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Regulation of TRAIL Expression by the Phosphatidylinositol 3-Kinase/Akt/GSK-3 Pathway in Human Colon Cancer Cells

2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês

10.1074/jbc.m206306200

ISSN

1083-351X

Autores

Qingding Wang, Xiaofu Wang, Ambrosio Hernandez, Mark R. Hellmich, Zoran Gatalica, B. Mark Evers,

Tópico(s)

Cancer-related Molecular Pathways

Resumo

The intestinal mucosa is a rapidly-renewing tissue characterized by cell proliferation, differentiation, and eventual apoptosis with progression up the vertical gut axis. The inhibition of phosphatidylinositol (PI) 3-kinase by specific chemical inhibitors or overexpression of the lipid phosphatase PTEN enhances enterocyte-like differentiation in human colon cancer cell models of intestinal differentiation. In this report, we examined the role of PI 3-kinase inhibition in the regulation of apoptotic gene expression in human colon cancer cell lines HT29, HCT-116, and Caco-2. Inhibition of PI 3-kinase with the chemical inhibitor wortmannin increased TNF-related apoptosis-inducing ligand (TRAIL; Apo2) mRNA and protein expression. Similarly, overexpression of the tumor suppressor protein PTEN, an antagonist of PI 3-kinase signaling, resulted in the increased expression of TRAIL. Activation of PI 3-kinase by pretreatment with IGF-1, a gut trophic factor, markedly attenuated the induction of TRAIL by wortmannin. Moreover, overexpression of active Akt, a downstream target of PI 3-kinase, or inhibition of GSK-3, a downstream target of active Akt, completely blocked the induction of TRAIL by wortmannin. Consistent with findings that TRAIL is induced by agents that enhance intestinal cell differentiation, TRAIL expression was specifically localized to the differentiated cells of the colon and small bowel. Adenovirus-mediated overexpression of TRAIL increased DNA fragmentation of HCT-116 cells, demonstrating the functional activity of TRAIL induction. Taken together, our findings demonstrate induction of the TRAIL by inhibition of PI 3-kinase in colon cancer cell lines. These results identify TRAIL, a novel TNF family member, as a downstream target of the PI 3-kinase/Akt/GSK-3 pathway and may have important implications for better understanding the role of the PI 3-kinase pathway in intestinal cell homeostasis. The intestinal mucosa is a rapidly-renewing tissue characterized by cell proliferation, differentiation, and eventual apoptosis with progression up the vertical gut axis. The inhibition of phosphatidylinositol (PI) 3-kinase by specific chemical inhibitors or overexpression of the lipid phosphatase PTEN enhances enterocyte-like differentiation in human colon cancer cell models of intestinal differentiation. In this report, we examined the role of PI 3-kinase inhibition in the regulation of apoptotic gene expression in human colon cancer cell lines HT29, HCT-116, and Caco-2. Inhibition of PI 3-kinase with the chemical inhibitor wortmannin increased TNF-related apoptosis-inducing ligand (TRAIL; Apo2) mRNA and protein expression. Similarly, overexpression of the tumor suppressor protein PTEN, an antagonist of PI 3-kinase signaling, resulted in the increased expression of TRAIL. Activation of PI 3-kinase by pretreatment with IGF-1, a gut trophic factor, markedly attenuated the induction of TRAIL by wortmannin. Moreover, overexpression of active Akt, a downstream target of PI 3-kinase, or inhibition of GSK-3, a downstream target of active Akt, completely blocked the induction of TRAIL by wortmannin. Consistent with findings that TRAIL is induced by agents that enhance intestinal cell differentiation, TRAIL expression was specifically localized to the differentiated cells of the colon and small bowel. Adenovirus-mediated overexpression of TRAIL increased DNA fragmentation of HCT-116 cells, demonstrating the functional activity of TRAIL induction. Taken together, our findings demonstrate induction of the TRAIL by inhibition of PI 3-kinase in colon cancer cell lines. These results identify TRAIL, a novel TNF family member, as a downstream target of the PI 3-kinase/Akt/GSK-3 