EpCAM antagonizes TGF B1 induced growth arrest TGF B1 acts on epithelial cells as potent growth inhibitory factor and promotes differentiation processes. Basal cells stimulated with TGF B1 stop proliferation within 3 days. In contrast to untreated control cells, displaying a small cell body Tofacitinib baldness and a strong light refracting morphology, TGF B1 treated cells changed morphology and acquired an enlarged and flat cell body. After EpCAM overexpression, TGF B1 stimulated HMECs showed a higher percentage of cells with a small, strongly light ref racting morphology. Moreover, HMECs treated with TGF B1 underwent a terminal growth arrest and stained positively for senescence associated beta galac tosidase, a marker of cellular sene scence.

In clear contrast, upon simultaneously EpCAM overexpression, we could observe many cell clusters that were negative for SA B Gal indicating that cells were not Inhibitors,Modulators,Libraries growth arrested and maintained a longer capacity to proliferate. EpCAM down regulates E cadherin and prolongs proliferative lifespan of HMECs Long term cultures of HMECs in culture medium containing TGF B1 were analyzed for differences between EpCAMGFP and GFP overexpression. In the presence of the differentiation factor TGF B1 EpCAM overexpressing cells were still able to proliferate and formed bigger cell clusters after 6 days in vitro. GFP transfected control cells stopped cell divisions after 3 days and con sisted predominantly of enlarged, flat and growth arrested cells. Analysis of cell numbers revealed a signifi cant increase in cell counts in EpCAM overexpressing cells 6 days after transfection.

Additionally, we analyzed a panel of epithelial to mesen chymal transition markers to define the phenotype Inhibitors,Modulators,Libraries of EpCAM overexpressing cells. In particular, HMECs showed down regulation of E cadherin after TGF B1 stimulation. In clear contrast to GFP con trol cells, EpCAM overexpressing cells showed an add itional down regulation of E cadherin, which would explain the more spindle shape phenotype of the cells. Another marker Inhibitors,Modulators,Libraries of EMT, vimentin expression, Inhibitors,Modulators,Libraries did not increase significantly after EpCAM overexpression in direct comparison to GFP transfected control cells. EpCAM overexpressing HMECs form bigger xenografts consisting of p63high progenitor cells and lack luminal structure formation Based on in vitro findings we analyzed Inhibitors,Modulators,Libraries effects of EpCAM overexpression in our in vivo model.

Therefore, we transplanted HMEC xenografts Imatinib Mesylate CAS onto chicken embryos to analyze in vivo growth. Chicken embryos have only innate immune responses and thus, tolerate growth of human cells. Transfected HMECs were transplanted as growth factor reduced matrigel drops containing TGF B1. After 6 days in vivo growth, xenografts became well vascularized and human onplants could be visualized by expression of GFP.