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Effects of truncated mutation of APC on cell-matrix and cell-cell adhesion in kidney epithelial cell lines

By Kim Wallace,2014-09-10 21:20
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Effects of truncated mutation of APC on cell-matrix and cell-cell adhesion in kidney epithelial cell lines

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    Effects of truncated mutation of APC on cell-matrix and

    cell-cell adhesion in kidney epithelial cell lines

    LI Wenling, ZHU Wensi, NIU Haibo, SONG Li, LI Zhuoyu

    5 (The Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry,

    Institute of Biotechnology, Shanxi University, TaiYuan 030006)

    Abstract: APC is associated with cell adhesion, but the effects of truncted APC and the mechanism are not well defined.To explore the impact of mutant adenomatous polyposis coli (APC) on cell-matrix, cell-cell adhesion and the relative mechanism. Cell-matrix and cell-cell adhesion assay were employed

    10 to determine the adhesion level of two stable cell lines MDCK-N2-APC and MDCK-GFP. The truncated APC of N2 fragment, which spans residues 449-781 was studies compared to control cells including GFP alone. The immunofluorescence staining, RT-PCR analysis and Western blotting were applied to check several adhesion molecular which have key roles in cell contacts process. In contract with control, cell-matrix adhesion was averagely increased to180% in N2 cells, whereas, cell-cell

    15 adhesion was reduced by about 30%. Our experiments indicated that N2 fragment of APC decreases cell adhesion via enervating E-cadherin expression level. It enhances cell adhesion by means of improving CD29 level respectively. These data suggest that full length APC plays a crucial role in cell-matrix adhesion and cell-cell adhesion. The truncation mutation of APC fragment, N2 restrained in the colon cancer cells, will alter the cell invasion and migration by the influence on cell adhesion and cell- matrix adhesion 20

    Keywords: cell-matrix adhesion; cell-cell adhesion; adenomatous polyposis coli (APC); truncated mutation

    0 Introduction

    25 Cellcell and cellmatrix adhesive interactions play key roles in many different aspects of cell invasion and migration. These interactions are involved in the process of cell localization,

    [1]effector recognition, and activation phenomena. Cadherins are calcium-dependent cell adhesion

    receptors with well established roles in morphogenesis. Several studies have suggested that cadherin family members have important roles in the malignant progression of various human

    30 cancers. In addition to their established functions in modulating cell adhesion, aggregation, cell polarity, and morphogenesis, the classic aggregation molecules, E-cadherin, has also been

    [2]implicated in the molecular pathogenesis of lung, breast, liver, and gastric cancers. In epithelial

    cells, E-cadherin is very important for compact association of the cells in epithelial sheets, and in this capability, E-cadherin might function as a suppressor of invasiveness and metastasis of [3]35 epithelial tumors.CD29 (Integrin beta-1) is an integrin unit associated with very late antigen receptors. It is the beta subunit of an integrin family of molecules expressed on diverse cell types which function as the major receptors for extracellular matrix and as cell-cell adhesion molecules.

    Adenomatous polyposis coli (APC), a tumor suppressor commonly mutated in cancer, is a cytoskeletal organizer for cell migration in the mammalian intestinal epithelium. Most human

    40 colorectal tumours carry mutations in the APC gene that result in the expression of truncated N-terminal APC fragments lacking sites required for the formation of the β-catenin targeting

    complex so that intracellular β-catenin is not regulated properly and the normal genetic programme is altered. In addition, APC participates in several other cellular processes, including

     cytoskeletal regulation, so that loss or truncation of APC also directly affects cell migration and

    Foundations: National Natural Science Foundation of China (No.31040018&8115026); Shanxi scholarship council of China (201010); International collaboration projects (2011081058); Special research foundation of the doctoral program of higher education (20111401110011)

     Brief author introduction:李文玲,,1986-!,女,硕士研究生,生物化学及细胞分子生物学。

     Correspondance author: 李卓玉,,1964-!,女,教授,博士生导师,细胞分子生物学。E-mail: lzy@sxu.edu.cn

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     [4]45 chromosome segregation . Notably, APC truncation can be associated with these adherens

    junctions and evidence points to a role for APC in cellular adhesion. The association of truncated

    mutation of APC with the cytoskeleton and the plasma membrane at cell-cell junctions has led to

    [5]suggestions of a role for APC in cell-cell adhesion , which could be relevant to its activity as a

    tumour suppressor. However, it is unknown whether there is a functional relationship between 50 APC truncation at cell cell contacts.

    Here, we report that truncated APC affects cell-cell adhesion and cell-matrix adhesin,

    especially MDCK-N2-APC, which contains N-terminal fragments 449-781 residues of APC. Our

    experiments show that cell adhesion is increased and cell-matrix adhesion is reduced respectively.

    These results implicate the roles of truncated N-APC fragment by the residues of 449-781 amino

     55 acids acts on cell cell contacts and further effects cancer cell invasion and

    migration.

     1 Materials and Methods

    1.1 Antibodies and cell culture

    Antibodies used in this study were: anti-E-cadherin (Bioworld), Monoclonal anti-α-tubulin

    60 antibody (Sigma), anti-GFP (Cell Signaling), anti-CD29 (BD Biosciences). HRP-conjugated goat

    anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) secondary antibodies were from Invitrogen.

