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• W2054292890 abstract "In the present study we investigated the actions of transforming growth factor (TGF)-β1 on gene induction and cyclin-dependent kinase inhibitors in relation to TGF-β receptor modulation in COLO-357 pancreatic cancer cells. TGF-β1 inhibited the growth of COLO-357 cells in a time- and dose-dependent manner and caused a rapid but transient increase in plasminogen activator inhibitor-I and insulin-like growth factor binding protein-3 mRNA levels. TGF-β1 caused a delayed but sustained increase in the protein levels of the cyclin-dependent kinase inhibitors p15Ink4B, p21Cip1, and p27Kip1 and a sustained increase in type I and II TGF-β receptors (TβRI and TβRII) mRNA and protein levels. The protein synthesis inhibitor cycloheximide (10 μg/ml) completely blocked the TGF-β1-mediated increase in TβRI and TβRII expression. Furthermore, a nuclear runoff transcription assay revealed that the increase in receptor mRNA levels was due to newly transcribed RNA. There was a significant increase in TβRI and TβRII mRNA levels in confluent cells in comparison to subconfluent (≤80% confluent) controls, as well as in serum- starved cells when compared with cells incubated in medium containing 10% fetal bovine serum. COLO-357 cells expressed a normal SMAD4 gene as determined by Northern blot analysis and sequencing. These results indicate that TGF-β1 modulates a variety of functions in COLO-357 cells and up-regulates TGF-β receptor expression via a transcriptional mechanism, which has the potential to maximize TGF-β1-dependent antiproliferative responses. In the present study we investigated the actions of transforming growth factor (TGF)-β1 on gene induction and cyclin-dependent kinase inhibitors in relation to TGF-β receptor modulation in COLO-357 pancreatic cancer cells. TGF-β1 inhibited the growth of COLO-357 cells in a time- and dose-dependent manner and caused a rapid but transient increase in plasminogen activator inhibitor-I and insulin-like growth factor binding protein-3 mRNA levels. TGF-β1 caused a delayed but sustained increase in the protein levels of the cyclin-dependent kinase inhibitors p15Ink4B, p21Cip1, and p27Kip1 and a sustained increase in type I and II TGF-β receptors (TβRI and TβRII) mRNA and protein levels. The protein synthesis inhibitor cycloheximide (10 μg/ml) completely blocked the TGF-β1-mediated increase in TβRI and TβRII expression. Furthermore, a nuclear runoff transcription assay revealed that the increase in receptor mRNA levels was due to newly transcribed RNA. There was a significant increase in TβRI and TβRII mRNA levels in confluent cells in comparison to subconfluent (≤80% confluent) controls, as well as in serum- starved cells when compared with cells incubated in medium containing 10% fetal bovine serum. COLO-357 cells expressed a normal SMAD4 gene as determined by Northern blot analysis and sequencing. These results indicate that TGF-β1 modulates a variety of functions in COLO-357 cells and up-regulates TGF-β receptor expression via a transcriptional mechanism, which has the potential to maximize TGF-β1-dependent antiproliferative responses. Transforming growth factor (TGF) 1The abbreviations used are: TGF, transforming growth factor; PAI, plasminogen activator inhibitor; IGF, insulin-like growth factor; IGFBP, IGF binding protein; TβRI and TβRII, type I and II TGF-β receptors; MTT, 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum; PCR, polymerase chain reaction; Cdk, cyclin-dependent kinase. 1The abbreviations used are: TGF, transforming growth factor; PAI, plasminogen activator inhibitor; IGF, insulin-like growth factor; IGFBP, IGF binding protein; TβRI and TβRII, type I and II TGF-β receptors; MTT, 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum; PCR, polymerase chain reaction; Cdk, cyclin-dependent kinase.-β1 is a multifunctional cytokine that plays an important role in regulating cellular growth and differentiation in many biological systems (1Attisano L. Wrana J.L. Cytokine Growth Factor Rev. 1996; 4: 327-339Crossref Scopus (143) Google Scholar). TGF-β1 induces growth inhibitory or stimulatory responses, depending on the cell type and growth conditions (2Sporn M.B. Roberts A.B. Nature. 1988; 332: 217-219Crossref PubMed Scopus (653) Google Scholar, 3Roberts A.B. Anzano M.A. Wakefield L.M. Roche N.S. Stern D.F. