preparations were observed by epifluorescence on an Olympus BX60 microscope. Luciferase reporter assays Cells were seeded in 24-well plates at 56104/well and transfected as previously [54] using EffecteneTM with 0.5 mg of either TOPflash (luciferase construct containing tandem repeats of the minimal TCF promoter) or FOPflash (negative reporter control construct that contains mutant TCF binding sites) plasmid (Millipore, Watford, UK). Reporter plasmids were transfected alongside 0.1 mg of Renilla luciferase expression vector pRL-tk as a transfection control and for normalisation of luciferase activity. Cell lysates were prepared and reporter activity assessed in a 96- well format by the Dual-LuciferaseTM Reporter Assay according to the manufacturer’s instructions (Promega, Southampton, UK) on a POLARstar OPTIMA microplate reader (BMG Labtech, Aylesbury, UK). References 1. Takeichi M (1993) Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Biol 5: 806–811. 2. Shapiro L, Fannon AM, Kwong PD, Thompson A, Lehmann MS, et al. (1995) Structural basis of cell-cell adhesion by cadherins. Nature 374: 327–337. 3. Takeichi M (1995) Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7: 619–627. 4. Nelson WJ, Nusse R (2004) Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303: 1483–1487. 5. Orsulic S, Huber O, Aberle H, Arnold S, Kemler R (1999) E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J Cell Sci 112(Pt 8): 1237–1245. 6. Stockinger A, Eger A, Wolf J, Beug H, Foisner R (2001) E-cadherin regulates cell growth by modulating proliferation-dependent beta-catenin transcriptional activity. J Cell Biol 154: 1185–1196. 7. Pece S, Gutkind JS (2000) Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell- cell contact formation. J Biol Chem 275: 41227–41233. 8. Andl CD, Mizushima T, Nakagawa H, Oyama K, Harada H, et al. (2003) Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo. J Biol Chem 278: 1824–1830. 9. Reddy P, Liu L, Ren C, Lindgren P, Boman K, et al. (2005) Formation of E- cadherin-mediated cell-cell adhesion activates AKT and mitogen activated protein kinase via phosphatidylinositol 3 kinase and ligand-independent activation of epidermal growth factor receptor in ovarian cancer cells. Mol Endocrinol 19: 2564–2578. 10. Takahashi K, Suzuki K (1996) Density-dependent inhibition of growth involves prevention of EGF receptor activation by E-cadherin-mediated cell-cell adhesion. Exp Cell Res 226: 214–222. 11. Qian X, Karpova T, Sheppard AM, McNally J, Lowy DR (2004) E-cadherin- mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases. Embo J 23: 1739–1748. 12. Southgate J, Masters JR, Trejdosiewicz LK (2002) Culture of Human Urothelium. In: Freshney RIaF, M.G (eds), eds. Culture of Epithelial Cells. New York: J Wiley and Sons, Inc. pp 381–400. 13. Varley C, Hill G, Pellegrin S, Shaw NJ, Selby PJ, et al. (2005) Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro. Exp Cell Res 306: 216–229. 14. Southgate J, Hutton KA, Thomas DF, Trejdosiewicz LK (1994) Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab Invest 71: 583–594. 15. Varley CL, Stahlschmidt J, Lee WC, Holder J, Diggle C, et al. (2004) Role of PPARgamma and EGFR signalling in the urothelial terminal differentiation programme. J Cell Sci 117: 2029–2036. 16. Cross WR, Eardley I, Leese HJ, Southgate J (2005) A biomimetic tissue from cultured normal human urothelial cells: analysis of physiological function. Am J Physiol Renal Physiol 289: F459–468. 17. Walker DC, Southgate J, Hill G, Holcombe M, Hose DR, et al. (2004) The epitheliome: agent-based modelling of the social behaviour of cells. Biosystems 76: 89–100. 18. Walker DC, Hill G, Wood SM, Smallwood RH, Southgate J (2004) Agent-based computational modeling of wounded epithelial cell monolayers. IEEE Trans Nanobioscience 3: 153–163. 