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Retinoic Acid Receptor α Mediates Growth Inhibition by Retinoids in Human Colon Carcinoma HT29 Cells
Biochemical and Biophysical Research Communications 261, 572–577 (1999) Article ID bbrc.1999.1086, available online at http://www.idealibrary.com on
Retinoic Acid Receptor a Mediates Growth Inhibition by Retinoids in Human Colon Carcinoma HT29 Cells Barbara Nicke,* Astrid Kaiser,* Bertram Wiedenmann,† Ernst-Otto Riecken,* and Stefan Rosewicz* ,† ,1 *Department of Gastroenterology, Klinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany; and †Medizinische Klinik m.S. Hepatologie/Gastroenterologie, Virchow-Klinikum, Charite´, Augustenburger Platz 1, 13353 Berlin, Germany
Received July 8, 1999
Although retinoids have been suggested to inhibit chemically induced colon carcinogenesis, the molecular mechanisms underlying retinoid-mediated growth regulation in colon carcinoma cells are unknown. Therefore, we investigated the biological effects of retinoids on growth in HT29 colon carcinoma cells. All-trans retinoic acid (ATRA) treatment of HT29 cells resulted in a profound inhibition of anchorageindependent growth without biochemical or morphological evidence for induction of differentiation. Treatment with the selective RARa agonist Ro 40-6055 completely mimicked the effects of ATRA on growth and transactivation of a bRAREx2-luciferase reporter construct, while RARb- and g-specific analogues were ineffective. Furthermore, ATRA-regulated growth and transactivation could be completely blocked by a RARa-selective receptor antagonist. Thus, ATRA potently inhibits anchorage-independent growth in HT29 cells and this effect is mainly if not exclusively mediated by the retinoic acid receptor a. © 1999 Academic Press
Natural and synthetic derivatives of vitamin A, which are biochemically summarized as retinoids, have been shown to inhibit growth and induce cellular differentiation in a wide variety of epithelial cells in vitro and in vivo (for review see 1). Subsequent clinical trials, which were either focused on chemoprevention or treatment of frank malignancies demonstrated con1 To whom correspondence should be addressed at Medizinische Klinik m.S., Hepatologie/Gastroenterologie, Virchow-Klinikum, Charite´, Augustenburger Platz 1, 13353 Berlin, Germany. Fax: 004930-45053905. E-mail: [email protected]. Abbreviations used: RAR a, retinoic acid receptor alpha; RAR, retinoic acid receptors; RXR, retinoid X receptors; ATRA, all-trans retinoic acid; RT-PCR, reverse-transcriptase polymerase chain reaction; RARE, retinoic acid responsive element; CEA, carcinoembryonic antigen; AP, alkaline phosphatase; NaB, sodium butyrate.
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
siderable therapeutic efficacy of retinoids in some cancers (cervical cancer, renal cell carcinoma, head and neck cancer), whereas others (malignant melanoma, lung cancer) proved to be unresponsive to retinoid therapy (2, 3). Experimental data regarding the potential chemopreventive effects of retinoids in colon carcinogenesis have revealed conflicting results. Whereas earlier studies failed to reproducibly demonstrate a chemopreventive effect of retinoids on chemically induced colon carcinogenesis (4, 5) more recent studies revealed promising activity of retinoids in preventing intestinal neoplasia (6 –9). These differences might be explained by the chemopreventive use of different retinoids as well as the heterogeneous models utilized to study chemically induced carcinogenesis (4 – 8). Much less is known about the biological effects of retinoids in malignant transformed colon carcinoma cells. Two studies have shown that retinoids inhibit mucosal ornithine decarboxylase activity in the rectum of colon cancer patients (10) and carcinogen induced DNA synthesis in rat colon carcinoma cells (8), suggesting that retinoids might also exert growth inhibitory actions in colon epithelial cells which have undergone malignant transformation. Thus, the current experimental evidence suggests that retinoids might have antiproliferative potential in a chemopreventive setting as well as treatment of colon carcinoma. However, no data are currently available as to the molecular mechanisms underlying retinoid action in colonic epithelial cells. Recently much has been learned about the molecular mechanisms by which retinoids modulate such pleiotropic effects as tumor cell proliferation and induction of cellular differentiation. The biological effects of retinoids are mediated by two families of nuclear retinoic acid receptors, each consisting of three receptor subtypes a, b and g: the retinoic acid receptors (RAR) and the retinoid X receptors (RXR). In addition, each RAR gene generates multiple isoforms by either alternative
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splicing of exons or differential use of internal promoters (11). Both receptor families act as ligand-activated transcription factors that control gene transcription initiated from promoters of retinoid regulated genes by interacting with cis-acting DNA elements, the so called RAREs (retinoic acid responsive elements) (reviewed in 11, 12). The naturally occurring ligand for RARs in vivo is all-trans retinoic acid (ATRA), while RXR preferentially bind to 9-cis retinoic acid (9-cis RA) (13). The large spectrum of biological processes affected by retinoic acid, as well as the tissue specific restricted expression of RAR/RXR subtypes during embryogenesis, and in the adult organism suggests that each RAR and RXR subtype exerts a unique biological function, and thereby determines whether a given cell or tissue is retinoid sensitive or resistant (11). Therefore, detailed knowledge of the cell-type specific expression pattern for each receptor subtype might provide a rationale why certain malignancies are retinoid responsive while others are not. Based on these observations, the current study was designed to evaluate the biological effects of retinoids on growth and differentiation in the human colon carcinoma cell line HT29 and to further determine the retinoid receptor subtype involved in mediating retinoid action in colon carcinoma cells. MATERIALS AND METHODS Materials. Human colon carcinoma HT29 cells were obtained from the American Type Tissue Culture Collection. Dulbecco’s modified Eagle medium was obtained from Gibco (Berlin, Germany), and fetal calf serum (FCS) from Biochrom (Berlin, Germany). All retinoids were kindly provided by Hoffman–LaRoche (Basel, Switzerland). [a- 32P]dCTP (6000 Ci/mmol) was obtained from DuPont (Bad Homburg, Germany). Random priming labeling kit was obtained from Amersham (Braunschweig, Germany); random hexamer primers and M-MLV were from Bethesda Research Laboratories (BRL, Bethesda, MD); Taq DNA polymerase from Promega (Heidelberg, Germany). Cell culture. HT29 cells were grown in DMEM supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 U/ml). The cells were kept under 95% air and 5% CO 2 at 37°C. Human tumor clonogenic assay (HTCA). For evaluation of clonal growth of HT29 cells we used a methylcellulose human tumor clonogenic assay (HTCA) exactly as previously described (14). Only colonies containing more than twenty cells were counted. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. RNA from HT29 cells was isolated exactly as previously described (14). Reverse transcription of was performed using 1 mg total RNA, 100 pM random hexamer primer, 1 mM DTT, 6 mM MgCl 2, 500 mM of each dNTP, 20 units RNAsin (Promega, Heidelberg, Germany), and MML-V reverse transcriptase. This RT mixture was used directly as a template for PCR in a 1:20 dilution. The reaction was carried out in 10 mM Tris–HCl buffer (pH 9.0) containing 50 mM KCl, 0.01% Triton X-100, 1 mM MgCl 2, 200 mM of each dNTP, 50 pM of each primer, and 2.5 U Taq DNA polymerase in a final volume of 50 ml. Amplification conditions for 35 cycles were carried out as follows: denaturation for 30 s at 92°C, annealing at 60°C for 90 s, and extension for 90 s at 72°C. Final extension was carried out for an additional 10 min after completion of all 35 cycles.