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 119-123Crossref PubMed Scopus (968) Google Scholar). In most epithelial cells, TGF-β1 inhibits growth while enhancing the production of extracellular matrix proteins (4Moses H.L. Tucker R.F. Leof E.B. Coffey Jr., R.J. Halper J. Shipley G.D. Feramisco J. Ozane B. Stiles C. Cancer Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1985: 65-75Google Scholar). TGF-β1 signals through a family of transmembrane receptors that have intrinsic serine/threonine kinase activity (1Attisano L. Wrana J.L. Cytokine Growth Factor Rev. 1996; 4: 327-339Crossref Scopus (143) Google Scholar). The type II TGF-β receptor (TβRII) binds TGF-β1 and then forms a heteromeric complex with the type I TGF-β receptor (TβRI). This complex consists most likely of two type I and type II receptors (1Attisano L. Wrana J.L. Cytokine Growth Factor Rev. 1996; 4: 327-339Crossref Scopus (143) Google Scholar). Activated TβRII transphosphorylates the glycine- and serine-rich domain of the type I receptor kinase, thereby activating TβRI, which then transiently associates with and phosphorylates SMAD2 and/or SMAD3. These proteins belong to a recently discovered family of intracellular signaling molecules (1Attisano L. Wrana J.L. Cytokine Growth Factor Rev. 1996; 4: 327-339Crossref Scopus (143) Google Scholar). Phosphorylated SMAD2 and/or SMAD-3 form a heteromeric complex with SMAD4, which is required for the translocation of both proteins into the nucleus, where they can act as transcriptional activators (1Attisano L. Wrana J.L. Cytokine Growth Factor Rev. 1996; 4: 327-339Crossref Scopus (143) Google Scholar, 5Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (808) Google Scholar, 6Massague J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar, 7Wu R.-Y. Zhang Y. Feng X.-H. Derynck R. Mol. Cell. Biol. 1997; 17: 2521-2528Crossref PubMed Scopus (186) Google Scholar). TGF-β1 is thus able to induce the expression of growth inhibitory proteins that suppress cell cycle progression, such as the cyclin-dependent kinase (Cdk) inhibitors p15Ink4B, p21Cip1, and p27Kip1(8Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar, 9Hannon G.J. Beach D. Nature. 1994; 371: 257-261Crossref PubMed Scopus (1884) Google Scholar, 10Polyak K. Kato J.-Y. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. Genes Dev. 1994; 8: 9-22Crossref PubMed Scopus (1826) Google Scholar, 11Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar) or proteins that interfere with mitogenic signaling, such as insulin-like growth factor binding protein-3 (IGFBP-3) (12Valentinis B. Bhala A. DeAngelis T. Baserga R. Cohen P. Mol. Endocrinol. 1995; 9: 361-367Crossref PubMed Google Scholar, 13Rajah R. Valentinis B. Cohen P. J. Biol. Chem. 1997; 272: 12181-12188Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). However, depending on the cell type, TGF-β1 activates or inhibits IGFBP-3 transcription (14Erondu N.E. Dake B.L. Moser D.R. Lin M. Boes M. Bar R.S. Growth Regul. 1996; 6: 1-9PubMed Google Scholar, 15Hembree J.R. Pampusch M.S. Yang F. Causey J.L. Hathaway M.R. Dayton W.R. J. Anim. Sci. 1996; 74: 1530-1540Crossref PubMed Scopus (32) Google Scholar). In some cell types growth inhibition may be mediated via a pathway that is distinct from the signaling pathway regulating expression of genes that modulate the extracellular matrix. For example, TGF-β1 is able to induce the expression of a number of genes such as PAI-I (16Carcamo J. Weis F.M.B. Ventura F. Wieser R. Wrana J.L. Attisano L. Massague J. Mol. Cell. Biol. 1994; 14: 3810-3821Crossref PubMed Google Scholar), which inhibits both tissue-type and urokinase-type plasminogen activators (17Sprengers E.D. Kluft C. Blood. 1987; 69: 381-387Crossref PubMed Google Scholar). In mink lung epithelial cells, expression of a truncated TβRII does not attenuate TGF-β1-mediated induction of PAI-I. In contrast, expression of the truncated TβRII renders these cells resistant to the antiproliferative effects of TGF-β1 (18Chen R.-H. Ebner R. Derynck R. Science. 1993; 260: 1335-1338Crossref PubMed Scopus (357) Google Scholar). Cultured human pancreatic cancer cell lines are usually resistant to the growth inhibitory effects of TGF-β1 (19Baldwin R.L. Friess H. Yokoyama M. Lopez M.E. Kobrin M.S. Büchler M.W. Korc M. Int. J. Cancer. 1996; 67: 283-288Crossref PubMed Scopus (116) Google Scholar). This resistance may be the consequence of a number of alterations, including the presence of mutated TGF-β receptors (20Takenoshita S. Hagiwara K. Nagashima M. Gemma A. Bennett W.P. Harris C.C. Genomics. 1996; 36: 341-344Crossref PubMed Scopus (39) Google Scholar), decreased expression of TβRI (19Baldwin R.L. Friess H. Yokoyama M. Lopez M.E. Kobrin M.S. Büchler M.W. Korc M. Int. J. Cancer. 1996; 67: 283-288Crossref PubMed Scopus (116) Google Scholar) or TβRII (21Freeman J.W. Mattingly C.A. Strodel W.E. J. Cell. Physiol. 1995; 165: 155-163Crossref PubMed Scopus (62) Google Scholar), and mutations in the SMAD4/DPC4 gene (22Hahn S.A. Schutte M. Hoque A.T.M. Moskaluk C.A. daCosta L.T. Rozenblum E. Weinstein C.L. Fischer A. Yeo C.J. Hruban R.H. Kern S.E. Science. 1996; 271: 350-353Crossref PubMed Scopus (2161) Google Scholar). Although, COLO-357 pancreatic cancer cells are relatively sensitive to TGF-β1-mediated growth inhibition (23Baldwin R.L. Korc M. Growth Factors. 