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

Open AccessPublished:July 24, 2002DOI:https://doi.org/10.1074/jbc.M206306200
      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.
      PI 3-kinase
      phosphatidylinositol 3-kinase
      TNF
      tumor necrosis factor
      TRAIL
      TNF-related apoptosis-inducing ligand
      GSK-3
      glycogen-synthase kinase-3
      IGF-1
      insulin-like growth factor 1
      PTEN
      phosphatase and tensin homologue, deleted on chromosome 10
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      MOI
      multiplicity of infection
      PBS
      phosphate-buffered saline
      pfu
      plaque-forming unit
      FITC
      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 (
      • Podolsky D.K.
      • Babyatsky M.W.
      ). 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 (
      • Cheng H.
      • Leblond C.P.
      ). 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 (
      • Pritchard D.M.
      • Watson A.J.
      ,
      • Potten C.S.
      ). 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) (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ). 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 (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Philpott K.L.
      • McCarthy M.J.
      • Klippel A.
      • Rubin L.L.
      ,
      • Davidson H.W.
      ,
      • Jones S.M.
      • Howell K.E.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ), 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 (
      • Wang Q.
      • Wang X.
      • Hernandez A.
      • Kim S.
      • Evers B.M.
      ). Glycogen synthase kinase-3 (GSK-3) is an Akt substrate shown to be inhibited upon phosphorylation by Akt (
      • Zheng W.H.
      • Kar S.
      • Quirion R.
      ). 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 (
      • Ali A.
      • Hoeflich K.P.
      • Woodgett J.R.
      ,
      • Plyte S.E.
      • Hughes K.
      • Nikolakaki E.
      • Pulverer B.J.
      • Woodgett J.R.
      ). As a downstream target of the PI 3-kinase/Akt pathway, GSK-3 activity suppresses cell proliferation and survival (
      • Kane L.P.
      • Shapiro V.S.
      • Stokoe D.
      • Weiss A.
      ,
      • Brunet 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.
      ). 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 (
      • Cantley L.C.
      • Neel B.G.
      ). 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 (
      • Maehama T.
      • Dixon J.E.
      ,
      • Sun H.
      • Lesche R., Li, D.M.
      • Liliental J.
      • Zhang H.
      • Gao J.
      • Gavrilova N.
      • Mueller B.
      • Liu X.
      • Wu H.
      ,
      • Di Cristofano A.
      • Pandolfi P.P.
      ).
      Members of the tumor necrosis factor (TNF) family interact with their cell surface receptors to directly engage the cellular apoptotic machinery (,
      • Ashkenazi A.
      • Dixit V.M.
      ). 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) (
      • Pitti R.M.
      • Marsters S.A.
      • Ruppert S.
      • Donahue C.J.
      • Moore A.
      • Ashkenazi A.
      ,
      • Wiley 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.
      ). Unlike TNF and FasL, TRAIL is expressed in a variety of cell types and is capable of inducing apoptosis in normal and neoplastic cells (
      • Walczak 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.
      ,
      • Ashkenazi 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.
      ). In addition, TRAIL blockade results in hyperproliferation of synovial cells and lymphocytes, whereas TRAIL inhibits DNA synthesis in lymphocytes by blocking cell cycle progression (
      • Song K.
      • Chen Y.
      • Goke R.
      • Wilmen A.
      • Seidel C.
      • Goke A.
      • Hilliard B.
      ). 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 (
      • Wang Q.
      • Wang X.
      • Hernandez A.
      • Kim S.
      • Evers B.M.
      ), which display a multipotent phenotype and are well-characterized models of intestinal differentiation (
      • Heerdt B.G.
      • Houston M.A.
      • Augenlicht L.H.
      ,
      • Basson M.D.
      • Emenaker N.J.
      • Hong F.
      ,
      • Zweibaum A.
      • Chantret L.
      ,
      • Evers B.M., Ko, T.C., Li, J.
      • Thompson E.A.
      ,
      • Litvak D.A.
      • Evers B.M.
      • Hwang K.O.
      • Hellmich M.R.
      • Ko T.C.
      • Townsend Jr., C.M.
      ,
      • Domon-Dell C.