pathway and may have important implications for better understanding the role of the PI 3-kinase pathway in intestinal cell homeostasis. phosphatidylinositol 3-kinase tumor necrosis factor TNF-related apoptosis-inducing ligand glycogen-synthase kinase-3 insulin-like growth factor 1 phosphatase and tensin homologue, deleted on chromosome 10 glyceraldehyde-3-phosphate dehydrogenase multiplicity of infection phosphate-buffered saline plaque-forming unit fluorescein isothiocyanate The epithelium of the mammalian intestine is a dynamic and continuously renewing tissue serving a number of critical physiologic functions which, depending upon the location along the cephalocaudal gut axis, include digestion and nutrient absorption, barrier and immune functions, and secretion (1Podolsky D.K. Babyatsky M.W. Yamada T. Textbook of Gastroenterology. Lippincott, Philadelphia, PA1995: 546-577Google Scholar). The intestinal mucosa is characterized by a remarkably efficient and highly regimented progression of proliferation and differentiation with progression of cells up the crypt axis of the colon and the crypt-villus axis of the small bowel (2Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 461-479Crossref PubMed Scopus (545) Google Scholar). Proliferating cells are localized to the lower crypt fractions with differentiated cells localized to the upper half of the colon and the villus fraction of the small bowel. Over a 3–5-day period, the differentiated colonocytes and enterocytes are extruded into the intestinal lumen (3Pritchard D.M. Watson A.J. Pharmacol. Ther. 1996; 72: 149-169Crossref PubMed Scopus (65) Google Scholar, 4Potten C.S. Am. J. Physiol. 1997; 273: G253-G257PubMed Google Scholar). The cellular mechanisms triggering the differentiation and subsequent extrusion of these epithelial cells are not entirely known. Phosphatidylinositol 3-kinase (PI 3-kinase),1 a ubiquitous lipid kinase that is involved in receptor signal transduction through tyrosine kinase receptors, is composed of a regulatory subunit (p85) and a 110-kDa catalytic subunit (p110) (5Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar, 6King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar). PI 3-kinase catalyzes the phosphorylation of phosphoinositol 4-phosphate and phosphoinositol 4,5-phosphate at the D3 position and activates various downstream elements including Akt/protein kinase B (PKB). PI 3-kinase regulates a number of important cellular processes such as cellular growth and transformation, membrane ruffling, actin rearrangement, vesicular trafficking, and cell survival. Promotion of cell survival by the activation of PI 3-kinase/Akt occurs by the inhibition of proapoptotic signals and the induction of survival signals (7Roche S. Koegl M. Courtneidge S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9185-9189Crossref PubMed Scopus (262) Google Scholar, 8Philpott K.L. McCarthy M.J. Klippel A. Rubin L.L. J. Cell Biol. 1997; 139: 809-815Crossref PubMed Scopus (220) Google Scholar, 9Davidson H.W. J. Cell Biol. 1995; 130: 797-805Crossref PubMed Scopus (184) Google Scholar, 10Jones S.M. Howell K.E. J. Cell Biol. 1997; 139: 339-349Crossref PubMed Scopus (78) Google Scholar, 11Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1375) Google Scholar), which may contribute to malignant transformation. Conversely, the inhibition of PI 3-kinase/Akt results in cell cycle arrest and differentiation in certain cell types, such as the human colon cancer cell lines HT29 and Caco-2 (12Wang Q. Wang X. Hernandez A. Kim S. Evers B.M. Gastroenterology. 