1993; 8: 23-34Crossref PubMed Scopus (68) Google Scholar), the mechanisms underlying this sensitivity are not known. Furthermore, it has not been established whether TGF-β1 modulates the expression of growth-regulating genes in these cells. Therefore, in the present study we characterized the effects of TGF-β1 on the expression of PAI-I, IGFBP-3, p15Ink4B, p21Cip1, and p27Kip1 in relation to its effects on the expression of TβRI and TβRII. We now report that TGF-β1 induces rapid but transient up-regulation of PAI-I and IGFBP-3 mRNA in COLO-357 cells and enhances p15Ink4B, p21Cip1, and p27Kip1 and TβRI/II expression in a sustained manner in these cells. The following materials were purchased: FBS, Dulbecco's modified Eagle's medium, trypsin solution, and penicillin-streptomycin solution from Irvine Scientific (Santa Ana, CA); Genescreen membranes from NEN Life Science Products; Immobilon P membranes from Millipore (Bedford, MA); restriction enzymes and random primed labeling kit from Boehringer Mannheim; Sequenase Version 1.0 DNA sequencing kit, and leupeptin from U. S. Biochemicals (Cleveland, OH); [α-32P]dCTP, [α-32P]UTP, γ-35S-labeled dATP, ECL blotting kit, and biotinylated goat anti-rabbit IgG from Amersham Corp.; anti- TβRI (V22), anti-TβRII (H567), anti-p15Ink4B (C20), and anti-p27Kip1 (C19) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-p21Cip1 (AB-3) antibodies from NeoMarkers (Fremont, CA); PCR primers from Bio Synthesis, Inc. (Lewisville, TX); reverse transcriptase kit from Life Technologies, Inc. All other reagents were from Sigma. TGF-β1 was a gift from Genentech, Inc. (South San Francisco, CA); COLO-357 cells were a gift from Dr. R. S. Metzger (Durham, NC). COLO-357 were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (complete medium). For TGF-β1 experiments, subconfluent cells were incubated overnight in serum-free medium (Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, and antibiotics), and subsequently incubated for the indicated time with the indicated additions. Cells were then harvested for protein or RNA extraction. To perform growth assays, COLO-357 cells were plated overnight at a density of 10,000 cells/well in 96-well plates and subsequently incubated in serum-free medium in the absence or presence of TGF-β1. Incubations were continued for the indicated time prior to adding 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 62.5 μg/well) for 4 h (19Baldwin R.L. Friess H. Yokoyama M. Lopez M.E. Kobrin M.S. Büchler M.W. Korc M. Int. J. Cancer. 1996; 67: 283-288Crossref PubMed Scopus (116) Google Scholar). Cellular MTT was solubilized with acidic isopropanol and optical density was measured at 570 nm with an enzyme-linked immunosorbent assay plate reader (Molecular Devices, Menlo Park, CA). In pancreatic cancer cells the results of the MTT assay correlate with results obtained by cell counting with a hemocymeter and by monitoring [3H]thymidine incorporation (23Baldwin R.L. Korc M. Growth Factors. 1993; 8: 23-34Crossref PubMed Scopus (68) Google Scholar, 24Raitano A.B. Korc M. J. Biol. Chem. 1990; 265: 10466-10472Abstract Full Text PDF PubMed Google Scholar). Total RNA was extracted by the single step acid guadinium thiocyanate phenol chloroform method. RNA was size fractionated on 1.2% agarose/1.8m formaldehyde gels, electrotransferred onto nylon membranes and cross-linked by UV irradiation (25Korc M. Chandrasekar B. Yamanaka Y. Friess H. Buechler M.W. Beger H.G. J. Clin. Invest. 1992; 90: 1352-1360Crossref PubMed Scopus (493) Google Scholar). Blots were prehybridized and hybridized with cDNA probes (TβRII, PAI-I, IGFBP-3, 7S) or a TβRI riboprobe and washed under high stringency conditions as previously reported (25Korc M. Chandrasekar B. Yamanaka Y. Friess H. Buechler M.W. Beger H.G. J. Clin. Invest. 1992; 90: 1352-1360Crossref PubMed Scopus (493) Google Scholar). Blots were then exposed at −80 °C to Kodak XAR-5 films and the intensity of the radiographic bands was quantified by densitometric analysis. The following cDNA probes were used: a human TβRI and TβRII fragment as previously reported (19Baldwin R.L. Friess H. Yokoyama M. Lopez M.E. Kobrin M.S. Büchler M.W. Korc M. Int. J. Cancer. 1996; 67: 283-288Crossref PubMed Scopus (116) Google Scholar), a 500-base pair SacII PstI fragment of PAI-I (obtained from Dr. H. Friess, University of Bern, Switzerland), a 475-base pair EcoRV fragment of IGFBP-3 (obtained from Dr. S. Shimasaki, University of California, San Diego), and a 1.7-kilobase pair SMAD4 construct (obtained from Dr. J. Massague, Memorial Sloan-Kettering Cancer Center, New York). ABamHI 190-base pair fragment of mouse 7S cDNA that hybridizes with human cytoplasmatic RNA was used to confirm equal RNA loading (25Korc M. Chandrasekar B. Yamanaka Y. Friess H. Buechler M.W. Beger H.G. J. Clin. Invest. 