Southern blot analysis. After amplification 10 ml of each RT-PCR were subjected to electrophoresis on a 1.5% agarose gel. The samples were denatured in 0.5 N NaOH and 1.5 M NaCl for 30 min and transferred to Hybond N 1 membranes (Amersham, Braunschweig, Germany). After blotting the membranes were neutralized in 50 mM NaH 2PO 4, pH 6.5, for 20 min and then baked at 80°C for 2 h. Hybridization was carried out using the respective receptor cDNA fragments. The cDNA inserts were radioactively labeled using the random primer labeling kit following the instructions provided by the supplier. Unincorporated nucleotides were removed by passing the reaction sample over a Sephadex G 50 column. The following plasmids were used: pSG5:RARa (14), pSG5:RARb (15), pSG5:RARg (14), pSG5:RXRa (16), pT11:RXRb (16), and pSG5:RXRg (16). Hybridization was carried out in a standard hybridization buffer containing 50% formamide at 42°C. After hybridization filters were washed and then exposed to X-ray films for 30 – 60 min. Differentiation assays. HT29 cells were incubated with ATRA, vehicle, or sodium butyrate. After 96 h supernatant was collected and cells were harvested and lysed in a lysis buffer (20 mM Tris, 10 mM EGTA, and 250 mM sucrose) by homogenization. Protein content was measured using Bio-Rad protein assay. Alkaline phosphatase (AP) activity was measured in aliquots ranging from 20 to 200 mg of protein using a colorimetric kit (Sigma, Deisenhofen, Germany) following the instructions of the supplier (17). The concentration of carcinoembryonic antigen (CEA) was measured in 1 ml of collected supernatant using an automated microparticle enzyme immunoassay (Abbott Laboratories, North Chicago, IL) following the instructions provided by the supplier. Transactivation assays. HT29 cells were transiently transfected with the pTK:bREx2-luc reporter construct (18) using the Lipofectamine reagent (Gibco, BRL, Eggenstein, Germany). After 12 h the indicated concentrations of retinoids or vehicle were added and cells were incubated for an additional 24 h. Luciferase activity of cell lysates was then determined using a luciferase assay system (Promega, Heidelberg, Germany). Luciferase activity was expressed as fold induction of vehicle treated control cells. Statistics. Results were evaluated statistically by analysis of variance (ANOVA) followed by a multiple contrast test. P values , 0.05 were considered to be significant.
RESULTS Effects of retinoids on growth and differentiation in HT29 cells. Initially, we investigated the effects of various naturally occurring retinoids on anchorageindependent growth in HT29 cells. All three retinoids tested revealed a potent and dose-dependent growth inhibition (Fig. 1). However, at lower, therapeutically relevant concentrations (,100 nM) only all-trans and its stereoisomer 13-cis retinoic acid demonstrated significant growth inhibition, whereas 9-cis retinoic acid was ineffective. On an equimolar basis maximal growth inhibition at 10 mM revealed the following gradient of biological potency: all-trans (9.7 6 3.4% of control) 5 13-cis (13.4 6 2.9% of control) . 9-cis retinoic acid (25.4 6 2.3% of control). We next investigated whether the retinoid mediated growth inhibition was paralleled by a concomitant induction of cellular differentiation in HT29 cells. Two biochemical markers closely associated with a more differentiated phenotype were examined: alkaline phosphatase (AP) and carcinoembryonic antigen (CEA). For these experiments we used sodium butyrate
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FIG. 1. Effects of retinoid analogues on anchorage-independent growth. HT29 cells were examined in the human tumor clonogenic assay with the indicated final concentrations of each retinoid analogue. After 10 days the number of colonies was evaluated and expressed as percentage of vehicle treated controls. Shown are the mean 6 SEM of three independent experiments, each performed in triplicate. *P , 0.05 versus controls.
(NaB) as a positive control, which has been repeatedly demonstrated to inhibit growth and induce cellular differentiation in HT29 cells (19, 20). Incubation of HT29 cells with NaB (5 mM) for 96 h resulted in a pronounced increase of cellular AP activity, whereas maximal concentrations of ATRA (10 mM) had no effect (Fig. 2). Similarly, NaB treatment resulted in an about six-fold increase of CEA concentrations in HT29 supernatants (325 6 8 mg/ml versus 54 6 5 mg/ml in controls) whereas ATRA had no significant effect (Fig. 2). Furthermore, HT29 cell morphology remained unchanged under ATRA treatment for up to five days (data not shown). Based on these experiments we have currently
FIG. 2. Effects of all-trans retinoic acid on HT29 cell differentiation. HT29 cells were incubated with vehicle (controls), 10 mM all-trans retinoic acid (ATRA), or sodium butyrate (5 mM) for 96 h. Thereafter, cellular AP activity or CEA concentrations in the supernatant were determined. Shown are the mean 6 SEM of three independent experiments, each performed in triplicate. *P , 0.05 versus vehicle treated controls.
