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• W2047880777 abstract "In view of the tumor suppressor role of the transforming growth factor-β (TGFβ) type II receptor (RII), the identification and characterization of agents that can induce the expression of this receptor are of potential importance to the development of chemoprevention approaches as well as treatment of cancer. To date, the identification of exogenous agents that control RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGFβ growth inhibition activity, was significantly inhibited by vitamin D3 and its analogue EB1089. In contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which have lost cell surface RII and are resistant to TGFβ, was not affected by these two compounds. TGFβ-neutralizing antibody was able to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGFβ activity. An RNase protection assay showed approximately a 3-fold induction of the RII mRNA, while a receptor cross-linking assay revealed a 3–4-fold induction of the RII protein. In contrast, there was no change in either RII mRNA or protein in the MCF-7 L cells. In view of the tumor suppressor role of the transforming growth factor-β (TGFβ) type II receptor (RII), the identification and characterization of agents that can induce the expression of this receptor are of potential importance to the development of chemoprevention approaches as well as treatment of cancer. To date, the identification of exogenous agents that control RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGFβ growth inhibition activity, was significantly inhibited by vitamin D3 and its analogue EB1089. In contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which have lost cell surface RII and are resistant to TGFβ, was not affected by these two compounds. TGFβ-neutralizing antibody was able to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGFβ activity. An RNase protection assay showed approximately a 3-fold induction of the RII mRNA, while a receptor cross-linking assay revealed a 3–4-fold induction of the RII protein. In contrast, there was no change in either RII mRNA or protein in the MCF-7 L cells. Transforming growth factor-β (TGFβ) 1The abbreviations used are: TGFβ, transforming growth factor-β; RA, retinoic acid; VDR, vitamin D receptor; RI, RII, and RIII, TGFβ receptor type I, II, and III, respectively. comprises a family of hormone-like polypeptides that affects cell growth, adhesion, and differentiation (1Roberts A.B. Sporn M.B. Peptide Growth Factors. 1990; 95: 419-472Google Scholar). They act as growth inhibitors for most epithelial cells and some cancer cells. Two pathways are primarily involved in mediating effects of TGFβ on cell growth and differentiation. One pathway involves blockade of cell cycle transit, while the other involves alteration of the extracellular matrix environment. TGFβs elicit their effects by binding to cell surface receptors. Three major types of receptors have been shown to be present in most TGFβ-responsive cell lines. They are designated as type I (RI), type II (RII), and type III (RIII), respectively. RIII is a 280–330-kDa glycoprotein that has no functional signaling domain but rather serves as a ligand storage protein and presents TGFβ to the signaling receptors (2Lopez-Casillas F. Wrana J.L. Massague J. Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (778) Google Scholar). RI and RII, which are glycoproteins of ∼55 and 85 kDa, respectively, form a heteromeric receptor complex. Both are serine/threonine kinases, and each appears to be indispensable for TGFβ signaling (3Lin H.Y. Wang X-F. Ng-Eaton E. Weinberg R.A. Lodish H.F. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (969) Google Scholar, 4Franzen P. Ten Dijke P. Ichijo H. Yamashita H. Schultz P. Heldin C-H. Miyazono K. Cell. 1993; 75: 681-692Abstract Full Text PDF PubMed Scopus (716) Google Scholar, 5Bassing C.H. Yingling J.M. Howe D.J. Wang T. He W.W. Gustafson M.L. Shah P. Donahoe K. Wang X-F. Science. 