2001; 120: 1381-1392Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Glycogen synthase kinase-3 (GSK-3) is an Akt substrate shown to be inhibited upon phosphorylation by Akt (13Zheng W.H. Kar S. Quirion R. J. Biol. Chem. 2000; 275: 39152-39158Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). GSK-3, a component of the Wnt signaling pathway, has been implicated in multiple biological processes by phosphorylation of a broad range of substrates, including several transcription factors such as c-Myc, c-Jun, and c-Myb and the translation factor eIF2B (14Ali A. Hoeflich K.P. Woodgett J.R. Chem. Rev. 2001; 101: 2527-2540Crossref PubMed Scopus (347) Google Scholar, 15Plyte S.E. Hughes K. Nikolakaki E. Pulverer B.J. Woodgett J.R. Biochim. Biophys. Acta. 1992; 1114: 147-162Crossref PubMed Scopus (336) Google Scholar). As a downstream target of the PI 3-kinase/Akt pathway, GSK-3 activity suppresses cell proliferation and survival (16Kane L.P. Shapiro V.S. Stokoe D. Weiss A. Curr. Biol. 1999; 9: 601-604Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar, 17Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P., Hu, L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). The tumor suppressor genePTEN (for Phosphatase and tensin homologue deleted on chromosome 10; also called MMAC1 orTEP1) encodes a 403-amino acid phosphatase that antagonizes the activity of PI 3-kinase by dephosphorylating the D3-phosphate group of lipid second messengers, thus serving as a negative regulator of the PI 3-kinase pathway (18Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Crossref PubMed Scopus (1752) Google Scholar). This effect of PTEN inhibits downstream functions mediated by the PI 3-kinase pathway, such as activation of Akt/PKB, cell survival, and cell proliferation (19Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2614) Google Scholar, 20Sun H. Lesche R., Li, D.M. Liliental J. Zhang H. Gao J. Gavrilova N. Mueller B. Liu X. Wu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6199-6204Crossref PubMed Scopus (692) Google Scholar, 21Di Cristofano A. Pandolfi P.P. Cell. 2000; 100: 387-390Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). Members of the tumor necrosis factor (TNF) family interact with their cell surface receptors to directly engage the cellular apoptotic machinery (22Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar, 23Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5166) Google Scholar). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; also called Apo-2 ligand), a novel member of the TNF family, is a type II membrane protein identified based on homology to the extracellular domains of TNF and FasL (CD95L) (24Pitti R.M. Marsters S.A. Ruppert S. Donahue C.J. Moore A. Ashkenazi A. J. Biol. Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1654) Google Scholar, 25Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2664) Google Scholar). Unlike TNF and FasL, TRAIL is expressed in a variety of cell types and is capable of inducing apoptosis in normal and neoplastic cells (26Walczak H. Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A., Le, T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2236) Google Scholar, 27Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Invest. 1999; 104: 155-162Crossref PubMed Scopus (2008) Google Scholar). In addition, TRAIL blockade results in hyperproliferation of synovial cells and lymphocytes, whereas TRAIL inhibits DNA synthesis in lymphocytes by blocking cell cycle progression (28Song K. Chen Y. Goke R. Wilmen A. Seidel C. Goke A. Hilliard B. J. Exp. Med. 2000; 191: 1095-1104Crossref PubMed Scopus (324) Google Scholar). Therefore, TRAIL appears to play important roles in cell proliferation and survival; however, little is known regarding the signaling pathways that regulate TRAIL expression. Recently, we have shown that inhibition of PI 3-kinase, using the chemical inhibitor wortmannin or PTEN overexpression significantly enhances enterocyte-like differentiation of the HT29 and Caco-2 human colon cancer cells (12Wang Q. Wang X. Hernandez A. Kim S. Evers B.M. Gastroenterology. 2001; 120: 1381-1392Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), which display a multipotent phenotype and are well-characterized models of intestinal differentiation (29Heerdt B.G. Houston M.A. Augenlicht L.H. Cancer Res. 1994; 54: 3288-3293PubMed Google Scholar, 30Basson M.D. Emenaker N.J. Hong F. Proc. Soc. Exp. Biol. Med. 1998; 217: 476-483Crossref PubMed Scopus (40) Google Scholar, 31Zweibaum A. Chantret L. Sepulveda F.B. Adaptation and Development of Gastrointestinal Function. Manchester University Press, Manchester, UK1989: 103-112Google Scholar, 32Evers B.M., Ko, T.C., Li, J. Thompson E.A. Am. J. Physiol. 