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
25 Cell–cell and cell–matrix adhesive interactions play key roles in many different aspects of cell invasion and migration. These interactions are involved in the process of cell localization,
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
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 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: email@example.com
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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
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
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 Dulbecco’s 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
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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130 ： Fig.1 Cell-cell adhesion of MDCK stable cells was influenced by truncated APC (A) Photographs of MDCK-GFP and N2 cells at different time points by microscope (B) Cell-cell adhesion assay of MDCK-N2-APC cell and the control. Bars represent the standard deviation of three
ndependent experiments conducted in triplicate. i135
2.2 Expression of N2-APC reduces cell aggregation by enervating E-cadherin expression level E-cadherin is an essential molecule which has functions on cell aggregation process. In order
to further elucidate the effects of N2 fragment on cell aggregation, we examined mRNA level of 140
E-cadherin in these two stable cell lines of MDCK-N2-APC and MDCK-GFP. As shown in Fig2A, N2 exhibited lower mRNA level of E-cadherin compared to control cells GFP. The decreased extent was about 60%. Similar results were observed in our subsequent immunostaining experiments. In N2-APC expressed cells, E-cadherin staining was sharp weaker than GFP cells
(Fig2B). Moreover, we examined the protein expression level of E-cadherin using Western 145
analysis. Consistent with the above-mentioned results, N2-APC expressed cells had a lower level
of E-cadherin (Fig2C). Together, these results suggest that reduced aggregation of N2-APC
expressed cell was possibly caused by diminishing E-cadherin expression level.
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155 ： ； Fig.2 Cell –cell adhesion was reduced in MDCK-N2-APC cells (A) Quantitative RT-PCR analysis of E-cadherin in MDCK-N2-APC and MDCK-GFP cells. Relative mRNA level 160 was normalized with MDCK-GFP mRNA. The data were obtained from three biologically independent experiments. The error bars represent the SD. (B) Immunostaining of E-cadherin protein (Red). Scale bars are 10 µm. (C) Western blots analysis of E-cadherin in MDCK-N2-APC and MDCK-GFP cells. Western analysis for α-tubulin from the same blot is shown as a loading control. 165 2.3 Truncated N2-APC promotes cell-matrix adhesion in MDCK epithelial cell line To determine the effects of N2-terminal APC on cell adhesion, we examined the cell
170 adhesion ratio of MDCK-N2-APC to MDCK-GFP control. MDCK-N2-APC is stable cells which
express residues 449-781 of N2-APC. Compare with control cells of MDCK-GFP, we found that
cells expressed N2-APC have relatively quick adhesion for varying periods of time (Fig3A). Then,
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we applied crystal violet to make further assay to the adhesion ratio. Consistent with our previous results, cell adhesion ratio was markedly increased in N2-APC stably expressed cells (p<0.001,
Fig3B). The rising extent was averagely 180%. Taking advantage of SPSS significance test, it 175 demonstrates that these variations of cell adhesion ratio are pretty much behavior significant
difference (Table1). Taken together, these results indicate that there is an intimate relationship between cell-matrix adhesion and N2-APC. The over-expression of N2-APC fragment of APC
affects the ability of MDCK cells to the matrix.
，180 ： Fig.3 Cell-matrix adhesion of MDCK stable cells was influenced by truncated APC. (A) Photographs of MDCK-N2-APC and MDCK-GFP cells at different time points by microscope. (B) Cell-matrix 185 adhesion assay in N2and GFP expressed stable cells. The data were obtained from three biologically independent
experiments. The data are expressed as the means of three independent experiments?SD. Tab.1 Cell-matrix adhesion ratio /Control AExperimental group A 570570Cell 0.5h 1h 1.5h 2h MDCK-GFP 1.00?0.017 1.00?0.047 1.00?0.030 1.00?0.029
MDCK-N2-APC 2.22?0.040** 2.25?0.018** 1.38?0.012** 1.37?0.014**
Mean values SEM from three independent experiments. Data were given as the mean?SEM. Significance was 190
established when P<0.05 by Student’s t test. ** P<0.01.
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2.4 N2 fragment of APC enhances cell adhesion by means of improving CD29 level It has been known that CD29 is a key molecule in the process of cell-matrix adhesion. To
195 further study how truncated APC does affect on cell adhesion, we examined the protein expression
level of CD29 in these two stable cell lines. As shown in Fig4, Western blot result illustrates that CD29 expression level was notable increased in MDCK-N2-APC cells. Our results suggested that
N2 fragment could enhance cell adhesion through improving CD29 expression. 200 Fig.4 Cell-matrix adhesion was increased in MDCK-N2-APC Western blot analysis of CD29 protein in MDCK-GFP and MDCK-N2-APC cells. Western analysis for α-tubulin from the same blot was shown as a loading control.
Adenomatous polyposis coli (APC) is a cytoskeletal organizer for cell migration and a 205
scaffold and it affects GSK3β/CKI-mediated phosphorylation and degradation of the Wnt key molecular β-catenin. A majority of colorectal cancers exhibit inactivating mutations in APC, and  loss of APC function is an early event if not initiating event during tumorigenesis . Abrogation of cell-adhesive properties is a hallmark of invasive carcinomas. Full length APC has more
recently shown to play a role in cell-cell adhesion. It has been detected at lateral plasma membrane 210
 in some cells, and loss of APC correlates with defective intercellular adhesion . Previous studies implicated APC in cell adhesion because expression of full-length APC in colon carcinoma cells  restored cell–cell adhesion . It was reported in another study, however, that loss of both APC1 and APC2 did not impair cadherin-based cell–cell adhesion in Drosophila . Our results, found
215 that APC had a crucial role in cell adhesion. Truncated APC could result in significant variation of
cell adhesion and aggregation in MDCK cells. the experiments implicated that N2-APC fragments has distinct effects on cell adhesion.