FIG. 3. Expression of nuclear retinoid acid receptors in HT29 cells. RT-PCR analysis was performed for HT29 cells using receptor specific oligonucleotide primers (see Ref. 14). The respective receptor subtype cDNA was used as a positive internal control. RT-PCR with (1) or without prior reverse transcription (2) (which served as a negative internal control) was performed as outlined in Methods. Aliquots from each RT-PCR product were electrophoresed through a 1% agarose gel (ethidium bromide staining, shown in the upper panel) and then subjected to Southern blot analysis using the respective cDNA probes for the RARs/RXRs to ensure specificity of the amplification product (lower panel).
no evidence that ATRA treatment of HT29 cells results in induction of cellular differentiation. Expression of retinoid receptors in HT29 cells. As an initial attempt to understand the molecular mechanisms involved in retinoid mediated growth inhibition of HT29 cells, we first had to characterize the expression pattern of nuclear retinoid receptors. Using a panel of retinoid receptor subtype specific primers in RT-PCR analysis, we detected mRNA transcripts for RAR a, b, g, and RXR a, and b (Fig. 3). In contrast, no amplification signal could be obtained for the RXR g. PCR amplification without prior reverse transcription, which served as an internal negative control, repeatedly failed to demonstrate an amplification signal. The specificity of the amplification products was confirmed by subsequent hybridization of the RT-PCR products using the respective receptor subtype specific cDNA probes in Southern blots (Fig. 3). The retinoic acid receptor a mediates growth inhibition in HT29 cells. We next investigated which retinoid receptor subtype mediates the growth inhibitory effects in HT29 cells. Based on our growth studies only ATRA and 13-cis retinoic acid were growth inhibitory at therapeutically relevant concentrations (#100 nM), whereas 9-cis RA was ineffective at these concentrations (Fig. 1). While 9-cis RA binds to RARs as well as RXRs, ATRA does not bind to RXRs, suggesting that
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(22) in growth and transactivation assays. The antiproliferative effects of 10 nM ATRA were blocked in a dose-dependent manner by the RARa antagonist Ro 41-5253 (Fig. 5A). Half-maximal inhibition was observed at a concentration of 100 nM antagonist (59 6 8% of ATRA-induced growth inhibition) and maximal inhibition occurred at 10 mM (6 6 1% of ATRA–induced growth inhibition. Very similar results were obtained when we examined the effects of the RARa antagonist on ATRA mediated transactivation in HT29 cells (Fig. 5B) with a complete inhibition of ATRA stimulated transactivation at a concentration of 10 mM of RARa receptor antagonist. The RARa antagonist alone had no significant effect in terms of growth inhibition or transactivation in a concentration range from 1 nM to 10 mM (data not shown). DISCUSSION In vivo experiments of chemically induced colon carcinogenesis indicated that retinoids might have chemopreventive effects on the development of colon cancer FIG. 4. Effects of RAR subtype specific retinoids on anchorageindependent growth and transactivation. (A) HT29 cells were examined in HTCA with the indicated final concentrations of each retinoid analogue. After 10 days the number of colonies was evaluated and expressed as percentage of vehicle treated controls. Shown are the mean 6 SEM of three independent experiments, each performed in triplicate. (B) HT29 cells were transiently transfected with the pTK:bREx2-luc reporter construct. After incubation with the indicated concentrations of each retinoid analogue for 24 h, luciferase activity was determined and expressed as fold increase over basal (luciferase activity in the absence of retinoids). Shown are the mean 6 SEM of six independent experiments, each performed in triplicate wells. Receptor subtype specificity of the analogues is shown in parenthesis.
the antiproliferative effect of ATRA is mediated by members of the RAR family. We therefore used a panel of retinoic acid receptor subtype specific analogues in growth and transactivation assays. Receptor subtype specificity of these analogues has recently been documented (21, 22). Of the three RAR subtype specific retinoids tested, only the RARa selective analogue Ro 40-6055 resulted in a significant dose-dependent growth inhibition in HT29 cells, which was quantitatively comparable to the antiproliferative effects of ATRA (Fig. 4A). In good agreement with the antiproliferative characteristics, only the RARa selective retinoid Ro 40-6055 resulted in a significant dosedependent transactivation of the pTK:bREx2-luc reporter construct transiently transfected into HT29 cells (Fig. 4B). The RARb (Ro 48-2249) and RARg (Ro 44-4753) specific retinoids were ineffective in terms of growth inhibition and transactivation (Fig. 4). To further substantiate the role of RARa in mediating the antiproliferative effects of ATRA in HT29 cells, we used the RARa specific receptor antagonist Ro 41-5253
FIG. 5. The effects of the RARa antagonist Ro 41-5253 on ATRA mediated growth inhibition and transactivation. (A) HT29 cells were examined in the HTCA with the indicated final concentrations of ATRA and the RARa antagonist. After 10 days the number of colonies was evaluated. Data are expressed as percentage of maximal growth inhibition observed at 10 nM ATRA. Shown are the mean 6 SEM of three independent experiments, each performed in triplicate. (B) HT29 cells were transiently transfected with the pTK:bREx2-luc reporter construct. After incubation with the indicated concentrations of ATRA and the RARa antagonist for 24 h, luciferase activity was determined and expressed as percentage of maximal transactivation observed at 10 nM ATRA. Shown are the mean 6 SEM of three independent experiments.