1994; 263: 87-89Crossref PubMed Scopus (275) Google Scholar). The direct involvement of both RI and RII in conferring TGFβ effects indicates that loss of either of the functional receptors would contribute to loss of autocrine TGFβ activity. Loss of negative autocrine TGFβ activity results in a growth advantage caused by an imbalance in positive and negative regulators, possibly leading to tumor formation and progression (6Sun L. Wu G. Willson J.K.V. Zborowska E. Yang J. Rajkarunanayake I. Wang J. Gentry L.E. Wang X-F. Brattain M.G. J. Biol. Chem. 1994; 269: 26449-26455Abstract Full Text PDF PubMed Google Scholar, 7Markowitz S. Wang J. Myeroff L. Parsons R. Sun L-Z. Lutterbaugh J. Fan R.S. Zborowska E. Kinzler K.W. Vogelstein B. Brattain M. Willson J.K.V. Science. 1995; 268: 1336-1338Crossref PubMed Scopus (2148) Google Scholar). Recent evidence has shown a loss of RII is often associated with the failure to respond to autocrine and exogenous TGFβ. We have previously demonstrated that re-expression of this receptor in an RII-deficient breast cancer cell line (late passage MCF-7) leads to restoration of TGFβ sensitivity and reduced malignancy in athymic nude mice (6Sun L. Wu G. Willson J.K.V. Zborowska E. Yang J. Rajkarunanayake I. Wang J. Gentry L.E. Wang X-F. Brattain M.G. J. Biol. Chem. 1994; 269: 26449-26455Abstract Full Text PDF PubMed Google Scholar). In addition, it has been shown that mutational inactivation of RII occurs frequently in a subset of colon tumors with microsatellite instability (7Markowitz S. Wang J. Myeroff L. Parsons R. Sun L-Z. Lutterbaugh J. Fan R.S. Zborowska E. Kinzler K.W. Vogelstein B. Brattain M. Willson J.K.V. Science. 1995; 268: 1336-1338Crossref PubMed Scopus (2148) Google Scholar), and reconstitution of RII expression by stable transfection also leads to reversal of malignancy in these cells (8Wang J. Sun L. Myerloff L. Wang X. Gentry L.E. Yang J. Liang J. Zborowska E. Markowitz S. Willson J.K.V. Brattain M.G. J. Biol. Chem. 1995; 270: 22044-22049Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Others have noted that loss of RII expression is important in other types of malignancies (9Garrigue-Antar L. Muñoz-Antonia T. Antonia S.J. Gesmonde J. Vellucci V.F. Reiss M. Cancer Res. 1995; 55: 3982-3987PubMed Google Scholar, 10Myeroff L.L. Parsons R. Kim S.-J. Hedrick L. Cho K.R. Orth K. Mathis M. Kinzler K.W. Lutterbaugh J. Park K. Bang Y.-J. Lee H.Y. Park J.-G. Lynch H.T. Roberts A.B. Vogelstein B. Markowitz S.D. Cancer Res. 1995; 55: 5545-5547PubMed Google Scholar, 11Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar, 12Park K. Kim S.J. Bang Y.J. Park J.G. Kim N.Y. Roberts A.B. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8772-8776Crossref PubMed Scopus (427) Google Scholar, 13Okamoto A. Jiang W. Kim S.J. Spillare E.A. Stoner G.O. Weistein B.I. Harris C.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11576-11580Crossref PubMed Scopus (84) Google Scholar, 14Geiser A.G. Burmester J.K. Webbink R. Roberts A.B. Sporn M.B. J. Biol. Chem. 1992; 267: 2588-2593Abstract Full Text PDF PubMed Google Scholar, 15Inagaki M. Moustakas A. Lin H.Y. Lodish H.F. Carr B.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5359-5363Crossref PubMed Scopus (184) Google Scholar). These lines of evidence suggest that RII is a critical determinant for conferring TGFβ tumor suppression as well as negative autocrine TGFβ growth function. Consequently, agents that can induce RII expression would be valuable in the development of approaches for cancer treatment and prevention where receptor expression appears to be repressed, such as estrogen receptor-positive (ER+) breast cancer (16Brattain M.G. Ko Y. Banerji S.S. Wu G. Willson J.K.V. J. Mammary Gland Biol. Neoplasia. 1996; 1: 365-372Crossref PubMed Scopus (24) Google Scholar, 17Kalkhoven E. Roelen B.A. de Winter J.P. Mummery C.L. van den Eijnden-van Raaij A.J. van der Saag P.T. van der Burg B. Cell Growth Differ. 1995; 6: 1151-1161PubMed Google Scholar). To date, no such agents have been carefully characterized for their ability to induce RII. Although Cohen et al. (55Cohen P.S. Letterio J.J. Gaetano C. Chan J. Matsumoto K. Sporn M.B. Thiele C.J. Cancer Res. 1995; 55: 2380-2386PubMed Google Scholar) showed increased RII mRNA in a human neuroblastoma cell line after retinoic acid (RA) treatment, they were not able to detect cell surface RII. In addition, they failed to test for increased autocrine activity and increased responsiveness/growth inhibition to TGFβ after RA treatment. A study by Turley et al. (56Turley J.M. Funakoshi S. Ruscetti F.W. Kasper J. Murphy W.J. Longo D.L. Birchenall-Roberts M.C. Cell Growth Differ. 