19. Walker D, Wood S, Southgate J, Holcombe M, Smallwood R (2006) An integrated agent-mathematical model of the effect of intercellular signalling via the epidermal growth factor receptor on cell proliferation. J Theor Biol 242: 774–789. E-Cadherin Growth Regulation Statistics Means and standard errors from the mean (S.E.M.) were used as descriptive statistics. Unless otherwise stated, Student’s t-test was used for evaluation of statistical significance. Acknowledgments We are grateful to all clinical colleagues who supplied urothelial tissues for our research. The authors thank Rod Smallwood for valuable discussions. Author Contributions Conceived and designed the experiments: NTG DCW JS. Performed the experiments: NTG LAK. Analyzed the data: NTG LAK DCW JS. Wrote the paper: NTG JS. 20. Walker DC, Georgopoulos NT, Southgate J (2008) From pathway to population—a multiscale model of juxtacrine EGFR-MAPK signalling. BMC Syst Biol 2: 102. 21. Pece S, Chiariello M, Murga C, Gutkind JS (1999) Activation of the protein kinase Akt/PKB by the formation of E-cadherin-mediated cell-cell junctions. Evidence for the association of phosphatidylinositol 3-kinase with the E-cadherin adhesion complex. J Biol Chem 274: 19347–19351. 22. Zhu AJ, Watt FM (1996) Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes. J Cell Sci 109 (Pt 13): 3013–3023. 23. Zhu AJ, Watt FM (1999) beta-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion. Development 126: 2285–2298. 24. Crallan RA, Georgopoulos NT, Southgate J (2006) Experimental models of human bladder carcinogenesis. Carcinogenesis 27: 374–381. 25. Deng J, Miller SA, Wang HY, Xia W, Wen Y, et al. (2002) beta-catenin interacts with and inhibits NF-kappa B in human colon and breast cancer. Cancer Cell 2: 323–334. 26. Zhu AJ, Haase I, Watt FM (1999) Signaling via beta1 integrins and mitogen- activated protein kinase determines human epidermal stem cell fate in vitro. Proc Natl Acad Sci U S A 96: 6728–6733. 27. Shen X, Kramer RH (2004) Adhesion-mediated squamous cell carcinoma survival through ligand-independent activation of epidermal growth factor receptor. Am J Pathol 165: 1315–1329. 28. St Croix B, Sheehan C, Rak JW, Florenes VA, Slingerland JM, et al. (1998) E- Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27(KIP1). J Cell Biol 142: 557–571. 29. Perrais M, Chen X, Perez-Moreno M, Gumbiner BM (2007) E-cadherin homophilic ligation inhibits cell growth and epidermal growth factor receptor signaling independently of other cell interactions. Mol Biol Cell 18: 2013–2025. 30. Liu WF, Nelson CM, Pirone DM, Chen CS (2006) E-cadherin engagement stimulates proliferation via Rac1. J Cell Biol 173: 431–441. 31. Nelson CM, Chen CS (2002) Cell-cell signaling by direct contact increases cell proliferation via a PI3K-dependent signal. FEBS Lett 514: 238–242. 32. Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411: 355–365. 33. MacLaine NJ, Wood MD, Holder JC, Rees RW, Southgate J (2008) Sensitivity of normal, paramalignant, and malignant human urothelial cells to inhibitors of the epidermal growth factor receptor signaling pathway. Mol Cancer Res 6: 53–63. 34. Margulis A, Zhang W, Alt-Holland A, Crawford HC, Fusenig NE, et al. (2005) E-cadherin suppression accelerates squamous cell carcinoma progression in three-dimensional, human tissue constructs. Cancer Res 65: 1783–1791. 35. Thievessen I, Seifert HH, Swiatkowski S, Florl AR, Schulz WA (2003) E- cadherin involved in inactivation of WNT/beta-catenin signalling in urothelial carcinoma and normal urothelial cells. Br J Cancer 88: 1932–1938. 36. Urakami S, Shiina H, Enokida H, Kawakami T, Tokizane T, et al. (2006) Epigenetic inactivation of Wnt inhibitory factor-1 plays an important role in bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway. Clin Cancer Res 12: 383–391. 37. Urakami S, Shiina H, Enokida H, Kawakami T, Kawamoto K, et al. (2006) Combination analysis of hypermethylated Wnt-antagonist family genes as a novel epigenetic biomarker panel for bladder cancer detection. Clin Cancer Res 12: 2109–2116. 38. Maruyama R, Toyooka S, Toyooka KO, Harada K, Virmani AK, et al. (2001) Aberrant promoter methylation profile of bladder cancer and its relationship to clinicopathological features. Cancer Res 61: 8659–8663. PLoS ONE | www.plosone.org 11 October 2010 | Volume 5 | Issue 10 | e13621