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(6 – 8). In addition, retinoids have been demonstrated to exert antiproliferative effects on some but not all colon carcinoma cells in vivo and in vitro (8, 9, 23–25, 35, 36). The underlying molecular mechanisms responsible for retinoid action in colon cancer cells are poorly understood. Therefore, the current study was designed to characterize the intracellular molecules involved in mediating the antiproliferative effects of retinoids in a retinoid sensitive human colon carcinoma cell model (HT29 cells). We chose a sensitive anchorage-independent growth assay (HTCA) to investigate the antiproliferative effects of retinoids on HT29 cells, because anchorageindependent growth has been shown to reflect more accurate and reliable growth regulatory effects in vitro with tumor growth behavior in vivo (26). Using the three clinically and therapeutically most relevant retinoid derivatives all-trans, 13-cis and 9-cis retinoic acid, we observed a profound dose-dependent growth inhibition of HT29 cells. At therapeutically relevant concentrations (#100 nM) (27) however, only all-trans and its stereoisomer 13-cis retinoic acid exerted antiproliferative effects, whereas the retinoid X receptor (RXR) family preferring ligand 9-cis RA was ineffective. Retinoid mediated growth inhibition is paralleled by induction of cellular differentiation in a wide variety of epithelial cells (1). We therefore examined the effects of retinoids on the expression of two biochemical markers closely associated with a more differentiated HT29 phenotype: alkaline phosphatase (AP) and carcinoembryonic antigen (CEA) (19, 20). In contrast to sodium butyrate, which has been shown to induce intestinal differentiation in HT29 cells, retinoids had no effect on AP and CEA expression. Furthermore, HT29 cell morphology remained unchanged under retinoid treatment. Although subtle alterations in the ultrastructure of HT29 cells cannot be ruled out by these experiments, we have currently no evidence that retinoid treatment results in a significant induction of cellular differentiation in HT29 cells. These data are in contrast to what has been observed in other gastrointestinal malignancies, e.g., human pancreatic carcinoma cells, where retinoid mediated growth inhibition is paralleled by induction of cellular differentiation as evidenced by biochemical and morphological criteria (13). These observations suggest that cell-type specific factors might determine the spectrum of retinoid action in a given tissue. In addition, differential regulation of growth and intestinal differentiation has recently also been described for the polar-planar compound HMBA in HT29 cells, indicating that regulation of these two fundamental processes is not always causally linked (19). To gain further insight into the molecular mechanisms, by which retinoids inhibit HT29 cell growth, we analyzed the expression of nuclear retinoid receptors
by RT-PCR using receptor subtype specific primers. With exception of the RXRg, which has previously been demonstrated to be expressed in a very tissue restricted manner (28), we detected mRNA transcripts for all major RAR isoforms as well as RXRa and b. The pleiotropic effects of retinoids are mediated by nuclear retinoid receptors, which act as ligandactivated transcription factors to control transcription of retinoid regulated genes or gene networks (10, 11). It recently became clear that each retinoid receptor subtype might serve a distinct biological function by activating a specific set of genes (10, 11). Based on these hypotheses, the antiproliferative potency of a given retinoid analogue should correlate with the capability to regulate transcription of certain retinoid responsive genes involved in cellular growth control. Therefore, we employed a panel of receptor subtype specific analogues and antagonists in growth and transactivation assays in order to investigate which one of the five retinoid receptor subtypes expressed in HT29 cells mediates the antiproliferative effects. We focused on members of the RAR family because all-trans retinoic acid, which only binds to members of the RARs but not RXRs (10 –12), had the most prominent growth inhibitory effects over a wide concentration range suggesting that RARs rather than RXRs are predominantly involved in retinoid mediated growth inhibition. We found that only treatment with the RARa selective agonist Ro 40-6055 resulted in a significant dosedependent inhibition of anchorage-independent growth in HT29 cells, whereas the RARb and RARg selective agonists were ineffective at a concentration range from 100 pM to 100 nM. Furthermore, the extent of growth inhibition was approximately comparable between alltrans retinoic acid and the RARa selective agonist. In good agreement with the antiproliferative characteristics, the RARa selective agonist resulted in a pronounced dose-dependent induction of a retinoid responsive reporter construct (pTK:bREx2-luc) in transactivation assays, whereas the RARb and RARg selective agonists again were essentially without effect. The tight correlation between the receptor subtype specific characteristics in growth and transactivation assays supports the notion that the antiproliferative action of retinoid analogues are tightly coupled to their potency to stimulate (or inhibit) transcription of growth-regulatory genes. To further explore the extent of RARa mediated pathways in ATRA induced growth inhibition we employed the highly selective RARa antagonist Ro 41-5253 (22) in growth and transactivation assays. While the RARa selective antagonist per se had no effect of growth or transactivation, it almost completely blocked in a dose-dependent manner ATRA mediated growth inhibition and transactivation supporting the observation that RARa predominantly it not exclusively mediates the antiproliferative effects of retinoic acid in HT29 cells.
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Previous studies in a variety of cells and tissues suggested that the specific biological function of a given retinoid receptor subtype is determined in a very restricted tissue- and cell-type specific manner. For example, RARg mediates growth inhibition and differentiation in human teratocarcinoma and neuroblastoma cells (29, 30), RXRb determines growth arrest in embryonic carcinoma cells (31), RARb exerts negative growth control in human fibroblasts (32) and RARa mediates growth inhibition in human breast cancer cells (33). In context with the current experimental evidence that RARa plays a key role in mediating the antiproliferative effects of retinoids in the human colon carcinoma cell line HT29, these data suggest that the biological significance of a given receptor subtype has to be determined separately for each cell type. In summary, the current study provides evidence, that the retinoic acid receptor alpha mediates the antiproliferative effects of retinoids in human colon carcinoma cells; therefore, RAR alpha selective agonists might be an attractive alternative to conventional nonselective retinoids for clinical chemoprevention trials of colon cancer. ACKNOWLEDGMENTS This work was supported by a grant from the Deutsche Krebshilfe (10-0954-Ro2) and the Maria Sonnenfeld Geda¨chtnisstiftung. We are grateful to Hoffman–LaRoche for providing us with the retinoids, Dr. R. Evans (Salk Institute, San Diego, CA) for the pTK:bREx2-luc construct, and Dr. P. Chambon (Strasbourg, France) for the retinoid receptor plasmids.