1995; 6: 655-663PubMed Google Scholar) in RL human B lymphoma cells demonstrated increased RII protein levels following treatment with RA and vitamin E succinate. However, they did not investigate whether this correlated with increased levels of cell surface RII. Development of effective therapeutic and preventive approaches for breast cancer remains an issue, since conventional treatment by antiestrogens such as tamoxifen often leads to resistance in estrogen receptor-positive tumors (18Jordan V.C. Robinson S.P. Welshons W.V. Kessel D. Resistance to Antineoplastic Drugs. CRC Press, Boca Raton, FL1989: 403-427Google Scholar), and chemotherapy of estrogen receptor-negative tumors is even less effective (19Pasqualini J.R. Sumida C. Giambiagi N. J. Steroid Biochem. 1988; 31: 613-643Crossref PubMed Scopus (83) Google Scholar). Since there is a high incidence of vitamin D receptors (VDRs) in human breast cancer tumors (21Eisman J.A. Suva L.J. Sher E. Pearce P.J. Funder J.W. Martin T.J. Cancer Res. 1981; 41: 5121-5124PubMed Google Scholar, 22Berger U. Wilson P. McClelland R.A. Colston K. Haussler M.R. Pike J.W. Coombes R.C. Cancer Res. 1987; 47: 6793-6799PubMed Google Scholar), vitamin D3 is an appealing candidate as a new therapeutic agent. Like other steroid hormones, it mediates its effect through interaction of its nuclear receptor (VDR) with DNA-responsive elements in the target genes (20Haussler M.R. Mangelsdorf D.J. Komm B.S. Terpening C.M. Yamaoka K. Allegretto E.A. Baker A.R. Shine J. McDonnell D.P. Hughes M. Weigel N.L. O'Malley B.W. Pike J.W. Recent Prog. Horm. Res. 1988; 44: 263-305PubMed Google Scholar). Moreover, many breast cancer cell lines are responsive to vitamin D3antiproliferative effects both in vitro and in athymic mice (23Abe-Hashimoto J. Kikuchi T. Matsumoto T. Nishii Y. Ogata E. Ikeda K. Cancer Res. 1993; 53: 2534-2537PubMed Google Scholar). However, a major drawback for its clinical application is that the doses effective for suppressing tumor growth often cause hypercalcemia. Consequently, analogues have been developed to reduce the calcemic effects while increasing the potency of inhibition of proliferation (23Abe-Hashimoto J. Kikuchi T. Matsumoto T. Nishii Y. Ogata E. Ikeda K. Cancer Res. 1993; 53: 2534-2537PubMed Google Scholar, 24Colston K.W. Mackay A.G. James S.Y. Binderup L. Chander S. Coombes R.C. Biochem. Pharmacol. 1992; 44: 2273-2280Crossref PubMed Scopus (232) Google Scholar). Two analogues, EB1089 and MC903, both of which are derived by modification of the C17 side chain of vitamin D3, have been shown to be effective against rat breast tumors in vivo (24Colston K.W. Mackay A.G. James S.Y. Binderup L. Chander S. Coombes R.C. Biochem. Pharmacol. 1992; 44: 2273-2280Crossref PubMed Scopus (232) Google Scholar) or as an antiproliferative agent when given topically for psoriasis as well as for cutaneous metastatic breast cancer (25Colston K.W. Chander S.K. Mackay A.G. Coombes R.C. Biochem. Pharmacol. 1992; 44: 693-702Crossref PubMed Scopus (232) Google Scholar). However, the mechanisms of vitamin D3-mediated growth inhibition and in particular its anti-tumor action remain largely unresolved. In this report, we show a correlation between RII expression and vitamin D3 inhibition in MCF-7 sublines that differ dramatically in their RII expression and hence their TGFβ sensitivity as well. We hypothesized that vitamin D3's mechanism of inhibition might involve induction of TGFβ autocrine activity through increased expression of RII. This hypothesis was confirmed by RNase protection assays showing approximately 3-fold induction of the RII mRNA and a 3–4-fold induction of cell surface RII protein. The increased inhibition by vitamin D3/analogues was blocked by TGFβ-neutralizing antibodies, indicating an induction of negative autocrine TGFβ activity. The use of an essential dietary nutrient with antiproliferative and anti-tumor properties represents an attractive approach for chemoprevention and/or therapy. This is particularly true of vitamin D compounds, since the high stress western style diet associated with colon and breast cancer is also associated with low levels of vitamin D and calcium (26Khan N. Yang K. Newmark H. Wong G. Telang N. Rivlin R. Lipkin M. Carcinogenesis. 