1996; 271: G722-G727PubMed Google Scholar, 33Litvak D.A. Evers B.M. Hwang K.O. Hellmich M.R. Ko T.C. Townsend Jr., C.M. Surgery. 1998; 124 (; discussion 169–170): 161-169Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 34Domon-Dell C. Wang Q. Kim S. Kedinger M. Evers B.M. Freund J.N. Gut. 2002; 50: 525-529Crossref PubMed Scopus (57) Google Scholar). The purpose of our present study was to identify potential downstream targets of PI 3-kinase inhibition, which may contribute to intestinal cell differentiation and/or apoptosis. Here, we report that the PI 3-kinase signaling pathway negatively regulates TRAIL expression in human colon cancer cell lines. The induction of TRAIL expression by PI 3-kinase inhibition was demonstrated with the PI 3-kinase inhibitor wortmannin or by the constitutive overexpression of the physiological antagonist, PTEN. Activation of PI 3-kinase by insulin-like growth factor 1 (IGF-1) markedly attenuated the induction of TRAIL by wortmannin. Furthermore, overexpression of Akt or inhibition of GSK-3 completely blocked the induction of TRAIL by wortmannin. Thus, our study identifies the TRAIL gene as a novel downstream target of PI 3-kinase inhibition. Wortmannin, actinomycin D, cycloheximide, and lithium chloride were purchased from Sigma Chemical Company. Ro-318220 and bis-indolylmaleimide (GF109203x) were from Calbiochem (San Diego, CA). The GSK-3 inhibitors SB-216763 and SB-415286 were gifts from GlaxoSmithKline Pharmaceuticals (Research Triangle Park, NC). IGF-1 was purchased from R&D systems (Minneapolis, MN). Mouse anti-human PTEN monoclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Akt, antiphospho-Akt (Ser-473) and rabbit antiphospho-GSK-3α/β (Ser-21/9) antibodies were purchased from Cell Signaling (Beverly, MA). Rabbit anti-β-actin antibody was from Sigma. Mouse antibody against human TRAIL (RIK-2) was a gift from Hideo Yagita (Juntendo University School of Medicine, Tokyo, Japan) (35Kayagaki N. Yamaguchi N. Nakayama M. Kawasaki A. Akiba H. Okumura K. Yagita H. J. Immunol. 1999; 162: 2639-2647PubMed Google Scholar). Recombinant human TRAIL-R2:Fc was purchased from Alexis Corporation (San Diego, CA). Adenovirus vectors encoding β-galactosidase (AdCA-LacZ; control) and PTEN (AdCA-PTEN) were from Akira Horii (Tohoku University School of Medicine, Sendai, Japan) (36Sakurada A. Hamada H. Fukushige S. Yokoyama T. Yoshinaga K. Furukawa T. Sato S. Yajima A. Sato M. Fujimura S. Horii A. Int. J. Oncol. 1999; 15: 1069-1074PubMed Google Scholar). The adenovirus vector encoding the myristoylated active form of Akt (AxCA-Myr-Akt) was from Wataru Ogawa (Kobe University School of Medicine, Chuo-ku, Japan) (37Kotani K. Ogawa W. Hino Y. Kitamura T. Ueno H. Sano W. Sutherland C. Granner D.K. Kasuga M. J. Biol. Chem. 1999; 274: 21305-21312Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The adenovirus vector-encoding TRAIL (Ad-TRAIL) was purchased from Thomas Griffith (University of Iowa, Iowa City, IA) (38Griffith T.S. Anderson R.D. Davidson B.L. Williams R.D. Ratliff T.L. J. Immunol. 2000; 165: 2886-2894Crossref PubMed Scopus (183) Google Scholar). The human apoptosis DNA template set (hAPO-3c) was from BD Pharmingen (San Diego, CA). [γ-32P]ATP (3,000 Ci/mmol) was fromAmersham Biosciences. Nitrocellulose filters for Northern blots were from Sartorius (Göttingen, Germany). The constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was obtained from Ambion (Austin, TX) and used to ensure the integrity of the RNA samples analyzed by Northern blot. Total RNA was isolated using Ultraspec RNA (Biotecx Laboratories, Houston, TX). Ribonuclease (RNase) protection experiments were performed using the RPA-III kit from Ambion. Immobilon-P nylon membranes for Western blots were purchased from Millipore (Bedford, MA), and x-ray film was purchased from Eastman Kodak (Rochester, NY). The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was from Amersham Biosciences. Tissue culture media and RT-PCR reagents were obtained from Invitrogen. All other reagents were of molecular biology grade and purchased from either Sigma or Amresco (Solon, OH). The