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Retinoic Acid Receptor a Mediates Growth Inhibition by Retinoids in Human Colon Carcinoma HT29 Cells Barbara Nicke,* Astrid Kaiser,* Bertram Wiedenmann,† Ernst-Otto Riecken,* and Stefan Rosewicz* ,† ,1 *Department of Gastroenterology, Klinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany; and †Medizinische Klinik m.S. Hepatologie/Gastroenterologie, Virchow-Klinikum, Charite´, Augustenburger Platz 1, 13353 Berlin, Germany
Received July 8, 1999
Although retinoids have been suggested to inhibit chemically induced colon carcinogenesis, the molecular mechanisms underlying retinoid-mediated growth regulation in colon carcinoma cells are unknown. Therefore, we investigated the biological effects of retinoids on growth in HT29 colon carcinoma cells. All-trans retinoic acid (ATRA) treatment of HT29 cells resulted in a profound inhibition of anchorageindependent growth without biochemical or morphological evidence for induction of differentiation. Treatment with the selective RARa agonist Ro 40-6055 completely mimicked the effects of ATRA on growth and transactivation of a bRAREx2-luciferase reporter construct, while RARb- and g-specific analogues were ineffective. Furthermore, ATRA-regulated growth and transactivation could be completely blocked by a RARa-selective receptor antagonist. Thus, ATRA potently inhibits anchorage-independent growth in HT29 cells and this effect is mainly if not exclusively mediated by the retinoic acid receptor a. © 1999 Academic Press
Natural and synthetic derivatives of vitamin A, which are biochemically summarized as retinoids, have been shown to inhibit growth and induce cellular differentiation in a wide variety of epithelial cells in vitro and in vivo (for review see 1). Subsequent clinical trials, which were either focused on chemoprevention or treatment of frank malignancies demonstrated con1 To whom correspondence should be addressed at Medizinische Klinik m.S., Hepatologie/Gastroenterologie, Virchow-Klinikum, Charite´, Augustenburger Platz 1, 13353 Berlin, Germany. Fax: 004930-45053905. E-mail: [email protected]. Abbreviations used: RAR a, retinoic acid receptor alpha; RAR, retinoic acid receptors; RXR, retinoid X receptors; ATRA, all-trans retinoic acid; RT-PCR, reverse-transcriptase polymerase chain reaction; RARE, retinoic acid responsive element; CEA, carcinoembryonic antigen; AP, alkaline phosphatase; NaB, sodium butyrate.
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
siderable therapeutic efficacy of retinoids in some cancers (cervical cancer, renal cell carcinoma, head and neck cancer), whereas others (malignant melanoma, lung cancer) proved to be unresponsive to retinoid therapy (2, 3). Experimental data regarding the potential chemopreventive effects of retinoids in colon carcinogenesis have revealed conflicting results. Whereas earlier studies failed to reproducibly demonstrate a chemopreventive effect of retinoids on chemically induced colon carcinogenesis (4, 5) more recent studies revealed promising activity of retinoids in preventing intestinal neoplasia (6 –9). These differences might be explained by the chemopreventive use of different retinoids as well as the heterogeneous models utilized to study chemically induced carcinogenesis (4 – 8). Much less is known about the biological effects of retinoids in malignant transformed colon carcinoma cells. Two studies have shown that retinoids inhibit mucosal ornithine decarboxylase activity in the rectum of colon cancer patients (10) and carcinogen induced DNA synthesis in rat colon carcinoma cells (8), suggesting that retinoids might also exert growth inhibitory actions in colon epithelial cells which have undergone malignant transformation. Thus, the current experimental evidence suggests that retinoids might have antiproliferative potential in a chemopreventive setting as well as treatment of colon carcinoma. However, no data are currently available as to the molecular mechanisms underlying retinoid action in colonic epithelial cells. Recently much has been learned about the molecular mechanisms by which retinoids modulate such pleiotropic effects as tumor cell proliferation and induction of cellular differentiation. The biological effects of retinoids are mediated by two families of nuclear retinoic acid receptors, each consisting of three receptor subtypes a, b and g: the retinoic acid receptors (RAR) and the retinoid X receptors (RXR). In addition, each RAR gene generates multiple isoforms by either alternative
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splicing of exons or differential use of internal promoters (11). Both receptor families act as ligand-activated transcription factors that control gene transcription initiated from promoters of retinoid regulated genes by interacting with cis-acting DNA elements, the so called RAREs (retinoic acid responsive elements) (reviewed in 11, 12). The naturally occurring ligand for RARs in vivo is all-trans retinoic acid (ATRA), while RXR preferentially bind to 9-cis retinoic acid (9-cis RA) (13). The large spectrum of biological processes affected by retinoic acid, as well as the tissue specific restricted expression of RAR/RXR subtypes during embryogenesis, and in the adult organism suggests that each RAR and RXR subtype exerts a unique biological function, and thereby determines whether a given cell or tissue is retinoid sensitive or resistant (11). Therefore, detailed knowledge of the cell-type specific expression pattern for each receptor subtype might provide a rationale why certain malignancies are retinoid responsive while others are not. Based on these observations, the current study was designed to evaluate the biological effects of retinoids on growth and differentiation in the human colon carcinoma cell line HT29 and to further determine the retinoid receptor subtype involved in mediating retinoid action in colon carcinoma cells. MATERIALS AND METHODS Materials. Human colon carcinoma HT29 cells were obtained from the American Type Tissue Culture Collection. Dulbecco’s modified Eagle medium was obtained from Gibco (Berlin, Germany), and fetal calf serum (FCS) from Biochrom (Berlin, Germany). All retinoids were kindly provided by Hoffman–LaRoche (Basel, Switzerland). [a- 32P]dCTP (6000 Ci/mmol) was obtained from DuPont (Bad Homburg, Germany). Random priming labeling kit was obtained from Amersham (Braunschweig, Germany); random hexamer primers and M-MLV were from Bethesda Research Laboratories (BRL, Bethesda, MD); Taq DNA polymerase from Promega (Heidelberg, Germany). Cell culture. HT29 cells were grown in DMEM supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 U/ml). The cells were kept under 95% air and 5% CO 2 at 37°C. Human tumor clonogenic assay (HTCA). For evaluation of clonal growth of HT29 cells we used a methylcellulose human tumor clonogenic assay (HTCA) exactly as previously described (14). Only colonies containing more than twenty cells were counted. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. RNA from HT29 cells was isolated exactly as previously described (14). Reverse transcription of was performed using 1 mg total RNA, 100 pM random hexamer primer, 1 mM DTT, 6 mM MgCl 2, 500 mM of each dNTP, 20 units RNAsin (Promega, Heidelberg, Germany), and MML-V reverse transcriptase. This RT mixture was used directly as a template for PCR in a 1:20 dilution. The reaction was carried out in 10 mM Tris–HCl buffer (pH 9.0) containing 50 mM KCl, 0.01% Triton X-100, 1 mM MgCl 2, 200 mM of each dNTP, 50 pM of each primer, and 2.5 U Taq DNA polymerase in a final volume of 50 ml. Amplification conditions for 35 cycles were carried out as follows: denaturation for 30 s at 92°C, annealing at 60°C for 90 s, and extension for 90 s at 72°C. Final extension was carried out for an additional 10 min after completion of all 35 cycles.
