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Hepatocytes convert to a fibroblastoid phenotype through the cooperation of TGF-ß1 and Ha-Ras: steps towards invasiveness

¹ Institute of Cancer Research, University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria
² Department of Clinical Pharmacology, Section of Experimental Oncology, Vienna General Hospital, Währinger Gürtel 18-20, A-1090 Vienna, Austria
³ Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria

View larger version (46K): [in a new window]   Fig. 1. MMH-D3 cells display a polarized phenotype and respond to the growth inhibitory function of TGF-ß1. (A) Phase contrast and confocal immunofluorescence microscopy of parental MMH-D3 cells stained with the adherens junction markers E-cadherin and ß-catenin and the tight junction marker ZO-1. (B) Proliferation kinetics of MMH-D3 cells (circles) versus MMH-D3 supplemented with 5 ng/ml TGF-ß1 (squares). (C) Flow cytometry determining the cell cycle distribution of MMH-D3 cells versus MMH-D3 at day 5 of TGF-ß1 (5 ng/ml) treatment.

View larger version (23K): [in a new window]   Fig. 3. Cell cycle progression of MMH cell types and expression of marker proteins. (A) Proliferation kinetics of epithelial (MMH-D3, circles; MMH-R, squares) versus fibroblastoid MMH-RT cells (triangles). (B) Protein abundance of representative components participating in intercellular communication in epithelial and fibroblastoid cells. Besides the exogenous expression of Ha-Ras in epithelial MMH-R and fibroblastoid MMH-RT cells, the downregulation and loss of respective markers is indicated in fibroblastoid cells by immunoblotting.

View larger version (131K): [in a new window]   Fig. 2. TGF-ß1 triggers an epithelial to fibroblastoid conversion of MMH-R cells expressing constitutive active Ha-Ras. Left panel (—TGF-ß1): MMH-R cells show a polarized epithelial phenotype as analyzed by phase contrast and confocal immunofluorescence microscopy. Right panel (+TGF-ß1): Epithelial MMH-R cells treated with 5 ng/ml TGF-ß1 undergo a conversion to a spindle-shaped fibroblastoid phenotype. The resulting depolarized MMH-RT cell type was processed for microscopical inspection. Exceptionally, cells were stained for Smad2 30 minutes after TGF-ß1 induction. Insets in panels of undetectable E-cadherin and desmoplakin staining indicate the presence of GFP-positive cells.

View larger version (55K): [in a new window]   Fig. 4. Malignant transformation of epithelial MMH-R and fibroblastoid MMH-RT cells analyzed in vitro and in vivo. (A) Phase contrast images depicting the typical colonies formed in vitro by anchorage-independent growth of MMH-R and MMH-RT cells in soft agar. (B) Kinetics of tumor formation in vivo after subcutaneous injection of MMH-R (circles) and MMH-RT cells (squares) into immunocompromized SCID/BALB/c recipient mice. (C) Visualization of endothelial cells in histological sections of tumors by immunological staining with anti-von Willebrand Factor. Insets represent lower magnifications (10x) of histological sections. (D) Dedifferentiation of epithelial MMH-R and fibroblastoid MMH-RT cells after tumor formation in vivo. Histological sections of tumors give rise to poorly differentiated cell carcinomas as shown by immunological staining with ZO-1. The cytoplasmic distribution of ZO-1 appears to be very weak in vascularized MMH-RT-derived tumors, and cell boundary staining is exclusively displayed by endothelial cells (white arrow). (E) Assessment of invasive properties in vitro. The ability of epithelial MMH-R and fibroblastoid MMH-RT cells to migrate through Matrigel matrices as reconstituted basement membranes is shown. Invaded cells on lower surfaces of membranes were visualized by immunofluorescence microscopy of GFP-positive MMH-R and MMH-RT cells.

View larger version (92K): [in a new window]   Fig. 5. Typical hepatocellular-derived cell structures in collagen gels. (A) A phase contrast image of epithelial MMH-R cells generating lumen-forming structures. (B) Treatment of epithelial MMH-R cells with exogenous TGF-ß1 (5 ng/ml) resulted in the formation of disordered branching cord-like structures.

View larger version (30K): [in a new window]   Fig. 6. Molecular characteristics of epithelial MMH-R versus established fibroblastoid MMH-RT cells. (A) TGF-ß1 production of epithelial and fibroblastoid cell types. The amount of latent TGF-ß1 secretion into the medium was determined by ELISA. (B) Semi-quantitative RT-PCR determining the selective decrease (left panel) and increase (right panel) of mRNA abundance in epithelial versus fibroblastoid cells. All samples contained equal amounts of cDNA. As a control, rhoA mRNA expression remained unaffected (right panel). Lane 1, epithelial MMH-R cells; lane 2, fibroblastoid MMH-RT cells.

View larger version (98K): [in a new window]   Fig. 7. Reversion of fibroblastoid MMH-RT cells to an epithelium-like phenotype. (A) Phase contrast microscopy of MMH-RT cells (control) grown on tissue culture plates and either treated with 30 µM UO126 or 5 µM LY294.002 for 24 hours. (B) Confocal immunofluorescence microscopy of MMH-RT cells treated with 5 µM LY294.002 for 24 hours. (C) Expression of E-cadherin and MMP-9 as determined by immunoblotting. Staining of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. Lane 1, fibroblastoid MMH-RT cells; lanes 2 and 3, MMH-RT cells treated with 5 µM LY294.002 for 24 and 72 hours, respectively; lane 4, epithelial MMH-R cells.