human colon cancer cell lines HT29 and HCT-116 (ATCC; Manassas, VA) were maintained in McCoy's 5A supplemented with 10% fetal calf serum. Caco-2 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum. Wortmannin was dissolved in dimethyl sulfoxide (Me2SO). In all experiments, the effects of wortmannin were compared with cells treated with vehicle (i.e.Me2SO at a concentration less than 0.05%). Cells were infected with adenovirus vectors AdCA-PTEN and AxCA-Akt at 10 plaque-forming units (pfu)/cell as described previously (25Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2664) Google Scholar) and Ad-TRAIL at 1000 pfu/cell (38Griffith T.S. Anderson R.D. Davidson B.L. Williams R.D. Ratliff T.L. J. Immunol. 2000; 165: 2886-2894Crossref PubMed Scopus (183) Google Scholar) and incubated for 24 h prior to initiating treatment. RNA was isolated from cells using Ultraspec RNA reagent according to the manufacturer's protocol. A 32P-labeled antisense RNA probe was prepared using the Human Apoptosis hAPO-3c Template Set (BD Pharmingen), which measures multiple mRNA species and RNA analyzed as we have previously described (39Dong Z. Wang X. Evers B.M. Am. J. Physiol. 2000; 279: G1139-G1147PubMed Google Scholar). Total RNA (40 μg) was run in 1.2% agarose/formaldehyde gels and transferred to supported nitrocellulose as previously described (40Evers B.M. Zhou Z. Celano P. Li J. J. Clin. Invest. 1995; 95: 2822-2830Crossref PubMed Scopus (45) Google Scholar). Membranes were hybridized to a random-primed 32P-labeled TRAIL cDNA probe overnight at 43 °C and then washed two times at room temperature with 2× SSC and 0.1% SDS and two times at 43 °C for 15 min with 0.1× SSC and 0.1% SDS. The human TRAIL cDNA probe was synthesized as previously described (41Wang Q., Ji, Y. Wang X. Evers B.M. Biochem. Biophys. Res. Commun. 2000; 276: 466-471Crossref PubMed Scopus (88) Google Scholar) by RT-PCR using the following primers: 5′-CTTCACAGTGCTCCTGCAGT-3′, which spans nucleotides 150–169 of the human TRAIL cDNA sequence, and 5′-TTAGCCAACTAAAAAGGCCCC-3′, which is complementary to nucleotides 913–933 of the cDNA sequence. The PCR fragment was sequenced and confirmed to be the correct sequence for human TRAIL. Blots were stripped and reprobed with GAPDH to ensure equal loading. Signals were detected by autoradiography. A 5-μg aliquot of total RNA was reverse transcribed with Maloney Murine Leukemia Virus (M-MLV) reverse transcriptase, and the resulting cDNA was combined with each primer pair and PCR reagents in a final reaction volume of 50 μl. PCR was carried out for 30 cycles (95 °C melting temperature for 45 s; 60 °C annealing temperature for 45 s; 72 °C extension temperature for 1 min). The following two primers were synthesized: 5′-CTTCACAGTGCTCCTGCAGT-3′, which spans nucleotides 150–169 of the human TRAIL cDNA sequence, and 5′-TTAGCCAACTAAAAAGGCCCC-3′, which is complementary to nucleotides 913–933 of the cDNA sequence. GAPDH was amplified to assess equal loading using the PCR primers that have been described previously (41Wang Q., Ji, Y. Wang X. Evers B.M. Biochem. Biophys. Res. Commun. 2000; 276: 466-471Crossref PubMed Scopus (88) Google Scholar). Western immunoblot analyses were performed as described previously (42Wang Q. Ding Q. Dong Z. Ehlers R.A. Evers B.M. Anticancer Res. 2000; 20: 75-83PubMed Google Scholar). Cells were lysed with TNN buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.5% Nonidet P-40, 50 mm NaF, 1 mm sodium orthovanadate, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride and 25 μg/ml each of aprotinin, leupeptin, and pepstatin A) at 4 °C for 30 min. Lysates were clarified by centrifugation (10,000 × gfor 30 min at 4 °C) and protein concentrations determined using the method of Bradford (43Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217450) Google Scholar). Briefly, total protein (100 μg) was resolved on a 10% polyacrylamide gel and transferred to immobilon-P nylon membranes. Filters were incubated overnight at 4 °C in blotting solution (Tris-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20). Akt, phosphorylated Akt, PTEN, β-actin, and phospho-GSK-3α/β were detected with specific antibodies to these proteins following blotting with a horseradish peroxidase-conjugated secondary antibody and visualized using ECL detection. HT29 cells were incubated with wortmannin or vehicle (i.e. Me2SO). 