      • Wang Q.
      • Kim S.
      • Kedinger M.
      • Evers B.M.
      • Freund J.N.
      ). 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.

      DISCUSSION

      Previously, we have shown that the inhibition of PI 3-kinase enhances enterocyte-like differentiation of the HT29 and Caco-2 human colon cancer cells suggesting a role for PI 3-kinase inhibition in intestinal cell differentiation (
      • Wang Q.
      • Wang X.
      • Hernandez A.
      • Kim S.
      • Evers B.M.
      ). In our current study, we show that the PI 3-kinase signaling pathway negatively regulates expression of TRAIL, a member of the TNF superfamily, in human colon cancers. Induction of TRAIL expression was demonstrated by PI 3-kinase inhibition using the chemical inhibitor wortmannin at dosages consistent with a specific inhibitory effect (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ). Conversely, activation of PI 3-kinase by IGF-1 markedly attenuated wortmannin-mediated TRAIL induction while overexpression of PTEN induced TRAIL expression, thus identifying the TRAIL gene as a downstream target of PI 3-kinase inhibition in human colon cancer cells. This induction was specific for TRAIL in these cells since the expression of FasL, another member of the TNF family, which, similar to TRAIL, induces apoptosis through a caspase-dependent pathway (
      • Suhara T.
      • Kim H.S.
      • Kirshenbaum L.A.
      • Walsh K.
      ), was not affected by wortmannin treatment. Similar to our findings showing induction of TRAIL by PI 3-kinase inhibition, Suharaet al. (
      • Suhara T.
      • Kim H.S.
      • Kirshenbaum L.A.
      • Walsh K.
      ) recently reported that inhibition of PI 3-kinase up-regulated FasL expression in vascular smooth muscle cells. Taken together with our current findings, these studies indicate that the inhibition of PI 3-kinase can result in the induction of apoptotic-related proteins, such as TRAIL or FasL in a cell type-dependent fashion. PI 3-kinase activation can promote cell survival by the activation of downstream effector proteins (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Philpott K.L.
      • McCarthy M.J.
      • Klippel A.
      • Rubin L.L.
      ,
      • Davidson H.W.
      ,
      • Jones S.M.
      • Howell K.E.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ); therefore, the finding that PI 3-kinase inhibition results in the induction of genes, which contribute to cell death is reasonable and further supports the notion that PI 3-kinase plays a major role in the regulation of proliferative signals in certain cells.
      The PTEN tumor suppressor gene encodes a multifunctional phosphatase that plays a critical physiologic role in inhibiting the PI 3-kinase pathway and downstream functions of PI 3-kinase such as cell survival and proliferation (
      • Di Cristofano A.
      • Pesce B.
      • Cordon-Cardo C.
      • Pandolfi P.P.
      ,
      • Suzuki A.
      • de la Pompa J.L.
      • Stambolic V.
      • Elia A.J.
      • Sasaki T.
      • del Barco Barrantes I., Ho, A.
      • Wakeham A.
      • Itie A.
      • Khoo W.
      • Fukumoto M.
      • Mak T.W.
      ,
      • Podsypanina K.
      • Ellenson L.H.
      • Nemes A., Gu, J.
      • Tamura M.
      • Yamada K.M.
      • Cordon-Cardo C.
      • Catoretti G.
      • Fisher P.E.
      • Parsons R.
      ). Moreover, current genetic data suggest that PTEN function is required for normal development and differentiation (
      • Wang Q.
      • Wang X.
      • Hernandez A.
      • Kim S.
      • Evers B.M.
      ,
      • Di Cristofano A.
      • Pesce B.
      • Cordon-Cardo C.
      • Pandolfi P.P.
      ). In this regard, our preliminary results suggest that PTEN expression is localized to the more differentiated cells of the colonic epithelium.
      S. Kim, unpublished observations.
      Overexpression of PTEN in HT29 and Caco-2 cells significantly augments the induction of brush border enzyme activity, which further identifies a role for PTEN in the process of intestinal cell differentiation (
      • Wang Q.
      • Wang X.
      • Hernandez A.
      • Kim S.
      • Evers B.M.
