at a density of 2000 cells in 6-cm diameter tissue culture dishes (Corning) and treated on the next day with retinoids. The medium was replaced with fresh medium containing retinoids every 3 days. After a 12-day treatment, colonies were stained with 0.5% methylene blue in 70% ethanol and counted. H157 cells express undetectable levels of RARβ (by Northern blotting) but a high level of induced RARβ after treatment with ATRA or other retinoids. This cell line was infected with the LNASβVI retroviral vector (Fig.1), and individual G418-resistant clones were isolated. These clones were designated H157-LNASβ. Vector-only control clones were obtained by infecting the same cell line with the retroviral vector LNSX, which does not contain the antisense RARβ sequence (Fig. 1). These control clones were designated H157-LNSX. Northern blotting was performed on transfectants to determine the expression level of antisense RARβ mRNA. The 4.3-kilobase antisense mRNA controlled by the SV40 promoter and the antisense mRNA larger than 5 kilobases driven by the long terminal repeat promoter were detected using a 385-base pairBamHI/SphI RARβ2 cDNA fragment as a probe in all of the LNASβVI-infected clones but not in the LNSX-infected control cells (Fig. 2 A). Western blot analysis using an RARβ-specific polyclonal antiserum (35.Rochette-Egly C. Gaub M.P. Lutz Y. Ali S. Scheuer L. Chambon P. Mol. Endocrinol. 1992; 6: 2197-2209Crossref PubMed Scopus (69) Google Scholar) revealed high levels of RARβ protein in ATRA-treated LNSX-P cells (vector only) (Fig. 2 B). In contrast, ATRA induced less RARβ protein in most antisense RARβ-transfected clones, indicating that antisense RARβ expression was effective in the cells. To examine the effect of stable transfection of antisense RARβ on the formation of complexes between nuclear receptors and RARE, we performed gel shift and supershift assays, which are very sensitive methods for detecting functional RAR proteins. Fig. 3 A shows that a weak signal of shifted band was observed with extract from untreated LNSX-P cells. This band was decreased in untreated LNASβ-9 cells. Antibodies against RARα and RARβ failed to supershift complexes in untreated LNSX-P or LNASβ-9 cell extracts. However, antibodies against RARγ and RXRs did supershift complexes in both cell types. Treatment with ATRA increased markedly the amount of shifted complexes in LNSX-P cells, but only a very small increase in the amount of shifted complex was observed in ATRA-treated LNASβ-9 cells. Importantly, antibodies against RARβ supershifted a major complex in the ATRA-treated LNSX-P cells but not at all in the LNASβ-9 cells (Fig. 3 A). These results clearly show that the antisense RARβ transfectants do not express functional RARβ even after ATRA treatment, whereas the vector controls exhibit a large increase in functional RARβ. The band specificity was assured by the fact that they were competed out by a 100-fold molar excess of nonradioactive wild-type RARE but not by the same amount of nonradioactive mutated RARE (data not shown). Furthermore, we found that induction of RARE transcriptional activation by ATRA was markedly decreased in antisense RARβ-transfected cells in comparison with LNSX-transfected cells. As shown in Fig. 3 B, RARE transactivation was increased 20–30-fold in both LNSX-transfected clones but only 8-fold in two LNASβ transfectants. One possible explanation for the residual transactivation capacity in LNASβ-transfected cells is the presence of other RARs and RXRs. Fig.3 A shows that RARγ and at least one RXR protein and traces of RARβ can be supershifted from complexes with RARE in LNASβ-9 cells after ATRA treatment. It is plausible to suggest that these receptors are responsible for the activation of RARE-Luc reporter in the antisense RARβ transfectants. The partial suppression of RARE transactivation in the LNASβ-transfected cells appeared to be specific for RARE, because the transactivation of another reporter construct mediated by AP-1 binding was not suppressed in these transfectants (Fig. 3 C). We next compared the responsiveness of LNASβVI-transfected clones to retinoid treatment with that of LNSX-transfected cells. Fig.4 A shows the effects of