1992; 90: 1352-1360Crossref PubMed Scopus (493) Google Scholar). Cells were washed with phosphate-buffered saline (4 °C) and solubilized in lysis buffer containing 50 mm Tris, 150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate, 1 mmsodium vanadate, 50 mm sodium fluoride, 100 μg/ml benzamidine, 10 μg/ml leupeptin, 100 μg/ml benzamidine, and 1 mm phenylmethylsulfonyl fluoride. Proteins were subjected to SDS-polyacrylamide gel electrophoresis and transferred to Immobilon P membranes. Membranes were incubated for 90 min with anti-TβRI (50 ng/ml), anti-TβRII (50 ng/ml), anti-p15Ink4B (200 ng/ml), anti-p21Cip1 (200 ng/ml), or anti-p27Kip1 (200 ng/ml) antibodies, washed, and incubated with a secondary antibody against mouse (anti-p21Cip1) or rabbit IgG for 60 min. After washing, visualization was performed by enhanced chemiluminescence. 2 μg of RNA, isolated as described earlier, were reversed transcribed using the manufacturer's recommended reaction conditions. PCR reactions were performed using 50-μl reaction mixtures containing 1 μl of cDNA mix, 5 μl of 10× PCR buffer, 1.5 mm MgCl2, 1 mm dNTP, 2 units of Taq polymerase, and 0.5 μm for each primer. The thermal cycling was programmed as follows: initial denaturation at 94 °C for 10 min; 40 cycles of 30 s at 94 °C for denaturation, 1 min at 55 °C for annealing, and 1 min at 72 °C for extension; the final extension was at 72 °C for 10 min. The following primers were used for SMAD4 amplification: sense 1, 5′-AGCAAGCTTGCTTCAGAAATTGGAGAC, antisense 1, 5′-AGCGAATTCCCCAAAGTCATGCACAT (26Takagi Y. Kohmura H. Futamura M. Kida H. Tanemura H. Shimokawa K. Saji S. Gastroenterology. 1996; 111: 1369-1372Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), sense 2, 5′-GAAGAATTCAAGTATGATGGTGAAGGATG, antisense 2, 5′-TACAAGCTTCATTCCAACTGCACACCT, sense 3, 5′-AGCAAGCTTCATTGAGAGAGCAAGGTTGCAC, antisense 3, 5′-AGCGGATCCCATCCTGATAAGGTTAAGGGC (26Takagi Y. Kohmura H. Futamura M. Kida H. Tanemura H. Shimokawa K. Saji S. Gastroenterology. 1996; 111: 1369-1372Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). PCR products were digested with the appropriate restriction enzymes and subcloned into pBluescript-IISK+ vector. Sequence analysis was performed using the Sequenase Version 1.0 DNA sequencing kit according to the recommended protocol. To measure specific gene transcription, COLO-357 cells (5 × 106 cells/sample) were trypsinized and resuspended in complete medium. After centrifugation at 500 × g for 5 min, the cell pellet was resuspended and washed in phosphate-buffered saline (4 °C) and centrifuged at 500 × g for 5 min. Cells were then lysed in 4 ml of lysis buffer containing 10 mm Tris (pH 7.4), 10 mm NaCl, 3 mm MgCl2, and 0.5% Nonidet P-40. After a second 500 × g (5 min) centrifugation step, the nuclear pellet was resuspended in 4 ml of lysis buffer and centrifuged again. The pellet was then resuspended in 200 μl of storage buffer containing 50 mm Tris (pH 8.3), 5 mm MgCl2, 0.1 mm EDTA, and 40% glycerol and stored at −80 °C for subsequent transcription. Nuclear runoff assays were initiated by incubating 200 μl of the frozen nuclei with 200 μl of reaction buffer containing 5 μl of 1m dithiothreitol, and 2 μl of 100 mm ATP, CTP, GTP, and 10 μl of 10 mCi/ml [α-32P]UTP for 30 min at 30 °C. Subsequently, 40 μl of 1 mg/ml DNase I and 1 ml of HSB buffer (0.5 m NaCl, 50 mmMgCl2, 2 mm CaCl2, 10 mm Tris, pH 7.4) were added and incubated for 5 min at 30 °C. After incubating samples for 30 min (42 °C) with 10 μl of proteinase K in 200 μl of SDS/Tris buffer consisting of 5% SDS, 0.5 m Tris, pH 7.4, and 0.125 m EDTA, nuclear RNA was extracted as described previously (25Korc M. Chandrasekar B. Yamanaka Y. Friess H. Buechler M.W. Beger H.G. J. Clin. Invest. 1992; 90: 1352-1360Crossref PubMed Scopus (493) Google Scholar) and dissolved in 0.5 ml of diethyl pyrocarbonate-H2O. When necessary, samples were diluted in diethyl pyrocarbonate-H2O to adjust32P-labeled RNA equally for each sample to ≥5 × 106 cpm/ml. Linearized TβRI and TβRII cDNA (100 μg) was denatured by incubating samples for 30 min (23 °C) in 0.2m NaOH and neutralized with 6× SSC. cDNA samples (5 μg) were slot blotted onto nylon membranes and UV cross-linked. Membranes were preincubated at 42 °C as described earlier and incubated at 42 °C with 32P-labeled RNA samples for 24 h. Membranes were washed twice in 2× SSC at 65 °C for 30 min, incubated for 30 min at 37 °C with 10 mg/ml RNase A in 2× SSC to remove unbound 32P-labeled RNA, washed again in 2× SSC at 37 °C for 1 h, and exposed at −80 °C to Kodak XAR-5 films using intensifying screens. To confirm that COLO-357 cells are sensitive to TGF-β1-mediated growth inhibition (19Baldwin R.L. Friess H. Yokoyama M. Lopez M.E. Kobrin M.S. Büchler M.W. Korc M. Int. J. Cancer. 