Southern blot analysis. After amplification 10 ml of each RT-PCR were subjected to electrophoresis on a 1.5% agarose gel. The samples were denatured in 0.5 N NaOH and 1.5 M NaCl for 30 min and transferred to Hybond N 1 membranes (Amersham, Braunschweig, Germany). After blotting the membranes were neutralized in 50 mM NaH 2PO 4, pH 6.5, for 20 min and then baked at 80°C for 2 h. Hybridization was carried out using the respective receptor cDNA fragments. The cDNA inserts were radioactively labeled using the random primer labeling kit following the instructions provided by the supplier. Unincorporated nucleotides were removed by passing the reaction sample over a Sephadex G 50 column. The following plasmids were used: pSG5:RARa (14), pSG5:RARb (15), pSG5:RARg (14), pSG5:RXRa (16), pT11:RXRb (16), and pSG5:RXRg (16). Hybridization was carried out in a standard hybridization buffer containing 50% formamide at 42°C. After hybridization filters were washed and then exposed to X-ray films for 30 – 60 min. Differentiation assays. HT29 cells were incubated with ATRA, vehicle, or sodium butyrate. After 96 h supernatant was collected and cells were harvested and lysed in a lysis buffer (20 mM Tris, 10 mM EGTA, and 250 mM sucrose) by homogenization. Protein content was measured using Bio-Rad protein assay. Alkaline phosphatase (AP) activity was measured in aliquots ranging from 20 to 200 mg of protein using a colorimetric kit (Sigma, Deisenhofen, Germany) following the instructions of the supplier (17). The concentration of carcinoembryonic antigen (CEA) was measured in 1 ml of collected supernatant using an automated microparticle enzyme immunoassay (Abbott Laboratories, North Chicago, IL) following the instructions provided by the supplier. Transactivation assays. HT29 cells were transiently transfected with the pTK:bREx2-luc reporter construct (18) using the Lipofectamine reagent (Gibco, BRL, Eggenstein, Germany). After 12 h the indicated concentrations of retinoids or vehicle were added and cells were incubated for an additional 24 h. Luciferase activity of cell lysates was then determined using a luciferase assay system (Promega, Heidelberg, Germany). Luciferase activity was expressed as fold induction of vehicle treated control cells. Statistics. Results were evaluated statistically by analysis of variance (ANOVA) followed by a multiple contrast test. P values , 0.05 were considered to be significant.
RESULTS Effects of retinoids on growth and differentiation in HT29 cells. Initially, we investigated the effects of various naturally occurring retinoids on anchorageindependent growth in HT29 cells. All three retinoids tested revealed a potent and dose-dependent growth inhibition (Fig. 1). However, at lower, therapeutically relevant concentrations (,100 nM) only all-trans and its stereoisomer 13-cis retinoic acid demonstrated significant growth inhibition, whereas 9-cis retinoic acid was ineffective. On an equimolar basis maximal growth inhibition at 10 mM revealed the following gradient of biological potency: all-trans (9.7 6 3.4% of control) 5 13-cis (13.4 6 2.9% of control) . 9-cis retinoic acid (25.4 6 2.3% of control). We next investigated whether the retinoid mediated growth inhibition was paralleled by a concomitant induction of cellular differentiation in HT29 cells. Two biochemical markers closely associated with a more differentiated phenotype were examined: alkaline phosphatase (AP) and carcinoembryonic antigen (CEA). For these experiments we used sodium butyrate
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FIG. 1. Effects of retinoid analogues on anchorage-independent growth. HT29 cells were examined in the human tumor clonogenic assay with the indicated final concentrations of each retinoid analogue. After 10 days the number of colonies was evaluated and expressed as percentage of vehicle treated controls. Shown are the mean 6 SEM of three independent experiments, each performed in triplicate. *P , 0.05 versus controls.
(NaB) as a positive control, which has been repeatedly demonstrated to inhibit growth and induce cellular differentiation in HT29 cells (19, 20). Incubation of HT29 cells with NaB (5 mM) for 96 h resulted in a pronounced increase of cellular AP activity, whereas maximal concentrations of ATRA (10 mM) had no effect (Fig. 2). Similarly, NaB treatment resulted in an about six-fold increase of CEA concentrations in HT29 supernatants (325 6 8 mg/ml versus 54 6 5 mg/ml in controls) whereas ATRA had no significant effect (Fig. 2). Furthermore, HT29 cell morphology remained unchanged under ATRA treatment for up to five days (data not shown). Based on these experiments we have currently
FIG. 2. Effects of all-trans retinoic acid on HT29 cell differentiation. HT29 cells were incubated with vehicle (controls), 10 mM all-trans retinoic acid (ATRA), or sodium butyrate (5 mM) for 96 h. Thereafter, cellular AP activity or CEA concentrations in the supernatant were determined. Shown are the mean 6 SEM of three independent experiments, each performed in triplicate. *P , 0.05 versus vehicle treated controls.