1994; 15: 2645-2648Crossref PubMed Scopus (35) Google Scholar). Thus, increased autocrine negative TGFβ activity mediated by vitamin D3 compounds in MCF-7 E cells may provide a novel mechanism for blocking malignant progression by chemopreventive approaches. MCF-7 E cells (passage number 150) were kindly provided by Drs. Robert J. Pauley and Herbert D. Soule from the Michigan Cancer Foundation. MCF-7 L cells were obtained from the ATCC and used at a passage number greater than 500. These cell lines were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum, pyruvate, vitamins, amino acids, and antibiotics. Working cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. 1,25-(OH)2vitamin D3 as well as its analogues EB1089 and MC903 were generous gifts from Dr. Lise Binderup of LEO Pharmaceutical Products (Ballerup, Denmark). Stock solutions were prepared in isopropyl alcohol at 4 mm. Serial dilutions were made in absolute ethanol and stored at −20 °C protected from light. These diluted solutions were added to the experimental culture media at a final ethanol concentration of 0.1%. Control cells received 0.1% ethanol vehicle, which had no effect on cell proliferation. [3H]thymidine incorporation into DNA was measured as described previously to determine TGFβ and vitamin D3 sensitivity (6Sun L. Wu G. Willson J.K.V. Zborowska E. Yang J. Rajkarunanayake I. Wang J. Gentry L.E. Wang X-F. Brattain M.G. J. Biol. Chem. 1994; 269: 26449-26455Abstract Full Text PDF PubMed Google Scholar). Briefly, MCF-7 cells were seeded in 24-well tissue culture plates at a density of 1.5 × 104 cells/well in 1 ml of medium. Various concentrations of compounds (1,25-(OH)2 D3, EB1089, MC903, or TGFβ) were added after cell attachment (approximately 2 h). Following 4 days of incubation, cells received a 2-h pulse with [3H]thymidine (7 μCi, 46 Ci/mmol, Amersham Pharmacia Biotech). DNA was then precipitated with 10% ice-cold trichloroacetic acid, and the amount of [3H]thymidine incorporated was analyzed by liquid scintillation counting in a Beckman LS 7500 scintillation counter as described previously (6Sun L. Wu G. Willson J.K.V. Zborowska E. Yang J. Rajkarunanayake I. Wang J. Gentry L.E. Wang X-F. Brattain M.G. J. Biol. Chem. 1994; 269: 26449-26455Abstract Full Text PDF PubMed Google Scholar). To determine whether there is an increase in the inhibitory effects by TGFβ1 following EB1089 treatment, MCF-7 E cells, which are TGFβ-responsive, were plated as described above. Various concentrations of EB1089 plus 0.1 ng/ml of TGFβ1 were added after attachment. Cells were incubated and [3H]thymidine incorporation was determined as described above. Cells were resuspended at a concentration of 1.5 × 104 cells/ml and plated into 24-well tissue culture plates (1 ml/well) either untreated or in the presence of 10 μg/ml TGFβ1 neutralizing antibody (R & D Systems) or control normal IgG. After 3 h of incubation, different concentrations of vitamin D3 compounds were added as indicated. Cells were allowed to grow for 72 h without changing the media, followed by determination of [3H]thymidine incorporation as described above. RNase protection assays were performed to determine RII RNA expression levels after vitamin D3treatment. A 476-base pair fragment of the RII cDNA within the cytoplasmic region was obtained by polymerase chain reaction with the following primers: 5′-TGGACCCTACTCTGTCTGTG-3′ and 5′-TGTTTAGGGAGCCGTCTTCA-3′. The fragment was subcloned into a pBSK (−) plasmid (Stratagene, La Jolla) for making the RII riboprobes. In vitro transcription using T3 RNA polymerase yields antisense riboprobes that protect a 476-base pair RII fragment. RNase protection assays were performed as described previously (27Wu S.P. Sun L-Z. Willson J.K.V. Humphrey L . E. Kerbel R. Brattain M.G. Cell Growth Differ. 1993; 4: 115-123PubMed Google Scholar). Briefly, exponentially growing cells were treated with EB1089 at 1 × 10−8m for the indicated time periods. Cells were solubilized in guanidine thiocyanate, and total RNA was obtained by cesium chloride gradient ultracentrifugation (28Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar). 