Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion gene into renal cultured epithelial cells. An orientation specific promoter for the AQP2 gene was found within the proximal 3 kb of 5'-flanking region. Minimal basal promoter activity of the AQP2 gene was found within 198 bp upstream from the transcription start site by deletion analysis. Sequencing the transcriptionally active region revealed a typical TATA box, adenosine 3',5'-cyclic monophosphate (cAMP) responsive element (CRE) and three putative CCAAT boxes in the proximal 1.2-kb region. Significantly, a GATA motif, AP1, AP2, and SP1 transcriptional factor consensus sites were also found in this region. Exposure to cAMP-enhancing agents (1 nM vasopressin or 20 mM forskolin and 250 mM 3-isobutyl-1-methylxanthine) showed that these agents increased luciferase activity in a parallel fashion, suggesting that vasopressin-induced AQP2 gene transcription is mediated through increases in intracellular cAMP in at least one renal cell type, the LLC-PK1 cells. The mechanism of cAMP responsiveness of AQP2 gene transcription was further studied using a series of deletion mutants in renal epithelial cells and other cell types. The cAMP regulatory motifs were shown to exist in a 50-bp sequence between -340 and -290 (containing CRE) and a 65-bp sequence (containing an AP2 site) between -150 and the ATG start site in LLC-PK1 cells. In rat inner medullary collecting duct (IMCD) cells, the cAMP regulatory motifs also exist in a 50-bp sequence between -340 and -290 (containing CRE) and in a 10-bp sequence between -160 and -150 (containing an SP1 site). These separate regions may cooperate to confer full cAMP inducibility to the AQP2 gene in a cell-specific manner.

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up-regulated E-cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF-b, SP cells changed their shape into mesenchymal-like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E-cadherin expression level. TGF-b induced EMT-associated gene alteration such as reduction of Ecadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)-2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF-b treatment, whereas MP cells did not respond to TGF-b-mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro-invasion, and in vivo metastasis, as compared to MP cells. Because micro-invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression. ' UICCKey words: TGF-beta; cancer stem cell; invasion; metastasis; E-cadherin Pancreatic cancer is a worldwide health problem with an increasing incidence and poor prognosis. 1 Because of highly metastatic and/or invasive properties of this cancer, the prognosis of the patients with unresectable disease is very poor. For example, the locally advanced disease is observed in 15-20% of pancreatic cancer patients, and associated with a median survival time (MST) of 6 to 10 months. 1,2 In addition, a much higher percentage (40 to 45%) of patients present with metastatic disease, which carries a shorter MST of only 3 to 6 months. 2 Thus, the key to improving the patient survival is to control their local invasion and distant metastasis.Execution of metastasis is composed of multiple processes including cancer cell detachment from the primary tumor, local invasion to disseminate through surrounding blood vessels or lymphatic vessels, and attachment and proliferation at the metastatic site. These processes are at least in part mediated by the phenotypic switch of cancer cells between epithelial phenotype and mesenchymal phenotype, the so-called epithelial to mesenchymal transition (EMT). 3 The EMT is an important process during embryonic development as well as cancer progression. 3 The loss of epithelial characteristics and the acquisition of mesenchymal phenotype lead to enhanced motility and invasive predisposition of cancer cells. The increased migratory activity is mediated by repression of cell to cell adhesion proteins such as E-cadherin, and the induction of invasion-associated integrins and matrix metalloproteinases (MMPs). 4 The cells which obtained...

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Our data suggest that Lefty is a novel TGF-β target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-β-Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-β.