    MDCK-GFP and MDCK-N2-APC stable cells (respectively expressing wild type and

    truncated APC spanning residues 449-781) were routinely maintained in Dulbeccos modified

    eagles medium (DMEM, HyClone) containing 250 µg/ml gentamycin (G418, Biosharp ) at 37?

    65 in a 5% COincubator. The media contained 10% fetal calf serum (Boster), 1% penicillin/ 2

    streptomycin (Solarbio)

    1.2 Immunofluorescence staining

    Cells were grown on cover glasses in media for appropriate at 37?C. After three washes with

    phosphate-buffered saline (PBS), cells were fixed in 4% paraformaldehyde for 20 min, 70 permeabilized with 0.3% Triton X-100/PBS for 3 min, and pre-blocked in 3% BSA/PBS for 1h.

    The slides were then incubated with a primary antibody (diluted 1:1000 in blocking solution) for

    4h at 4?C, washed three times with PBS, and incubated with a secondary antibody with a

    TRITC-conjugated anti-rabbit antibody (diluted 1:100) for 1h. After further washes, the slides

    were mounted with Sealed tablet (Solarbio), and cells were examined with fluorescence optics on 75 a confocal microscope.

    1.3 Cell adhesion

    MDCK-N2-APC and MDCK-GDP cell adhesion was performed by a pre-coated 96-well

    plate with fibronectin (10 g/ml; Sigma) at 37?C for 1 hour. The cells were digested with 0.05%

    5 trypsin and resuspended at 5×10cells/ml in DMEM media. Cells (100 µl) were added to each

    80 well and allowed to adhere for 0.5 or 2.5 h. At the end of the incubation period, cells were washed

    three times with PBS, and each well was added in 50 µl 4% for 10min at room temperature. Then

    the wells were washed three times, stained with 0.5% crystal violet for 20 min at room

    temperature. After these, 50 µl of 1% sodium dodecyl sulfate was added to each well. The number

    of adherent cells was quantified by absorbance at 570 nm. Each experiment was repeated at least 85 three times with identical results

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     1.4 Cell aggregation assay Cells were washed with PBS twice and digested with 0.05% trypsin. The cells were 5 resuspended at 5×10per 100 µl with DMEM media, and 100 µl of the cell suspension was seeded in each well of 96-well plates. The plates were placed in a 37?C shaker and rotated at 80 rpm for 1 hour. The wells were then gently stirred. The number of aggregates and single cells were 90

     counted with a hemacytometer. The rate of aggregation was calculated by the percentage of decrease in the number of single cells using the formula [(N-N)/N]/100. Where Nis the number 0i00

     of single cells at time 0, and Nis the number of single cells detected in cultures at various time i points after incubation. Each value is the average of at least three independent experiments 1.5 RNA preparation and RT-PCR analysis 95 To identify E-cadherin RNA transcripts, quantitative real time PCR (qRT-PCR) was performed using the Applied Biosystems. In brief, Total RNA isolation from cultured cells was performed using Trizol on the basis of manufacturer’s instructions (TAKARA). 1 µg of DNA-free total RNAs was reverse transcribed using PrimeScript RT Master Mix according to the manufacturer's instructions (TAKARA). For the qRT-PCR assay, 25 µl reaction containing 4 µl 100 TM cDNA, 1 µl of each primer and 12.5 µl SYBR Premix Ex Taq(TAKARA) were used to monitor double-strand DNA synthesis. Primers used were 5AGGACCAGGTGACCACCCTAGA 3’ (forward), 5’ ATGCCCAAGATGGCAGGAAC 3(reverse) for E-cadherin and 5 GCACCGTCAAGGCTGAGAAC 3’ (forward), 5’ TGGTGAAGACGCCAGTGGA 3(reverse) for GAPDH. 105 1.6 Western blotting Lysates were separated by 8% sodium dodecyl sulphatepolyacrylamide gels and transferred

     to nitrocellulose membranes. Anti-E-cadherin (1:1000; Bioworld), anti-a-tubulin (1:1000; Sigma), anti-CD29 (1:1000; BD Biosciences) antibodies were as primary antibodies. After three times washing, membranes were incubated with the appropriate horseradish peroxidase conjugated 110

     secondary antibody. Blots were developed with ECL Plus Western Blotting Detection System.

     1.7 Statistical methods Statistical analysis was carried out using the SPSS software program. Data, derived from three or four independent experiments, are presented as the means +SD, and were analyzed with analysis of variance followed by the t test, with significance (P) set at 0.01. 115 2 Results 2.1 Expression truncated N2-APC affects cell-cell adhesion in MDCK cells To verify whether N2 fragment of APC is also involved in the regulation of cell-cell adhesion (cell aggregation), we examined the cell aggregation ratio of MDCK-N2-APC and the concrol of MDCK-GFP cells. From Fig1A, the data indicated clearly that cell aggregation ratio was 120

    decreased in N2-APC expressed cells. The followed calculated result showed that aggregation

    ratio with N2-APC was reduced by about 30% compared with GFP control cells (Fig1B). These

     data suggest that truncated N2-APC could affects cell-cell adhesion in MDCK cells.

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