1996; 67: 283-288Crossref PubMed Scopus (116) Google Scholar), cells were incubated in the absence (control) or presence of TGF-β1 (Fig. 1). TGF-β1 significantly inhibited growth after a 48-h incubation, maximal effects occurring at 100 pm TGF-β1 (−35.8% ± 6.3%, p < 0.01). Following a more prolonged incubation (72 h), 100 pmTGF-β1 inhibited the growth of COLO-357 cells by 47.7% ± 4.5% (p < 0.01). To study the effects of TGF-β1 on the expression of PAI-I and IGFBP-3 mRNA, Northern blot analysis was performed using total RNA extracted from control and TGF-β1-treated (200 pm) COLO 357 cells. TGF-β1 caused a time-dependent increase in PAI-I and IGFBP-3 mRNA levels (Fig. 2). Densitometric analysis revealed a rapid increase in PAI-I mRNA levels, with maximal stimulation (11-fold) occurring within 3 h. PAI-I mRNA levels then decreased gradually but were still elevated above baseline levels (5-fold) after 48 h of incubation. IGFBP-3 mRNA levels also increased maximally (4-fold) after 3 h of TGF-β1 stimulation. In contrast to PAI-I mRNA levels, IGFBP-3 mRNA returned to control levels after 6 h. To investigate the effects of TGF-β1 on the levels of the Cdk inhibitors p15Ink4B, p21Cip1, and p27Kip1, 60% confluent COLO-357 cells were initially incubated in serum-free medium for 12 h. Cells were then incubated in serum-free medium for an additional 48 h in the absence or presence of 200 pm TGF-β1, which was present for 12, 24, and 48 h prior to analysis. Lysates were then analyzed by immunoblotting with specific antibodies. Initially, TGF-β1 did not increase p15Ink4B, p21Cip1, and p27Kip1 protein levels. The mild decrease observed after 12 h (Fig. 3) was inconsistent. In contrast, after 24 h, TGF-β1 caused a marked and consistent increase in all three cyclin-dependent kinase inhibitors, and this effect was consistently sustained for at least 48 h (Fig. 3). To investigate the effects of TGF-β1 on the expression of TGF-β receptors, we first sought to determine the effects of serum and cell confluency on TβRI and TβRII mRNA expression. COLO-357 cells were incubated in complete medium until they were 70% confluent and then incubated an additional 12 h in serum-free or complete medium. Densitometric analysis revealed that following 12 h of serum starvation there was a 5- and 6-fold increase in TβRI and TβRII mRNA levels, respectively, compared with control cells incubated with medium containing 10% FBS (Fig. 4). Prolonged incubation (24, 48, and 72 h) of COLO-357 cells in serum-free medium did not significantly alter TβRI/II mRNA levels by comparison with the 12-h incubation (data not shown), suggesting that a steady state of TβRI/II expression was achieved within the first 12 h of serum starvation. Next, COLO-357 cells were incubated in complete medium and grown to 60, 80, and 100% confluency. There was a 6- and 2-fold increase in TβRI and TβRII mRNA levels, respectively, in 100% confluent cells compared with that of the 60% confluent cells (Fig. 4). In contrast, receptor expression was comparable in 60 and 80% confluent cells. To assure that serum starvation did not contribute to any ligand-induced change in receptor expression, all subsequent experiments were carried out after 12 h of serum starvation, so that the effects of serum starvation were already maximal prior to ligand addition. Conversely, to minimize the effects of cell confluency and serum on TβRI/II expression, all subsequent experiments were carried with cell confluency not exceeding 80% during the entire course of the experiments. Accordingly, COLO-357 cells were grown to 60% confluency and initially incubated in serum-free medium for 12 h. Cells were subsequently incubated in serum-free medium for 48 h in the absence or presence of 200 pm TGF-β1, which was added 3, 6, 12, 24, and 48 h prior to RNA extraction. Under these experimental conditions, TGF-β1 caused a 4-fold increase in TβRI mRNA after 24 h and a further increase (6-fold) after 48 h in comparison to the unstimulated control (Fig. 5 A). Similarly, TβRII mRNA levels exhibited a 3-fold increase after 24 h of incubation with 200 pm TGF-β1, and this increase was sustained for at least 48 h (4-fold). To determine whether the TGF-β1-induced increase in steady-state TβRI and TβRII mRNA levels was associated with an increased in the corresponding protein levels, COLO-357 cells were incubated in the absence or presence of 200 pm TGF-β1 under the same conditions, and protein lysates were analyzed by SDS-polyacrylamide gel electrophoresis. TβRI protein levels were slightly elevated after 24 h and markedly increased after 48 h in comparison with control cells. In contrast, TβRII protein up-regulation was evident within 12 h of TGF-β1 addition and maximal after 48 h (Fig. 5 B). Thus, the