      ). Similar to our findings demonstrating induction of TRAIL by wortmannin, we show that TRAIL induction in the colon cancer cell lines occurs by PTEN overexpression thus further confirming a role for PI 3-kinase inhibition in the induction of TRAIL expression. Recently, Matsushima-Nishiu et al. (
      • Matsushima-Nishiu M.
      • Unoki M.
      • Ono K.
      • Tsunoda T.
      • Minaguchi T.
      • Kuramoto H.
      • Nishida M.
      • Satoh T.
      • Tanaka T.
      • Nakamura Y.
      ) analyzed genes that were up-regulated with overexpression ofPTEN in endometrial cancer cell lines by cDNA microarray. Notably, induction of members of the TNF-receptor family and TNF-associated genes was identified. Although TRAIL was not specifically analyzed and not all of the gene changes identified by gene array were confirmed by RT-PCR or conventional hybridization methods, this study suggests that PTEN-mediated up-regulation of TNF superfamily members may represent an important cellular function of PTEN. This up-regulation of TNF-associated genes may contribute to the subsequent apoptosis noted in some cells by PTEN overexpression. Furthermore, the regulation of the TNF-associated genes by PI 3-kinase inhibition may represent an important cellular mechanism for regulating proliferation and cell death.
      TRAIL is expressed in a number of tissues and displays potent apoptotic activity against selected targets including a variety of cancers (,
      • Ashkenazi A.
      • Dixit V.M.
      ,
      • Walczak 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.
      ,
      • Ashkenazi 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.
      ). In addition to its well-described effects on cell death, TRAIL can inhibit cell cycle progression whereas blockade of TRAIL results in hyperproliferation in autoreactive lymphocytes, which are resistant to TRAIL-induced apoptosis (
      • Song K.
      • Chen Y.
      • Goke R.
      • Wilmen A.
      • Seidel C.
      • Goke A.
      • Hilliard B.
      ,
      • Lunemann J.D.
      • Waiczies S.
      • Ehrlich S.
      • Wendling U.
      • Seeger B.
      • Kamradt T.
      • Zipp F.
      ) thus further implying a physiologic role for TRAIL in certain cells. In our present study, we demonstrate that overexpression of TRAIL in the HCT-116 cell line, which is sensitive to exogenous TRAIL treatment (
      • Lacour S.
      • Hammann A.
      • Wotawa A.
      • Corcos L.
      • Solary E.
      • Dimanche-Boitrel M.T.
      ), results in enhanced DNA fragmentation and cell death, which was blocked by caspase inhibition. These results, in combination with our findings of a spatial-specific pattern of TRAIL expression along the vertical axis of the small bowel and colon, strongly suggests a role for TRAIL in intestinal homeostasis. Consistent with our findings, Strater et al. (
      • Strater J.
      • Walczak H.
      • Pukrop T.
      • Von Muller L.
      • Hasel C.
      • Kornmann M.
      • Mertens T.
      • Moller P.
      ) recently demonstrated a similar pattern of TRAIL expression localized predominantly to the luminal surface epithelium of the colon. In addition, the TRAIL receptor (TRAIL-R2/DR-5) was coexpressed with TRAIL, and it was postulated that TRAIL may play a role in the early elimination of virus-infected epithelial cells in the normal gut. Collectively, our present study as well as the findings by Strater et al. (
      • Strater J.
      • Walczak H.
      • Pukrop T.
      • Von Muller L.
      • Hasel C.
      • Kornmann M.
      • Mertens T.
      • Moller P.
      ) identify TRAIL as a potentially important protein for intestinal cell homeostasis. The precise role for TRAIL in the intestine remains to be fully delineated.
      The PI 3-kinase signaling pathway has been implicated in the growth and apoptosis of various cell types (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Philpott K.L.
      • McCarthy M.J.
      • Klippel A.
      • Rubin L.L.
      ,
      • Davidson H.W.
      ,
      • Jones S.M.
      • Howell K.E.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ). Activation of PI 3-kinase by growth factors, such as IGF-1, results in the local accumulation of PtdIns-3,4,5-P3 at the plasma membrane. Newly synthesized PtdIns-3,4,5-P3 recruits Akt/PKB to the plasma membrane where the combination of lipid binding and phosphorylation by PDK-1 serves to further phosphorylate the downstream substrates, such as GSK-3 (
      • Le Good J.A.