40 μg of total RNA was used for overnight hybridization with 32P-labeled antisense riboprobes. Following RNase A and T1 treatment, the protected double-stranded RNA fragments were heat-denatured at 95 °C and analyzed by urea-polyacrylamide gel electrophoresis, and the radioactive probes were visualized by autoradiography. Actin was used as an internal control for normalizing the amount of sample loading. Simian recombinant TGFβ1 was purified as described (29Gentry L.E. Lioubin M.N. Purchio A.F. Marquardt H. Mol. Cell. Biol. 1988; 8: 4162-4168Crossref PubMed Scopus (210) Google Scholar) and iodinated by the chloramine T method (30Ruff E. Rizzino A. Biochem. Biophys. Res. Commun. 1986; 138: 714-719Crossref PubMed Scopus (58) Google Scholar). MCF-7 cells were seeded into 35-mm2 tissue culture wells at a density of 105cells/well. In the kinetic studies, exponentially growing cells were treated with various concentrations of the compounds for 24 h or with a single concentration for the indicated time periods. Cell monolayers were then incubated with 200 pm125I-labeled TGFβ1 at 4 °C for 4 h followed by chemical cross-linking with disuccinimidyl suberate for 15 min (31Segarini P.R. Roberts A.B. Rosen D.M. Seyedin S.M. J. Biol. Chem. 1987; 262: 14655-14662Abstract Full Text PDF PubMed Google Scholar). Labeled cell monolayers were solubilized in 200 μl of 1% Triton X-100 with 1 mm phenylmethylsulfonyl fluoride. Equal amounts of cell lysate protein were separated by 4–10% gradient SDS-polyacrylamide gel electrophoresis under reducing conditions and exposed for autoradiography. MCF-7 E cells were plated into 100-mm2 tissue culture dishes and allowed to reach 70–80% confluency. The medium was then removed and replaced with 5 ml of fresh McCoy's 5A medium supplemented with pyruvate, vitamins, amino acids, and antibiotics (SM). The cells were then treated with EB1089 (1 × 10−8m) or with vehicle only for 24 h. Following treatment, the conditioned medium was collected, and the indicated volumes were used to treat mink lung epithelial cells plated in 96-well tissue culture plates at a density of 1500 cells/well. In addition, a standard TGFβ growth inhibition curve was generated by treating the cells with various concentrations of TGFβ1. The mink lung epithelial cells were allowed to incubate for 3 days at which time the medium was removed and replaced by 100 μl of fresh SM. Colonies were immediately visualized by staining with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma) for 2 h. The stained cells were solubilized with Me2SO (dimethyl sulfoxide) (Mallinckrodt) and the relative cell numbers were then determined by the resultant absorbance at 595 nm. The TGFβ-responsive cyclin A promoter in tandem with a luciferase reporter construct (−133/−2) was used as described previously (32Yoshizumi M. Wang H. Hsieh C-M. Sibinga N.E.S. Perrella M.A. Lee M-E. J. Biol. Chem. 1997; 272: 22259-22264Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The reporter construct (−133/−2) contains only the activating transcription factor site, which has been shown to be the site required to mediate down-regulation of cyclin A promoter activity by TGFβ1 in mink lung epithelial cells (32Yoshizumi M. Wang H. Hsieh C-M. Sibinga N.E.S. Perrella M.A. Lee M-E. J. Biol. Chem. 1997; 272: 22259-22264Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). MCF-7 E cells were transiently transfected with 30 μg of luciferase reporter construct and 7 μg of β-galactosidase plasmid by electroporation with a Bio-Rad gene pulser at 250 mV and 960 microfarads. Cells were plated into a six-well tissue culture plate and treated with TGFβ-neutralizing antibody (10 μg/ml) or control normal IgG and allowed to attach for 3 h. Following attachment, cells were treated with EB1089 (1 × 10−8m), while control cells were treated with vehicle only. At 51 h post-transfection, cells were harvested with 100 μl of lysis buffer (Luciferase assay system, Promega). Luciferase activity was determined according to the manufacturer's instruction using a luminometer (Berthold Lumat LB 0501) and expressed as relative units after normalization to β-galactosidase. Inconsistent response of MCF-7 cells to TGFβ has been observed in several laboratories (34Zugmaier G. Ennis B.W. Deschauer B. Katz D. Knabbe C. Wilding G. Daly P. Lippman M.E. Dickson R.B. J. Cell. Physiol. 