different retinoids on the population growth of LNASβVI-transfected and LNSX-transfected cells. Four LNASβVI-transfected clones exhibited much lower sensitivity to ATRA treatment than three LNSX-transfected cells did. ATRA at concentrations of 1 and 2.5 μm caused 20–40% growth inhibition in LNSX-transfected cells but less than 20% growth inhibition in all LNASβ VI-transfected clones (Fig.4 A). The synthetic retinoid Ch55 is a pan-RAR-selective agonist and has better receptor binding affinities, especially to RARβ, than ATRA, but it is much more active than ATRA in inhibiting the growth of H157 and other lung cancer cells (32.Sun S-Y. Yue P. Dawson M.I. Shroot B. Michel S. Lamph W.W. Heyman R.A. Teng M. Chandraratna R.A.S. Shudo K. Hong W.K. Lotan R. Cancer Res. 1997; 57: 4931-4939PubMed Google Scholar). To better demonstrate the low responsiveness of antisense RARβ-transfected cells, we further examined the effects of Ch55 on the growth of different LNSX-transfected cells and LNASβVI-transfected clones. As shown in Fig. 4 A, Ch55 caused dose- and time-dependent growth-inhibitory effects in all LNSX-transfected cells. However, the responsiveness of all LNASβVI-transfected cells to Ch55 was lower than that of LNSX-transfected cells; Ch55 at 1 and 2.5 μm inhibited cell growth by 40–60 and 65–80%, respectively in LNSX-transfected cells but by less than 20 and 35%, respectively, in LNASβVI-transfected cells after a 6-day treatment (Fig.4 A). In addition, we analyzed the effects of ATRA and Ch55 on the colony formation of different transfectants. Similar to the population growth inhibition results, all LNASβVI-transfected cells exhibited less sensitivity (about 50%) than LNSX-transfected cells to inhibition of colony formation by ATRA and Ch55 (Fig. 4 B). These results clearly show that blockage of RARβ induction decreased the cell responsiveness to retinoids. It has been suggested that RARβ may play a role as a tumor suppressor. This hypothesis was based on the observation that RARβ levels decreased in a variety of tumor cell lines including lung carcinomas (9.Gebert J.F. Moghal N. Frangioni J.V. Sugarbaker D.J. Neel B.G. Oncogene. 1991; 6: 1859-1868PubMed Google Scholar, 10.Houle B. Ledue F. Bradley W.E.C. Choromosomes Cancer. 1991; 3: 358-366Crossref PubMed Scopus (86) Google Scholar, 11.Hu L. Crowe D.L. Rheinwald J.G. Chambon P. Gudas L. Cancer Res. 1991; 51: 3972-3981PubMed Google Scholar, 12.Nervi C. Volleberg T.M. Gerore M.D. Zelent A. Chambon P. Jetten A.M. Exp. Cell. Res. 1991; 195: 163-170Crossref PubMed Scopus (101) Google Scholar, 13.Swisshelm, K., Ryan, K., Lee, X., Tsou, H. C., Beacocke, M., and Sager, R. Cell Growth Differ. 5, 133–141Google Scholar) as well as in premalignant and malignant epithelial tissues in vivo (14.Xu X-C. Ro J.Y. Lee J.S. Shin D.M. Hong W.K. Lotan R. Cancer Res. 1994; 54: 3580-3587PubMed Google Scholar, 15.Xu X.C. Sozzi G. Lee J.S. Lee J.J. Pastorino U. Pilotti S. Kurie J.M. Hong W.K. Lotan R. J. Natl. Cancer Inst. 1997; 89: 624-629Crossref PubMed Scopus (199) Google Scholar, 16.Berg W.J. Nanus D.M. Leung A. Brown K.T. Hutchinson B. Mazumdar M. Xu X.C. Lotan R. Reuter V.E. Motzer R.J. Clin. Cancer Res. 1999; 5: 1671-1675PubMed Google Scholar, 17.Xu X.C. Liu X. Tahara E. Lippman S.M. Lotan R. Cancer Res. 1999; 59: 2477-2483PubMed Google Scholar, 27.Lotan R. Xu X.C. Lippman S.M. Ro J.Y. Lee J.S. Lee J.J. Hong W.K. N. Engl. J. Med. 1995; 332: 1405-1410Crossref PubMed Scopus (378) Google Scholar, 28.Xu X.C. Lee J.S. Lee J.J. Morice R.C. Liu X. Lippman S.M. Hong W.K. Lotan R. J. Natl. Cancer Inst. 1999; 91: 1317-1321Crossref PubMed Scopus (71) Google Scholar). It has been shown that RARβ expression, which is suppressed at early stages of head and neck and lung carcinogenesis, can be induced by retinoid treatment (27.Lotan R. Xu X.C. Lippman S.M. Ro J.Y. Lee J.S. Lee J.J. Hong W.K. N. Engl. J. Med. 1995; 332: 1405-1410Crossref PubMed Scopus (378) Google Scholar, 28.Xu X.C. Lee J.S. Lee J.J. Morice R.C. Liu X. Lippman S.M. Hong W.K. Lotan R. J. Natl. 