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Co-culturing human prostate carcinoma cells with hepatocytes leads to increased expression of E-cadherin
C C Yates, C R Shepard, D B Stolz and A Wells
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Figure 1.
Co-culture of human prostate cancer cell with rat hepatocytes reversed E-cadherin expression. DU-145 or PC-3 (A) cells were co-cultured in the presence of primary rat hepatocytes over a 6-day period. Hepatocytes and single cultures were lysed before co-cultures. On days 1, 2, 4 and 6, co-culture lysates were immunoblotted with indicated human selective antibodies: anti-E-cadherin, anti-EGFR antibody, anti-cytokeratin 18 and anti-tubulin (as the loading control). (B) Densitometry of immunoblots in DU-145 and PC-3 cells co-cultures (
) EGFR, (
) E-cadherin () and Cytokeratin 18; shown are the mean
s.d. of three blots with day 0 being at 100 (*
P<0.05 from C0). Cellular levels of E-cadherin mRNA in DU-145 or PC-3 (C) cells were analysed by RT-PCR using GAPDH as a loading control. In A and B, Co (for control) are an equal number of prostate carcinoma cells incubated for 1 day in the absence of hepatocytes. The Hep (hepatocytes) were an equal number of hepatocytes as for the co-cultures. The first three lanes were all lysed at the same time. Shown are representative of at least three experiments.
Figure 2.
Immunofluorescence of co-cultures show subcellular location of E-cadherin re-expression. (A) Immunofluorescence of DU-145 RFP (red) and GFP (green) primary rat hepatocytes were stained with human-specific anti-E-cadherin, pan-species anti-
-catenin or human-specific anti-EGFR antibody. Cy5 secondary antibody (blue) was used for respective primary antibodies. Note the gain of blue (E-cadherin) in the RFP/red prostate cells in the top and middle rows, and the loss of blue (EGFR) in the bottom row. (B) The blue channel only of the lower left inset in the E-cadherin and -catenin staining on day 2 is shown in black and white to demonstrate the localisation of the human E-cadherin in the prostate carcinoma cells to the interface with the hepatocytes. In the middle row, the hepatocytes were from WT and not GFP rats, so as not to interfere with the antibody staining as the anti--catenin detected both human and rat. However, the presence of -catenin upregulation in the DU-145 cells is noted by a violet color and the membranous pattern at the hepatocyte–prostate carcinoma cell interface. Shown are representative of at least three experiments.
Figure 3.
Human prostate cancer cells form E-cadherin-mediated heterotypic interactions with hepatocytes. DU-145 (A and B) cells or PC-3 (C–E) cells fluorescently labelled with Calcein A were incubated for 48 h with PD153035 or E-cadherin blocking antibody and seeded onto a monolayer of hepatocytes from non-GFP-expressing rats to analyse their ability to adhere. Cell binding was assessed by fluorescent intensity using a plate fluorometer and visually verified under a fluorescent light microscope. Y-axis is arbitrary fluorescent units. Data represent mean of three experiments performed in triplicate; s.e. * P<0.05. Shown are sample representative fields to show the bound tumour cells (converted to white dots) overlying the hepatocytes in B and E. (C) PC-3 cells were exposed to PD153035 for 48 h with a resultant upregulation of E-cadherin as shown by immunoblotting. This is similar to our previously published finding with DU145 cells (Yates et al, 2005).
Full figure and legend (121K)
Figure 4.
DU-145 cells expressing a PKC transattenuation-resistant EGFR (A654) are resistant to hepatocyte-induced E-cadherin re-expression. DU-145 A654 cells co-culture lysates were immunoblotted with an antibody selective for human E-cadherin, EGFR, cytokeratin 18 or tubulin. The legend is as with Figure 1, Co (control) DU145 A654 cells and Hep (hepatocytes) only. Shown is one of two representative blot series.
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Figure 5.
Human prostate cancer metastases to liver show expression of cell–cell adhesion molecules. Formalin-fixed, paraffin-embedded tissues were obtained from two well-defined prostate adenocarcinomas with liver metastasis. Tissues were stained with indicated antibodies, Secondary antibody, anti-mouse only as the staining control (top left; 3700 
m2), anti-E-cadherin (top centre; 3700 m2 and top right; 300 m2), anti-
-catenin (bottom right; 300 m2), anti--catenin (bottom centre; 300 m2) and anti-p120 (bottom right; 300 m2). Shown are representative of repeated stainings; the other metastasis presented similar findings.
Figure 6.
Human prostate cancer metastases show reversion of metastatic markers. Tissues were stained with anti-rabbit (top left; 1400 m2) anti-EGFR (top centre; 1400 m2), anti-phosphotyrosyl-EGFR (activated EGFR) (top right; 1400 m2), anti-vimentin (bottom left; 1400 m2) and anti-cytokeratin 18 (bottom left; 1400 m2). Shown are representative of repeated stainings; the other metastasis presented similar findings.