5 × 105 cells in a final volume of 100 μl of PBS were incubated with l μg of RIK-2 antibody or isotype control (mouse IgG) for 1 h at 4 °C, washed twice, and resuspended in 100 μl of PBS. For secondary staining, cells were incubated for 45 min at 4 °C in the dark with 1 μg of fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG. After washing with PBS, the cells were fixed with 1% paraformaldehyde in PBS; specific fluorescence was measured using a FACScan. HT29 cells were seeded onto sterile glass coverslips in 60-mm dishes and cultured for 16–24 h before treatment. Cells were treated with wortmannin (500 nm) or Me2SO at 37 °C for 4 h. Cells were washed in PBS, fixed in cold methanol for 5 min, and washed in three changes of PBS followed by a solution of 10% normal goat serum (Sigma). After washing, cells were then incubated with the TRAIL receptor, TRAIL-R2 (also known as DR5), fused with human FC (TRAIL-R2:FC) (1 μg/ml diluted in PBS with 1.5% normal goat serum) for 60 min. After washing with PBS, cells were incubated with FITC-conjugated goat anti-human FC antibody (2 μg/ml) in PBS with 1.5% normal goat serum for 45 min. After three final washes, the slides were viewed with a fluorescence and phase contrast microscope. Cells were plated in 96-well plates 24 h before treatment. After treatment, DNA fragmentation was evaluated by examination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) using a Cell death Detection ELISAPlus kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Formalin-fixed, paraffin-embedded tissue samples of normal human colon and small bowel were used. Sections (5-μm thick) were fixed to the slide by incubation in a dry oven at 58 °C for 30 min, and then sequentially transferred to xylene (5 min, 2 changes), 100% ethanol (3 min, 2 changes), 95% ethanol (3 min, 2 changes) and rinsed with deionized water. A standard heat-induced epitope retrieval procedure (20 min, 98 °C) was employed by placing slides in commercially available retrieval solution (Target Retrieval Solution, pH 6.0; DAKO, Carpinteria, CA). Slides were allowed to cool at room temperature and rinsed twice with deionized water. Endogenous peroxidase was blocked by placing slides in 3% H2O2/methanol block solution for 10 min, washed with deionized water, and placed in phosphate-buffered saline for 5 min. Slides were incubated at room temperature with primary mouse monoclonal anti-human TRAIL antibody (1:400, BD Pharmingen, cat. 556468) for 30 min. Avidin-biotin peroxidase complex amplification and detection system (LSAB2, DAKO) with diaminobenzidine as chromagen was used. All steps were performed on the automated stainer (DAKO). Negative controls (including no primary antibody or isotype matched mouse IgG) were used in each assessment. Previously, we have shown that inhibition of PI 3-kinase augments the enterocyte-like differentiation of the HT29 and Caco-2 human colon cancer cells (12Wang Q. Wang X. Hernandez A. Kim S. Evers B.M. Gastroenterology. 2001; 120: 1381-1392Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). These cells undergo differentiation to a small bowel-like phenotype as noted by the induction of brush border enzymes and the presence of microvilli and have been extensively utilized to address mechanistic questions regarding intestinal differentiation (29Heerdt B.G. Houston M.A. Augenlicht L.H. Cancer Res. 1994; 54: 3288-3293PubMed Google Scholar, 30Basson M.D. Emenaker N.J. Hong F. Proc. Soc. Exp. Biol. Med. 1998; 217: 476-483Crossref PubMed Scopus (40) Google Scholar, 31Zweibaum A. Chantret L. Sepulveda F.B. Adaptation and Development of Gastrointestinal Function. Manchester University Press, Manchester, UK1989: 103-112Google Scholar, 32Evers B.M., Ko, T.C., Li, J. Thompson E.A. Am. J. Physiol. 1996; 271: G722-G727PubMed Google Scholar, 33Litvak D.A. Evers B.M. Hwang K.O. Hellmich M.R. Ko T.C. Townsend Jr., C.M. Surgery. 