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However, it was not clear whether this induction is important for the overall effect of ATRA on cell phenotype, namely whether RARβ increase plays a role in growth inhibition. Our study has addressed this question by blocking RARβ induction by ATRA using a retroviral vector harboring antisense RARβ. The results show clearly that the antisense vector was effective in decreasing the level of the RARβ protein and that this was accompanied by a decrease in the response of the transfected cells to two RAR-selective retinoids (ATRA and Ch55). Thus, our data support the conclusion that the induction of RARβ by retinoids may be an early step in the cascade of events leading to growth inhibition. We used H157 lung carcinoma cells, which do not express RARβ constitutively but can be induced to express this receptor after ATRA treatment. These cells were, therefore, a good system to explore the role of RARβ induction in mediating the growth-inhibitory effects of retinoids. We used for the first time antisense RARβ to block induction of RARβ by retinoids. This approach does not involve severe selective pressure as isolation of transfectants expressing sense RARβ would, because the H157 cells do not express endogenous sense RARβ. Therefore, the expression of exogenous antisense RARβ should not affect them until they are treated with ATRA. Our present study provides the first direct evidence supporting the important role of RARβ induction in mediating growth-inhibitory effects of retinoids in human cancer cells. Our in vitro finding that induction of RARβ by retinoids is important for growth inhibition may explain the correlation that we had established previously in vivobetween the induction of RARβ and clinical response in patients with oral premalignant lesions (27.Lotan R. Xu X.C. Lippman S.M. Ro J.Y. Lee J.S. Lee J.J. Hong W.K. N. Engl. J. 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In addition, the cells were somewhat compromised in their ability to undergo differentiation by ATRA and showed a markedly lower induction of several ATRA-responsive genes (38.Faria T.N. Mendelsohn C. Chambon P. Gudas L.J. J. Biol. Chem. 1999; 274: 26783-26788Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These results suggest that RARβ is required for ATRA-induced growth arrest in F9 cells. Severe defects in RARβ expression have been observed in cells where ATRA treatment cannot induce RARβ expression (39.Geradts J. Chen J.Y. Russell E.K. Yankaskas J.R. Nieves L. Minna J.D. Cell Growth Differ. 1993; 4: 799-809PubMed Google Scholar, 40.Zhang X.K. Liu Y. Lee M.O. Pfahl M. Cancer Res. 1994; 54: 5663-5669PubMed Google Scholar, 41.Moghal N. Neel B.G. Mol. Cell. Biol. 1995; 15: 3945-3959Crossref PubMed Scopus (68) Google Scholar). Previous studies have demonstrated that transfection of RARβ sense expression vectors into cells deficient in this receptor resulted in restoration of growth inhibition, apoptosis, and decreased tumorigenicity by retinoids (21.Li Y. Dawson M.I. Agadir A. Lee M.O. Jong L. Hobbs P.D. Zhang X.K. Int. J. Cancer. 1998; 75: 88-95Crossref PubMed Scopus (79) Google Scholar, 22.Si S.P. Lee X. Tsou H.C. Buchsbaum R. Tibaduiza E. Peacocke M. Cell Exp. Res. 1996; 223: 102-111Crossref PubMed Scopus (50) Google Scholar, 23.Seewaldt V. Johnson B.S. Parker M.B. Collins S.J. Swisshelm K. Cell Growth Differ. 1995; 6: 1077-1088PubMed Google Scholar, 24.Liu Y. Lee M-O. Wang H-G. Li Y. Hashimoto Y. Klaus M. Reed J.C. Zhang X-K. Mol. Cell. Biol. 1996; 16: 1138-1149Crossref PubMed Scopus (330) Google Scholar, 25.Weber E. Ravi R.K. Knudsen E.S. Williams J.R. Dillehay L.E. Nelkin B.D. Kalemkerian G.P. Feramisco J.R. Mabry M. Int. J. Cancer. 1999; 80: 935-943Crossref PubMed Scopus (38) Google Scholar). Thus, both induction of endogenous RARβ by retinoids (this study) and expression of exogenous RARβ (studies by others) (21-25) may lead to a similar result, namely growth inhibition by retinoids. However, the role of induction of endogenous RARβ by retinoids may be more relevant to explain ongoing clinical chemoprevention trials with retinoids. We thank Drs. W. Bollag and K. Shudo for providing the retinoids used in this study. We are also grateful to Drs. D. Miller, P. Chambon, and R. A. Heyman for providing the plasmids and antibodies used in this study.
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