effects of TGFβ1 on TβRI/II protein levels paralleled its effects on the corresponding mRNA levels. To determine whether TGF-β1-mediated receptor up-regulation requires protein synthesis, COLO-357 cells were incubated 24–48 h in the presence or absence of 10 μg/ml cycloheximide or 1 nmTGF-β1. Cycloheximide did not cause cell death and did not significantly alter basal TβRI or TβRII mRNA levels (Fig. 6, A and B). In contrast, after both 24 h (Fig. 6 A) and 48 h (Fig. 6 B), cycloheximide completely blocked the TGF-β1-mediated increase in TβRI/II mRNA levels. Cycloheximide also markedly attenuated the TGF-β1-induced increase in TβRI/II protein levels (Fig. 6 C). Thus, TGF-β1-mediated up-regulation of TβRI/II is dependent on new protein synthesis. To determine whether up-regulation of TβRI/II expression was due to increased RNA synthesis or enhanced stability of the mRNA moieties, the effects of TGF-β1 on newly transcribed RNA were examined next with the nuclear runoff transcription assay. COLO-357 cells were grown to 60% confluency, serum-starved for 12 h, and incubated in serum-free medium for 48 h in the absence or presence of 1 nm TGF-β1 for 3, 6, 12, 24, and 48 h prior to analysis. No increase of newly transcribed TβRI/II mRNA was observed during the first 12 h of stimulation. However, there was a marked increase in newly transcribed TβRI and TβRII mRNA after a 24- or 48-h incubation with TGF-β1 (Fig. 7). COLO-357 cells were recently reported to harbor a homozygous deletion involving exons 1–4 of SMAD4(27Schutte M. Hruban R.H. Hedrick L. Cho K.R. Nadasdy G.M. Weinstein C.L. Bova G.S. Isaacs W.B. Cairns P. Nawroz H. Sidransky D. Casero Jr., R.A. Meltzer P.S. Hahn S.A. Kern S.E. Cancer Res. 1996; 56: 2527-2530PubMed Google Scholar). In view of the importance of SMAD4 in TGF-β1-dependent signaling, we examined next the status of SMAD4 in our COLO 357 cells. Northern blot analysis of total RNA from COLO-357 cells clearly demonstrated a SMAD4transcript in these cells that had the same size (approximately 4.5 kilobase pairs) as the SMAD4 transcript in human placenta (Fig. 8). Next, three reverse transcriptase-PCR fragments of the SMAD4 gene covering its entire coding region were sequenced, revealing that COLO-357 cells did not harbor deletions or mutations in the SMAD4 gene (data not shown). An important mechanism for regulating the cellular response to cytokines and hormones resides at the level of receptor expression. It has been shown by Northern blot analysis and binding studies that 1,25-dihydroxyvitamin D3 and prostaglandin E2down-regulate TβRII expression in human osteoblastic cells and human fibroblasts, respectively (28Fine A. Panchenko M.P. Smith B.D. Yu Q. Goldstein R.H. Biochim. Biophys. Acta. 1995; 1261: 19-24Crossref PubMed Scopus (15) Google Scholar, 29Iimura T. Oida S. Ichijo H. Goseki M. Maruoka Y. Takeda K. Sasaki S. Biochem. Biophys. Res. Commun. 1994; 204: 918-923Crossref PubMed Scopus (26) Google Scholar). Furthermore binding studies revealed down-regulation of TβRI in human monocytes by interferon-γ (30Brandes M.E. Wakefield L.M. Wahl S.M. J. Biol. Chem. 1990; 266: 19697-19703Google Scholar). Conversely, in human lung fibroblasts (31Bloom B.B. Humphries D.E. Kuang P.-P. Fine A. Goldstein R.H. Biochim. Biophys. Acta. 1996; 1312: 243-248Crossref PubMed Scopus (57) Google Scholar) and human corpus carvernosum smooth muscle cells (32Moreland R.B. Traish A. McMillan M.A. Smith B. Goldstein I. de Tejada I.S. J. Urol. 1985; 153: 826-834Crossref Scopus (218) Google Scholar), TGF-β1 increases steady-state levels of TβRI mRNA, potentially by increasing TβRI promotor activity (31Bloom B.B. Humphries D.E. Kuang P.-P. Fine A. Goldstein R.H. Biochim. Biophys. Acta. 1996; 1312: 243-248Crossref PubMed Scopus (57) Google Scholar). However, little is known about the regulation of TβRII by TGF-β1. In the present study we determined that TGF-β1 enhances both TβRI and TβRII expression levels in COLO-357 pancreatic cancer cells. This effect occurred in a time-dependent manner with a marked increase after 24 and 48 h. This increase in mRNA levels was associated with enhanced protein synthesis of both receptors. Two lines of evidence indicate that the TGF-β1-induced increase in TβRI and TβRII mRNA levels was effected at the level of transcription. First, blocking protein synthesis with cycloheximide completely abrogated the TGF-β1- induced TβRI/II up-regulation. Second, the nuclear runoff transcription assay demonstrated that TGF-β1 acted to enhance transcription of both receptors. In the present study we also determined that TβRI/II is up-regulated following serum starvation, reaching a new steady-state level within 12 h. Moreover confluent COLO-357 cells also exhibited increased TβRI/II mRNA levels compared with subconfluent control cells. It has been shown for the epidermal growth factor receptor, that serum starvation leads to its down-regulation (33Suarez-Quian C.A. Byers S.W. Tissue Cell. 1993; 25: 1-17Crossref PubMed Scopus (14) Google Scholar). Similarly, cell density-dependent down-regulation of several growth factor receptors, such as vascular endothelial growth factor receptor or hepatocyte growth factor receptor, has been demonstrated in a variety of cell types (34Koura A.N. Liu W. Kitadai Y. Singh R.K. Radinsky R. Ellis L.M. Cancer Res. 1996; 56: 3891-3894PubMed Google Scholar, 35Mizuno K. Higuchi O. Tajima H. Yonemasu T. Nakamura T. J. Biochem. (Tokyo). 