1998; 124 (; discussion 169–170): 161-169Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 34Domon-Dell C. Wang Q. Kim S. Kedinger M. Evers B.M. Freund J.N. Gut. 2002; 50: 525-529Crossref PubMed Scopus (57) Google Scholar). In this study, we have investigated potential downstream targets of PI 3-kinase inhibition in these intestinal-derived cell lines. HT29 cells were treated for 4 h with the PI 3-kinase inhibitor wortmannin (250 nm), or vehicle control and then the RNA was analyzed by an RNase protection analysis using a multiprobe template (hAPO-3c; BD Pharmingen), which assesses the expression of eleven different genes that contribute to the apoptotic pathway in cells (e.g.TRAIL, Fas, FasL, and TRAIL receptors); L32 and GAPDH are included to ensure equality of loading (Fig.1 A). Wortmannin treatment resulted in the induction of TRAIL gene expression compared with control cells treated with vehicle (i.e. Me2SO) with no marked change in the expression of the other apoptotic-related genes contained in this probe set. To confirm the induction of TRAIL by wortmannin, HT29 cells were treated for 4 h with different concentrations of wortmannin (2.5 nm to 1 μm) and Northern blot analysis performed using a human TRAIL cDNA probe (Fig. 1 B). Wortmannin treatment increased TRAIL mRNA levels in a dose-dependent fashion with TRAIL induction noted using a dosage of only 2.5 nm. To next assess the time course for TRAIL mRNA induction, HT29 cells were treated with wortmannin (250 nm) for 0.5–8 h (Fig. 1 C). Induction of TRAIL mRNA was noted at 2 h after wortmannin treatment with further increases demonstrated at 4 and 8 h. To determine whether TRAIL induction requires RNA transcription or new protein synthesis, HT29 cells were treated with either actinomycin D (10 μg/ml), which inhibits transcription, or cycloheximide (9 μm), which inhibits protein synthesis, in combination with wortmannin (250 nm) for 4 h, total RNA was extracted and Northern blot analysis was performed (Fig.1 D). Induction of TRAIL by wortmannin was completely blocked by actinomycin D. In contrast, treatment with cycloheximide had no effect on TRAIL induction. Therefore, these findings suggest that TRAIL induction by PI 3-kinase inhibition is regulated at the level of transcription and is not dependent on de novo protein synthesis. Our findings using the HT29 human colon cancer cell line demonstrate induction of TRAIL expression associated with PI 3-kinase inhibition, which we have previously shown enhances the differentiation of the human colon cancer cell lines HT29 and Caco-2 (12Wang Q. Wang X. Hernandez A. Kim S. Evers B.M. Gastroenterology. 2001; 120: 1381-1392Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To determine the location of TRAIL expression in vivo, sections of normal small bowel and colon were obtained from adult patients and analyzed (Fig. 1 E). Interestingly, TRAIL expression was specifically localized to the intestinal cells in the differentiated fractions of the intestinal mucosa. That is, intense staining for TRAIL was located in upper crypt portion of the colon (Fig. 1 E, left panel) and the villus fraction of the jejunum (Fig. 1 E,right panel). Little to no staining of TRAIL was noted in the lower crypt of the colon or crypt cells of the jejunum. Therefore, these findings confirm the induction of TRAIL expression specifically in the more differentiated portions of the intestinal mucosa thus further suggesting the association of TRAIL induction in the HT29 cells with treatments that result in a more differentiated phenotype. TRAIL is a type II membrane protein; therefore, we next assessed whether PI 3-kinase inhibition affects TRAIL protein expression on HT29 cells using both flow cytometry and immunofluorescence (Fig. 2). HT29 cells were treated with wortmannin (500 nm) for 4 h and then assessed by flow cytometry using an anti-TRAIL antibody (RIK2) (35Kayagaki N. Yamaguchi N. Nakayama M. Kawasaki A. Akiba H. Okumura K. Yagita H. J. Immunol. 1999; 162: 2639-2647PubMed Google Scholar). TRAIL expression was detected on the surface of HT29 cells

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