FIG. 3. Expression of nuclear retinoid acid receptors in HT29 cells. RT-PCR analysis was performed for HT29 cells using receptor specific oligonucleotide primers (see Ref. 14). The respective receptor subtype cDNA was used as a positive internal control. RT-PCR with (1) or without prior reverse transcription (2) (which served as a negative internal control) was performed as outlined in Methods. Aliquots from each RT-PCR product were electrophoresed through a 1% agarose gel (ethidium bromide staining, shown in the upper panel) and then subjected to Southern blot analysis using the respective cDNA probes for the RARs/RXRs to ensure specificity of the amplification product (lower panel).
no evidence that ATRA treatment of HT29 cells results in induction of cellular differentiation. Expression of retinoid receptors in HT29 cells. As an initial attempt to understand the molecular mechanisms involved in retinoid mediated growth inhibition of HT29 cells, we first had to characterize the expression pattern of nuclear retinoid receptors. Using a panel of retinoid receptor subtype specific primers in RT-PCR analysis, we detected mRNA transcripts for RAR a, b, g, and RXR a, and b (Fig. 3). In contrast, no amplification signal could be obtained for the RXR g. PCR amplification without prior reverse transcription, which served as an internal negative control, repeatedly failed to demonstrate an amplification signal. The specificity of the amplification products was confirmed by subsequent hybridization of the RT-PCR products using the respective receptor subtype specific cDNA probes in Southern blots (Fig. 3). The retinoic acid receptor a mediates growth inhibition in HT29 cells. We next investigated which retinoid receptor subtype mediates the growth inhibitory effects in HT29 cells. Based on our growth studies only ATRA and 13-cis retinoic acid were growth inhibitory at therapeutically relevant concentrations (#100 nM), whereas 9-cis RA was ineffective at these concentrations (Fig. 1). While 9-cis RA binds to RARs as well as RXRs, ATRA does not bind to RXRs, suggesting that
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(22) in growth and transactivation assays. The antiproliferative effects of 10 nM ATRA were blocked in a dose-dependent manner by the RARa antagonist Ro 41-5253 (Fig. 5A). Half-maximal inhibition was observed at a concentration of 100 nM antagonist (59 6 8% of ATRA-induced growth inhibition) and maximal inhibition occurred at 10 mM (6 6 1% of ATRA–induced growth inhibition. Very similar results were obtained when we examined the effects of the RARa antagonist on ATRA mediated transactivation in HT29 cells (Fig. 5B) with a complete inhibition of ATRA stimulated transactivation at a concentration of 10 mM of RARa receptor antagonist. The RARa antagonist alone had no significant effect in terms of growth inhibition or transactivation in a concentration range from 1 nM to 10 mM (data not shown). DISCUSSION In vivo experiments of chemically induced colon carcinogenesis indicated that retinoids might have chemopreventive effects on the development of colon cancer FIG. 4. Effects of RAR subtype specific retinoids on anchorageindependent growth and transactivation. (A) HT29 cells were examined in HTCA with the indicated final concentrations of each retinoid analogue. After 10 days the number of colonies was evaluated and expressed as percentage of vehicle treated controls. Shown are the mean 6 SEM of three independent experiments, each performed in triplicate. (B) HT29 cells were transiently transfected with the pTK:bREx2-luc reporter construct. After incubation with the indicated concentrations of each retinoid analogue for 24 h, luciferase activity was determined and expressed as fold increase over basal (luciferase activity in the absence of retinoids). Shown are the mean 6 SEM of six independent experiments, each performed in triplicate wells. Receptor subtype specificity of the analogues is shown in parenthesis.
the antiproliferative effect of ATRA is mediated by members of the RAR family. We therefore used a panel of retinoic acid receptor subtype specific analogues in growth and transactivation assays. Receptor subtype specificity of these analogues has recently been documented (21, 22). Of the three RAR subtype specific retinoids tested, only the RARa selective analogue Ro 40-6055 resulted in a significant dose-dependent growth inhibition in HT29 cells, which was quantitatively comparable to the antiproliferative effects of ATRA (Fig. 4A). In good agreement with the antiproliferative characteristics, only the RARa selective retinoid Ro 40-6055 resulted in a significant dosedependent transactivation of the pTK:bREx2-luc reporter construct transiently transfected into HT29 cells (Fig. 4B). The RARb (Ro 48-2249) and RARg (Ro 44-4753) specific retinoids were ineffective in terms of growth inhibition and transactivation (Fig. 4). To further substantiate the role of RARa in mediating the antiproliferative effects of ATRA in HT29 cells, we used the RARa specific receptor antagonist Ro 41-5253
FIG. 5. The effects of the RARa antagonist Ro 41-5253 on ATRA mediated growth inhibition and transactivation. (A) HT29 cells were examined in the HTCA with the indicated final concentrations of ATRA and the RARa antagonist. After 10 days the number of colonies was evaluated. Data are expressed as percentage of maximal growth inhibition observed at 10 nM ATRA. Shown are the mean 6 SEM of three independent experiments, each performed in triplicate. (B) HT29 cells were transiently transfected with the pTK:bREx2-luc reporter construct. After incubation with the indicated concentrations of ATRA and the RARa antagonist for 24 h, luciferase activity was determined and expressed as percentage of maximal transactivation observed at 10 nM ATRA. Shown are the mean 6 SEM of three independent experiments.