1989; 141: 353-361Crossref PubMed Scopus (122) Google Scholar,35Jeng M.H. Dijke P.T. Iwata K.K. Jordan V.C. Mol. Cell. Endocrinol. 1993; 97: 115-123Crossref PubMed Scopus (47) Google Scholar), probably due to growth selection during long term passage of cultures. Having obtained both early (150) and late (>500) passage MCF-7 cells, we decided to first test whether they responded differently to TGFβ (Fig. 1). MCF-7 E cells showed a significant dose-dependent inhibition by TGFβ with an IC50 of 0.2 ng/ml. In contrast, MCF-7 L cells demonstrated complete resistance to TGFβ up to 25 ng/ml (Fig. 1). As described below, MCF-7 E cells expressed RII mRNA and protein in contrast to MCF-7 L cells, which had 5-fold less mRNA and no detectable cell surface protein. Effects of 1,25-(OH)2 D3 and its analogues on cell proliferation of MCF-7 cells were investigated by assessing [3H]thymidine incorporation following treatment by these compounds as described under “Materials and Methods.” MCF-7 E cells showed a dose-dependent inhibition by vitamin D3 with an IC50 of 5 × 10−8m. In contrast, MCF-7 L cells were not affected by vitamin D3 (Fig. 2 A). Vitamin D3 analogues EB1089 and MC903 demonstrate similar growth-inhibitory patterns (Fig. 2, B and C). The overall potency of growth inhibition by EB1089 was approximately 2 orders of magnitude higher than vitamin D3. MCF-7 E cells showed an IC50 of 2.5 × 10−10m, and MCF-7 L cells did not respond to EB1089 treatment up to 1 × 10−7m. The correlation between TGFβ and vitamin D3 sensitivity suggested that vitamin D3 may function through increasing TGFβ autocrine-negative activity in MCF-7 E cells. To test this hypothesis, TGFβ-neutralizing antibodies were used to determine whether they were capable of blocking the growth inhibition induced by these compounds (Fig. 3). At 10 μg/ml, TGFβ1-neutralizing antibody reversed the inhibitory effect of vitamin D3 and its analogues, generating an approximately 60% increase in DNA synthesis as compared with the normal chicken IgG treatment. In contrast, MCF-7 L cells did not respond to TGFβ1-neutralizing antibody, indicating a lack of induction of autocrine TGFβ activity. These results indicate that the growth-inhibitory mechanism of vitamin D3 involves induction of TGFβ autocrine-negative activity in MCF-7 E cells. Increased autocrine TGFβ activity could result from enhanced expression of TGFβ isoforms and/or their receptors. To test these possibilities, RNase protection assays were initially carried out on MCF-7 E cells to determine whether there were alterations of TGFβ isoform expression upon treatment with vitamin D3 compounds. MCF-7 E cells expressed high levels of TGFβ1 mRNA and low levels of TGFβ2and TGFβ3 mRNA. Treatment with EB1089 did not generate altered mRNA expression for any of the three TGFβ isoforms (data not shown). In addition, enzyme-linked immunosorbent assay analysis of the conditioned medium showed no significant increase in the levels of activated TGFβ1 protein (data not shown). Since the levels of activated TGFβ cannot be determined by enzyme-linked immunosorbent assay analysis, a growth inhibition bioassay on mink lung epithelial cells was performed. The condition medium from EB1089-treated and -untreated MCF-7 E cells was added to mink lung epithelial cells as described under “Materials and Methods.” After exposure to either treated or untreated conditioned medium, no significant difference in growth inhibition was observed in the mink lung epithelial cells (Fig. 4). These results indicate that EB1089 treatment did not alter the activation of secreted growth and/or inhibitory peptides from MCF-7 E cells, one of which is likely to be TGFβ1, as demonstrated by enzyme-linked immunosorbent assay analysis. Taken together, these results suggest that the enhanced TGFβ autocrine activity by vitamin D3 did not result from modulation of ligand expression or activation. The other possibility for increased autocrine TGFβ activity upon treatment with vitamin D3 compounds was induction of receptor expression; therefore, we determined whether vitamin D3 analogue treatment modulated