Technical Reference #1628

1628.

E-Cadherin Mutations and Cell Motility: A Genotype-Phenotype Correlation Ana Rita Mateus; Joana Simoes-Correia; Joana Figueiredo; Stefan Heindl; Catarina Castro Alves; Gianpaolo Suriano; Birgit Luber; Raquel Seruca, University of Porto, Experimental Cell Research, 315(1628), (2009)

Abstract:
E-cadherin has a determinant role in tumour progression acting as an invasion and metastasissuppressor. Germline mutations of E-cadherin gene (CDH1) occur in 30% of families withHereditary Diffuse Gastric Cancer (HDGC); of these 23% are missense muta

Keywords:
gastric cancer; HDGC; germline mutations; E-cadherin; cell motility; cell signaling; EGFR; CDH1; diffuse carcinoma; familial cancer

Materials & Methods:
Cell culture and transduction CHO-K1 (Chinese Hamster Ovary) cells were transduced with a pcDNA3 mammalian expression vector encoding the E-cadherin variants A298T T340A P799R and V832M as well as wild-type Ecadherin or the empty vector alone as previously described [1314]. The transfected cells were selected by antibiotic resistance to geneticin G418 500 mg/ml. ViraPower Lentiviral Expression kit (Invitrogen Carlsbad CA USA) was used for the expression of the E-cadherin variants T118R L214P G239R P373L R749W E757K and E781D as well as wild-type and empty vector alone as previously described [2526]. The transduced cells were selected by antibiotic resistance to blasticidin (5 μg/ml). Cells were grown at 37 °C under 5% CO2 humidified air in α- MEM (+) medium (Invitrogen) supplemented with 5% foetal bovine serum (HyClone) 2 mM L-glutamine 1% penicillin/ streptomycin and geneticin or blasticidin (Invitrogen) according to the expressing vector. Wild-type and mock controls from both expression methods have been used in all experiments with equal results therefore excluding any dependence of the results on the transduction method used. Cell motility assay The migratory/motility behaviour of the transfected cells was analysed by time-lapse microscopy. Cells were seeded in full medium at a density of 1.5×105 cells in 3.5 cm glass bottom plates (MatTek Corporation Ashland MA) coated overnight at 4 °C with Fibronectin (10 μg/ml Sigma). Plates were kept in a microscopecoupled incubation chamber (Leica) at 37 °C under 5% CO2 humidified air. Phase-contrast images were taken with a DC- 300F Leica camera at 3 min intervals for 7 h starting 2 h after cell seeding using a 20× objective. The percentage of motile cells was determined by drawing the outlines of cells and counting those cells which had completely left the initial area within the observation time of 7 h [27]. Statistical analysis Student's t test for independent samples has been used to calculate the difference between the motility behaviour of cells expressing wild-type E-cadherin and cells negative for E-cadherin or expressing the different E-cadherin mutations. Null hypothesis was considered as existing no difference in cell motility and rejected for p values lower than 0.05. Immunofluorescence staining Cells were seeded on 6-well plates on top of glass coverslips at a density of 1.5×105 per well and starved overnight 24 h after seeding. Prior to fixation cells were stimulated with EGF 100 ng/ ml for 15 min. Fixation was performed in 4% Formaldehyde in PBS for 30 min at 4 °C followed by blocking of the aldehyde groups with NH4Cl 50 mM in PBS for 10 min at room temperature. Permeabilization was done with Triton X-100 0.25% in PBS for 5 min at room temperature before blocking with BSA 1% for 30 min at room temperature. The Tyr1086 phospho-EGFR (Zymed Laboratories Invitrogen San Francisco CA USA) was used at 1:150 dilution in PBS with BSA 1% and incubated overnight at 4 °C. An anti-rabbit FITC coupled antibody (DakoCytomation Glostrup Denmark) was applied as secondary antibody at 1:50 dilution together with DAPI (1:1000) to counterstain the nucleus. Antibody was diluted in PBS containing 1% BSA and incubated for 1 h at room temperature in the dark. Coverslips were mounted on slides using an antifading reagent (Vectashield Vector) and examined on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope using 20× and 40× objectives. Images were taken with an Axiocam HRm camera and processed with the Zeiss Apotome software. Immunoprecipitation and immunoblotting Cells were seeded on 10 cm diameter cell culture dishes coated with 10 μg/ml Fibronectin (Sigma) according to the manufacturer's instructions at a density of 1.5×106 cells in full medium to allow an approximate confluence of 70% after 24 h. Cell lysis was performed 24 h after seeding with 300 μl of L-CAM buffer (140 mM NaCl 4.7 mM KCl 0.7 mM MgSO4 1.2 mM CaCl2 10 mM Hepes pH 7.4 containing 1% (v/v) Triton X-100 [28]. The lysis buffer contained 2 mM phenylmethylsulfonylfluoride 2 mM orthovanadate 19 mg/ml aprotinin 20 mg/ml leupeptin 10 mM sodium phosphate and 100 mM sodium fluoride. Total protein was quantified by following the Bradford dye-binding procedure [29]. Immunoprecipitation studies were performed by using the ‘Catch and Release v2.0’ kit (Upstate). An aliquot of 500 μg of protein was immunoprecipitated according to the manufacturer's instructions; 2 μg of monoclonal antibody against EGFR (Santa Cruz) were used. Immunoprecipitated proteins were separated on a 7.5% SDS-polyacrylamide gel electrophoresis followed by transfer onto a nitrocellulose membrane (Hybond C-extra Amersham Bioscience). Immunoblotting was performed with an antibody against E-cadherin (BD Biosciences 1:5000). Twenty micrograms of total cell lysates was immunoblotted with antibodies against Src kinase (1:2000) phospho-Src Tyr 416 (1:1000) p38 MAPK (1:2000) and phospho-p38 Thr 180/Tyr 182 (1:1000) from Cell Signaling Technologies (New England Biolabs Frankfurt Germany) phospho-c-Met Tyr 1230/1234/1235 (1:1000) from Calbiochem (Merck KGaA Darmstadt Germany) and α-tubulin (1:10000) from Sigma (Sigma Deisenhofen Germany) as a loading control. For signal detection the ECL western blotting detection kit (Amersham Bioscience) was used. Immunoblots were quantified with the Scion Image Software from Scion Corporation (Version Beta 4.0.2; Frederick). Cell treatments To address the role of EGFR signalling cells were cultivated in the presence of the EGFR inhibitor Tyrphostin AG 1478 (9.45 mM) (SIGMA) or the same volume of DMSO to exclude the effect of the solvent. For protein analysis Tyrphostin AG 1478 was added to the cells for 4 h prior to lysis; for motility analysis Tyrphostin AG 1478 was added to the cells immediately after seeding. To inhibit the proteosome and restore E-cadherin expression in mutants R749W and E757K cells were treated with DMSO 2% during 24 h prior to lysis [26].

Microscopic Technique
Time Lapse Microscopy

Cell Type(s)
CHO-K1