Hepatocytes convert to a fibroblastoid phenotype through the cooperation of TGF-ß1 and Ha-Ras: steps towards invasiveness
Josef Gotzmann1,
Heidemarie Huber1,
Christiane Thallinger2,
Markus Wolschek2,
Burkhard Jansen2,
Rolf Schulte-Hermann1,
Hartmut Beug3 and
Wolfgang Mikulits1,*
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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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© The Company of Biologists Ltd 2002