The specific mechanism of APC regulates cell adhesion and aggregation has not been  completely clear. Harris et al considered that it seems unlikely that APC regulates cell–cell  220 adhesion directly, as the interaction of APC with V / E-cadherin is minimal. However, Faux et
al. also observed changes in the post-transcriptional modification of E-cadherin in APC truncated  cells, which could be linked to its APC-induced redistribution to the plasma membrane. Kamal
and colleagues implicated that APC could dominate cell adhesion indirectly by regulating the validity of β-catenin for connecting into adherens junctions or by regulating vesicle transporting to
and from the plasma membrane by tethering microtubules to cell–cell contacts. In keeping with 225
recently reported studies, we also found that truncated APC has the powerful influence on cell adhesion and aggregation. N2 fragment affects cell adhesion by regulation certain molecular
which participate in cell–cell contacts process. In other words, intracellular distribution and expression level of this adhesion molecular were changed owing to loss of APC function. This study demonstrated that there is an intimate relationship between N2-APC and cell 230
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adhesion. In MDCK cell, N2 enhances cell adhesion via ascend of CD29, and reduces cell aggregation through lowing E-cadherin expression level. Evidence is beginning to emerge that
APC proteins may be another example of a dual-function protein with dividable roles in Wnt . However, up to now, it has not been shown that APC signaling pathway and cell adhesion
proteins participate directly in the two processes. There are multiple comprehensions to explain 235
the mechanism how does APC control cell adhesion. Our research concretely focuses on the N2 ment of APC. This fragment affects cell adhesion and aggregation. This study demonstrated frag that N-terminal of APC, especially N2 fragment, play an important role in the process of cell adhesion. Our data’s apply novel evidence for studying the effects of APC truncation on cell
adhesion in colon cancer cells. Further understanding on the mechanism of APC and cell adhesion 240 will have a significant impact on promoting the fundamental study in the areas pertaining to APC
and carcinomas, which in turn will hold great potential for clinical therapy of patients with tumors. Acknowledgements This project was supported by grants of National Natural Science Foundation of China (No.
31040018 & 8115026); Shanxi scholarship council of China (201010); International collaboration 245 projects (2011081058); Specialized Research Fund for the Doctoral Program of Higher Education
References  Madri JA, Graesser D. Cell migration in the immune system: the evolving inter-related roles of adhesion 250 molecules and pretenses [J]. Dev Immunol, 2000, 7(2-4):103-116.  HUANG Zhi-yong, Wu Yan-Li, Hedrick N, et al. Gutmann. T-Cadherin-Mediated Cell Growth Regulation Involves G2 Phase Arrest and Requires p21CIP1/WAF1 Expression [J]. Mol Cell Biol, 2003, 23(2): 566-578.  LI Zhi, Gallin WJ, Lauzon G, et al.L-CAM expression induces fibroblast-epidermoid transition in squamous
carcinoma cells and down-regulates the endogenous N-cadherin [J]. J Cell Sci, 1998, 111 (7):1005-1019. 255  LI Zhuo-yu, Kroboth K, Newton IP,et al.Novel self-association of the APC molecule affects APC clusters and cell migration [J]. J Cell Sci,2008,121(11):1916-1925.
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 Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer [J]. Cell, 1996, 87(2):159-170. 260  Rosin-Arbesfeld R, Ihrke G, and Bienz M. Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells [J]. EMBO J, 2001, 20(21), 5929-5939.  Faux MC, Ross JL, Meeker C, et al.Restoration of full-length adenomatous polyposis coli (APC) protein in a
colon cancer cell line enhances cell adhesion [J]. J Cell Sci, 2004, 117(3): 427-439.
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APC 截短突变对 MDCK 细胞-基质、细胞-细胞粘附 的影响作用
李文玲，祝文思，牛海波，宋莉，李卓玉 ，山西大学生物技术研究所，化学生物学与分子工程教育部重点实验室，太原 030006！ 摘要(目的 探讨结肠腺瘤息肉蛋白 APC 截短突变对细胞-细胞、细胞-基质之间粘附的影响 285 作用及具体的机制。方法 应用细胞粘附测定实验检验 MDCK-N2-APC 及 MDCK-GFP 两稳定表 达株系的粘附率。相对与对照细胞 MDCK-GFP,MDCK-N2-APC 细胞为稳定突变株，表达 APC 蛋 白 N 端 449-781 氨基酸片段。免疫荧光染色、荧光定量 PCR 及 Western Blot 测定一些在细 胞粘附过程中发挥重要作用的粘附分子的表达情况。结果 与对照相比，N2 中细胞-基质 之间粘附率增加，平均升高约 180%；而细胞-细胞间粘附率约下降 30%。研究表明，N2 片段
通过降低 E-cadherin 的表达水平来减少细胞-细胞间粘附力，通过提高 CD29 的表达水平增 290 加细胞-基质间粘附力。结论 APC 在细胞-基质及细胞-细胞间粘附中发挥着重要的作用。 APC 截短突变片段 N2 片段通过影响一些粘附分子的表达量对 MDCK 细胞的粘附产生作用。 关键词(细胞-基质间粘附；细胞-细胞粘附；结肠腺瘤息肉蛋白，APC！；截短突变 中图分 类号(Q2
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