expression of RII mRNA. EB1089 (10−8m) was utilized to determine the kinetic effects on RII expression. MCF-7 E cells expressed 5-fold higher RII mRNA than MCF-7 L cells. After exposure to EB1089, a 3-fold increase in the RII mRNA levels of MCF-7 E cells was observed. In contrast, no significant modulation was noted for the MCF-7 L cells after exposure to EB1089 (Fig. 5). EB1089 treatment did not effect the levels of RI or RIII mRNA (data not shown). The increase in MCF-7 E RII mRNA expression led us to examine whether this corresponded to an increase in cell surface RII protein. This was tested by receptor cross-linking with 125I-labeled TGFβ (Fig. 6 A). The GEO cell line, which expresses all three types of TGFβ receptors, was used as a positive control (lane 1). The specificity of cross-linking was demonstrated by competing with 100-fold cold TGFβ1 (lane 2). MCF-7 E cells expressed all three types of receptors (lane 3). Treatment of these cells with the indicated concentrations of vitamin D3 or EB1089 resulted in a 3–4-fold induction of RII, while expression levels of RI remained relatively unchanged (Fig. 6 A). Compared with the MCF-7 E cells, no cell surface RII protein was detected in MCF-7 L cells. Treatment with the vitamin D3 compounds did not result in any change in receptor expression of these cells (Fig. 6 B). To determine if the induction of RII protein in MCF-7 E cells was also time-dependent, a kinetic study was performed. Receptor cross-linking assays revealed a time-dependent increase of cell surface RII protein (Fig. 7 A). Induction was detected as early as 8 h with a 3–4-fold increase after 24 h of treatment.Figure 7Kinetics of induction of cell surface TGFβ RII. Exponentially growing MCF-7 E cells were treated with EB1089 (10−8m) for the indicated time periods.A, receptor cross-linking was performed as described under “Materials and Methods.” RI, RII, and RIII were visualized by autoradiography. B, the RII bands were quantified densitometrically with an Ambis scanning system and presented as -fold increase compared with the control at 0 h. The autoradiograph is representative of two separate experiments.View Large Image Figure ViewerDownload (PPT) To evaluate whether RII induction by treatment with EB1089 enhanced autocrine TGFβ sensitivity, TGFβ-dependent promoter activity was analyzed using the TGFβ-responsive cyclin A luciferase reporter construct (32Yoshizumi M. Wang H. Hsieh C-M. Sibinga N.E.S. Perrella M.A. Lee M-E. J. Biol. Chem. 1997; 272: 22259-22264Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). TGFβ induces down-regulation of cyclin A promoter activity but requires a functional TGFβ type I and II receptor complex (32Yoshizumi M. Wang H. Hsieh C-M. Sibinga N.E.S. Perrella M.A. Lee M-E. J. Biol. Chem. 1997; 272: 22259-22264Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 33Feng X.-H. Filvaroff E.H. Derynck R. J. Biol. Chem. 1995; 270: 24237-24245Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Thus, an increase in functional receptor levels would result in enhanced down-regulation of cyclin A promoter activity. The cyclin A reporter construct (−133/−2) contains only the activating transcription factor site, which has been shown to mediate down-regulation of cyclin A promoter activity by TGFβ1 in mink lung epithelial cells (32Yoshizumi M. Wang H. Hsieh C-M. Sibinga N.E.S. Perrella M.A. Lee M-E. J. Biol. Chem. 1997; 272: 22259-22264Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This reporter construct was transiently transfected into MCF-7 E cells, which are sensitive to TGFβ, followed by treatment with TGFβ-neutralizing antibody and EB1089 as described under “Materials" @default.
• W2047880777 created "2016-06-24" @default.
• W2047880777 creator A5008511483 @default.
• W2047880777 creator A5012581623 @default.
• W2047880777 creator A5037014608 @default.
• W2047880777 creator A5042562989 @default.
• W2047880777 creator A5090038165 @default.
• W2047880777 date "1998-03-01" @default.
• W2047880777 modified "2023-10-17" @default.
• W2047880777 title "Regulation of Transforming Growth Factor-β Type II Receptor Expression in Human Breast Cancer MCF-7 Cells by Vitamin D3and Its Analogues" @default.
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