1993; 114: 96-102Crossref PubMed Scopus (32) Google Scholar). Ostensibly, this down-regulation is an important component of the signaling mechanism that leads to suppression of growth when cells are either deprived of nutrients or approaching confluency. In this context, up-regulation of TβRI and TβRII may serve to enhance growth inhibitory pathways under the same culture conditions (serum-free medium, confluent cells). While the mechanisms that contribute to cell density-dependent TGF-β receptor up-regulation are not known, it has been recently shown that activation of focal adhesion kinase by α2β1 integrins causes decreased surface expression of TβRI and TβRII (36Takeuchi Y. Suzawa M. Kikuchi T. Nishida E. Fujita T. Matsumoto T. J. Biol. Chem. 1997; 272: 29309-29316Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Inasmuch as α2β1 integrins and focal adhesion kinase participate in extracellular matrix-initiated intracellular signaling, our findings raise the possibility that cell-cell contact may also act to activate pathways that modulate TβRI/II gene expression. The mammalian Cdk inhibitors p21Cip1, and p27Kip1 inhibit the activities of cyclin D-Cdk4, cyclin D-Cdk6, cyclin E-Cdk2, and cyclin A-Cdk2, whereas p15Ink4Binterferes specifically with cyclin D binding to Cdk4 and Cdk6 (8Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar, 9Hannon G.J. Beach D. Nature. 1994; 371: 257-261Crossref PubMed Scopus (1884) Google Scholar, 10Polyak K. Kato J.-Y. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. Genes Dev. 1994; 8: 9-22Crossref PubMed Scopus (1826) Google Scholar, 11Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar,37Ravitz M.J. Wenner C.E. Adv. Cancer Res. 1997; 71: 165-207Crossref PubMed Google Scholar). Previous studies in a variety of epithelial cells have demonstrated that TGF-β1 can markedly up-regulate the expression of p21Cip1 and p15Ink4B, but exerts only a small stimulatory effect on p27Kip1 levels (11Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar). In cell lines that are highly sensitive to TGF-β1-mediated growth inhibition, the effect of TGF-β1 on p21Cip1 and p15Ink4Bup-regulation are relatively rapid (11Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar, 37Ravitz M.J. Wenner C.E. Adv. Cancer Res. 1997; 71: 165-207Crossref PubMed Google Scholar). Subsequently, TGF-β1 acts via the increase in p15Ink4B levels to displace p27Kip1 from Cdk4 and Cdk6 (11Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar). However, in normal mouse B lymphocytes, TGF-β1 increases p27Kip1 protein levels (38Bouchard C. Fridman W.H. Sautes C. J. Immunol. 1997; 159: 4155-4164PubMed Google Scholar), and such an effect may be due in part to decreased degradation of the protein (37Ravitz M.J. Wenner C.E. Adv. Cancer Res. 1997; 71: 165-207Crossref PubMed Google Scholar). In the present study, we determined that TGF-β1 causes a delayed but sustained increase in p15Ink4B, p21Cip1, and p27Kip1 protein levels, which was readily and consistently evident only after 24 h. While the molecular mechanisms that lead to this increase of three different Cdk inhibitors in COLO-357 cells are not known, the relatively slow kinetics of this up-regulation underscore our observation that these cells are insensitive to the TGF-β1-mediated growth inhibition during the initial 24 h incubation period (Fig. 1). Conversely, the marked increase in p15Ink4B, p21Cip1, and p27Kip1 protein levels that occurs 24 and 48 h after the addition of TGF-β1 suggests that these Cdk inhibitors are then able to contribute to the inhibitory effect of TGF-β1 on cell growth. These observations also raise the possibility that the delayed up-regulation of the Cdk inhibitors is dependent on the TGF-β1-induced increase in TβR-I/II expression. TGF-β1 caused a rapid increase in PAI-I mRNA levels in COLO-357 cells followed by a less pronounced but sustained increase during the subsequent 48 h. PAI-I is the main inhibitor of the urokinase plasminogen activator system, which is thought to play an important role in cancer cell invasion (40Grondahl-Hansen J. Hilsenbeck S.G. Christensen I.J. Clark G.M. Osborne C.K. Brunner N. Breast Cancer Res. Treat. 