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(6 – 8). In addition, retinoids have been demonstrated to exert antiproliferative effects on some but not all colon carcinoma cells in vivo and in vitro (8, 9, 23–25, 35, 36). The underlying molecular mechanisms responsible for retinoid action in colon cancer cells are poorly understood. Therefore, the current study was designed to characterize the intracellular molecules involved in mediating the antiproliferative effects of retinoids in a retinoid sensitive human colon carcinoma cell model (HT29 cells). We chose a sensitive anchorage-independent growth assay (HTCA) to investigate the antiproliferative effects of retinoids on HT29 cells, because anchorageindependent growth has been shown to reflect more accurate and reliable growth regulatory effects in vitro with tumor growth behavior in vivo (26). Using the three clinically and therapeutically most relevant retinoid derivatives all-trans, 13-cis and 9-cis retinoic acid, we observed a profound dose-dependent growth inhibition of HT29 cells. At therapeutically relevant concentrations (#100 nM) (27) however, only all-trans and its stereoisomer 13-cis retinoic acid exerted antiproliferative effects, whereas the retinoid X receptor (RXR) family preferring ligand 9-cis RA was ineffective. Retinoid mediated growth inhibition is paralleled by induction of cellular differentiation in a wide variety of epithelial cells (1). We therefore examined the effects of retinoids on the expression of two biochemical markers closely associated with a more differentiated HT29 phenotype: alkaline phosphatase (AP) and carcinoembryonic antigen (CEA) (19, 20). In contrast to sodium butyrate, which has been shown to induce intestinal differentiation in HT29 cells, retinoids had no effect on AP and CEA expression. Furthermore, HT29 cell morphology remained unchanged under retinoid treatment. Although subtle alterations in the ultrastructure of HT29 cells cannot be ruled out by these experiments, we have currently no evidence that retinoid treatment results in a significant induction of cellular differentiation in HT29 cells. These data are in contrast to what has been observed in other gastrointestinal malignancies, e.g., human pancreatic carcinoma cells, where retinoid mediated growth inhibition is paralleled by induction of cellular differentiation as evidenced by biochemical and morphological criteria (13). These observations suggest that cell-type specific factors might determine the spectrum of retinoid action in a given tissue. In addition, differential regulation of growth and intestinal differentiation has recently also been described for the polar-planar compound HMBA in HT29 cells, indicating that regulation of these two fundamental processes is not always causally linked (19). To gain further insight into the molecular mechanisms, by which retinoids inhibit HT29 cell growth, we analyzed the expression of nuclear retinoid receptors
by RT-PCR using receptor subtype specific primers. With exception of the RXRg, which has previously been demonstrated to be expressed in a very tissue restricted manner (28), we detected mRNA transcripts for all major RAR isoforms as well as RXRa and b. The pleiotropic effects of retinoids are mediated by nuclear retinoid receptors, which act as ligandactivated transcription factors to control transcription of retinoid regulated genes or gene networks (10, 11). It recently became clear that each retinoid receptor subtype might serve a distinct biological function by activating a specific set of genes (10, 11). Based on these hypotheses, the antiproliferative potency of a given retinoid analogue should correlate with the capability to regulate transcription of certain retinoid responsive genes involved in cellular growth control. Therefore, we employed a panel of receptor subtype specific analogues and antagonists in growth and transactivation assays in order to investigate which one of the five retinoid receptor subtypes expressed in HT29 cells mediates the antiproliferative effects. We focused on members of the RAR family because all-trans retinoic acid, which only binds to members of the RARs but not RXRs (10 –12), had the most prominent growth inhibitory effects over a wide concentration range suggesting that RARs rather than RXRs are predominantly involved in retinoid mediated growth inhibition. We found that only treatment with the RARa selective agonist Ro 40-6055 resulted in a significant dosedependent inhibition of anchorage-independent growth in HT29 cells, whereas the RARb and RARg selective agonists were ineffective at a concentration range from 100 pM to 100 nM. Furthermore, the extent of growth inhibition was approximately comparable between alltrans retinoic acid and the RARa selective agonist. In good agreement with the antiproliferative characteristics, the RARa selective agonist resulted in a pronounced dose-dependent induction of a retinoid responsive reporter construct (pTK:bREx2-luc) in transactivation assays, whereas the RARb and RARg selective agonists again were essentially without effect. The tight correlation between the receptor subtype specific characteristics in growth and transactivation assays supports the notion that the antiproliferative action of retinoid analogues are tightly coupled to their potency to stimulate (or inhibit) transcription of growth-regulatory genes. To further explore the extent of RARa mediated pathways in ATRA induced growth inhibition we employed the highly selective RARa antagonist Ro 41-5253 (22) in growth and transactivation assays. While the RARa selective antagonist per se had no effect of growth or transactivation, it almost completely blocked in a dose-dependent manner ATRA mediated growth inhibition and transactivation supporting the observation that RARa predominantly it not exclusively mediates the antiproliferative effects of retinoic acid in HT29 cells.
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Previous studies in a variety of cells and tissues suggested that the specific biological function of a given retinoid receptor subtype is determined in a very restricted tissue- and cell-type specific manner. For example, RARg mediates growth inhibition and differentiation in human teratocarcinoma and neuroblastoma cells (29, 30), RXRb determines growth arrest in embryonic carcinoma cells (31), RARb exerts negative growth control in human fibroblasts (32) and RARa mediates growth inhibition in human breast cancer cells (33). In context with the current experimental evidence that RARa plays a key role in mediating the antiproliferative effects of retinoids in the human colon carcinoma cell line HT29, these data suggest that the biological significance of a given receptor subtype has to be determined separately for each cell type. In summary, the current study provides evidence, that the retinoic acid receptor alpha mediates the antiproliferative effects of retinoids in human colon carcinoma cells; therefore, RAR alpha selective agonists might be an attractive alternative to conventional nonselective retinoids for clinical chemoprevention trials of colon cancer. ACKNOWLEDGMENTS This work was supported by a grant from the Deutsche Krebshilfe (10-0954-Ro2) and the Maria Sonnenfeld Geda¨chtnisstiftung. We are grateful to Hoffman–LaRoche for providing us with the retinoids, Dr. R. Evans (Salk Institute, San Diego, CA) for the pTK:bREx2-luc construct, and Dr. P. Chambon (Strasbourg, France) for the retinoid receptor plasmids.
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