      • Ziegler W.H.
      • Parekh D.B.
      • Alessi D.R.
      • Cohen P.
      • Parker P.J.
      ,
      • Toker A.
      ). We found that constitutively active Akt completely prevented the TRAIL induction observed with PI 3-kinase blockade, implicating the regulation of TRAIL expression by PI 3-kinase passes through Akt. Phosphorylation and activation of Akt contribute to increased cell survival and malignant transformation acting through downstream effector proteins such as the pro-apoptotic BAD protein (
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ), caspase-9 (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ), and the transcription factors CREB (
      • Du K.
      • Montminy M.
      ), NF-κB (
      • Kane L.P.
      • Shapiro V.S.
      • Stokoe D.
      • Weiss A.
      ), and Forkhead (
      • Brunet 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.
      ). Furthermore, Akt is involved in the phosphorylation and inactivation of GSK-3. Activation of GSK-3 induces apoptosis while inhibition has been shown to reduce apoptosis and enhance cell survival (
      • Pap M.
      • Cooper G.M.
      ,
      • Crowder R.J.
      • Freeman R.S.
      ), implicating GSK-3 as a central element in the PI 3-kinase/Akt survival pathway. Overexpression of Akt or inhibition of GSK-3 completely blocked TRAIL induction by wortmannin, thus demonstrating regulation of TRAIL expression through the PI 3-kinase/Akt/GSK-3 pathway.
      Our current study demonstrates a role for PI 3-kinase inhibition in the induction of TRAIL expression in human colon cancer cells. However, in contrast to our findings, Musgrave et al. (
      • Musgrave B.L.
      • Phu T.
      • Butler J.J.
      • Makrigiannis A.P.
      • Hoskin D.W.
      ) reported that anti-CD3-induced TRAIL expression in T-cells was blocked by the PI 3-kinase inhibitors, wortmannin and LY294002. These conflicting results may be explained by differences in cell type and the fact that common signaling mechanisms may be interpreted differently depending on the cellular context. This is further supported by the fact that PI 3-kinase activation may result in cell survival or differentiation depending upon the particular cell type. For example, the inhibition of PI 3-kinase enhances enterocyte-like differentiation of colon cancer cells and induces B16 melanoma cell differentiation (
      • Wang Q.
      • Wang X.
      • Hernandez A.
      • Kim S.
      • Evers B.M.
      ,
      • Busca R.
      • Bertolotto C.
      • Ortonne J.P.
      • Ballotti R.
      ). However, in contrast, PI 3-kinase inhibition blocks myogenic and adipocyte differentiation (
      • Jiang B.H.
      • Zheng J.Z.
      • Vogt P.K.
      ,
      • Sakaue H.
      • Ogawa W.
      • Matsumoto M.
      • Kuroda S.
      • Takata M.
      • Sugimoto T.
      • Spiegelman B.M.
      • Kasuga M.
      ).
      In conclusion, our results indicate that TRAIL expression is regulated in human colon cancer cells by the PI 3-kinase/Akt/GSK-3 signaling pathway. Importantly, these findings add the TNF-related TRAIL gene to the growing list of apoptosis-related proteins regulated by the PI 3-kinase pathway. Moreover, our results provide a better understanding of the potential role of the PI 3-kinase pathway in intestinal cell homeostasis.

      Acknowledgments

      We thank Hideo Yagita (Juntendo University School of Medicine, Tokyo, Japan) for the TRAIL antibody (RIK-2), Akira Horii (Tohoku University School of Medicine, Sendai, Japan) for adenoviruses encoding β-galactosidase (AdCA-LacZ) and PTEN (AdCA-PTEN) and Wataru Ogawa (Kobe University School of Medicine, Chuo-ku, Japan) for the adenovirus encoding the myristoylated-activated form of Akt (AxCA-Myr-Akt). We also thank Kathleen O'Connor for helpful discussions and reviewing the article and Eileen Figueroa and Karen Martin for article preparation.

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