1997; 43: 153-163Crossref PubMed Scopus (45) Google Scholar, 41Ito H. Yonemura Y. Fujita H. Tsuchihara K. Kawamura T. Nojima N. Fujimura T. Nose H. Endo Y. Sasaki T. Virchows Arch. 1996; 427: 487-496Crossref PubMed Scopus (50) Google Scholar, 42Liu G. Shuman M.A. Cohen R.L. Int. J. Cancer. 1995; 60: 501-506Crossref PubMed Scopus (158) Google Scholar). Our observation that TGF-β1 up-regulates PAI-I expression in COLO-357 cells suggests that TGFβ-1 derived from the cells may act via PAI-I to enhance their metastatic potential. TGF-β1 also caused a rapid increase in IGFBP-3 mRNA in COLO-357 cells with a maximal response occurring within 3 h of TGF-β1 addition. Growth inhibitory effects of IGFBP-3 may be caused by inhibition of IGF-1-dependent mitogenesis (43Bertherat J. Eur. J. Endocrinol. 1996; 134: 426-427Crossref PubMed Scopus (10) Google Scholar) or by IGF-1-independent mechanisms (12Valentinis B. Bhala A. DeAngelis T. Baserga R. Cohen P. Mol. Endocrinol. 1995; 9: 361-367Crossref PubMed Google Scholar, 44Oh Y. Gucev Z. Ng L. Muller H.L. Rosenfeld R.G. Prog. Growth Factor Res. 1995; 6: 503-512Abstract Full Text PDF PubMed Scopus (71) Google Scholar). TGF-β1 is known to induce divergent effects on IGFBP-3 expression. In endothelial cells, TGF-β1 reduces IGFBP-3 mRNA levels (14Erondu N.E. Dake B.L. Moser D.R. Lin M. Boes M. Bar R.S. Growth Regul. 1996; 6: 1-9PubMed Google Scholar), whereas it causes a dose-dependent increase of IGFBP-3 in porcine myogenic cells (15Hembree J.R. Pampusch M.S. Yang F. Causey J.L. Hathaway M.R. Dayton W.R. J. Anim. Sci. 1996; 74: 1530-1540Crossref PubMed Scopus (32) Google Scholar). In human breast cancer cells TGF-β1 stimulates IGFBP-3 production and induces binding of IGFBP-3 to the cell surface (44Oh Y. Gucev Z. Ng L. Muller H.L. Rosenfeld R.G. Prog. Growth Factor Res. 1995; 6: 503-512Abstract Full Text PDF PubMed Scopus (71) Google Scholar). Irrespective of its role in other cell lines, the transient nature of IGFBP-3 induction observed in the present study suggests that IGFBP-3 does not play a major role in the TGF-β1-induced antiproliferative response in COLO-357 cells. SMAD4 is an important signaling molecule that is downstream of the TβRI and TβRII signaling pathway. It is crucial in mediating TGF-β1 responses (5Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (808) Google Scholar). Pancreatic cancers and cultured pancreatic cancer cell lines often harbor SMAD4 mutations, which lead to loss of TGF-β1-dependent growth suppression (22Hahn S.A. Schutte M. Hoque A.T.M. Moskaluk C.A. daCosta L.T. Rozenblum E. Weinstein C.L. Fischer A. Yeo C.J. Hruban R.H. Kern S.E. Science. 1996; 271: 350-353Crossref PubMed Scopus (2161) Google Scholar, 27Schutte M. Hruban R.H. Hedrick L. Cho K.R. Nadasdy G.M. Weinstein C.L. Bova G.S. Isaacs W.B. Cairns P. Nawroz H. Sidransky D. Casero Jr., R.A. Meltzer P.S. Hahn S.A. Kern S.E. Cancer Res. 1996; 56: 2527-2530PubMed Google Scholar,45Moskaluk C.A. Kern S.E. Biochim. Biophys. Acta. 1996; 1288: M31-M33PubMed Google Scholar). Although COLO-357 cells have been reported to harbor a homozygous deletion in the SMAD4 gene (27Schutte M. Hruban R.H. Hedrick L. Cho K.R. Nadasdy G.M. Weinstein C.L. Bova G.S. Isaacs W.B. Cairns P. Nawroz H. Sidransky D. Casero Jr., R.A. Meltzer P.S. Hahn S.A. Kern S.E. Cancer Res. 1996; 56: 2527-2530PubMed Google Scholar), four lines of evidence indicate that COLO-357 cells used in the present study express functional SMAD4. First, COLO-357 cells exhibited a SMAD4transcript by Northern blot analysis, which was the same size as the SMAD4 transcript in human placenta. Second, the SMAD4 transcript was readily demonstrated by reverse transcriptase-PCR analysis, using specific SMAD4 primers (27Schutte M. Hruban R.H. Hedrick L. Cho K.R. Nadasdy G.M. Weinstein C.L. Bova G.S. Isaacs W.B. Cairns P. Nawroz H. Sidransky D. Casero Jr., R.A. Meltzer P.S. Hahn S.A. Kern S.E. Cancer Res. 1996; 56: 2527-2530PubMed Google Scholar). Third, complete sequencing of the SMAD4 gene in COLO-357 cells did not reveal any mutation. Fourth, COLO-357 cells exhibited rapid, intermediate, and delayed responses to TGF-β1, including our previous finding of autoinduction of TGF-β1 (23Baldwin R.L. Korc M. Growth Factors. 1993; 8: 23-34Crossref PubMed Scopus (68) Google Scholar) and the present results demonstrating increased expression of IGFBP-3, PAI-I, TβRI, and TβRII. Taken together, these observations suggest that up-regulation of TβRI and TβRII may be part of an overall gene response in certain cells that serves to maximize the antiproliferative actions of TGFβ-1." @default.
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• W2054292890 title "Up-regulation of Transforming Growth Factor (TGF)-β Receptors by TGF-β1 in COLO-357 Cells" @default.
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