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First published online 16 December 2003
doi: 10.1242/jcs.00862

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Restoration of full-length adenomatous polyposis coli (APC) protein in a colon cancer cell line enhances cell adhesion

¹ Ludwig Institute for Cancer Research, Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia
² Joint ProteomicS Laboratory, Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia
³ CRC for Cellular Growth Factors, Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia

View larger version (53K): [in a new window]   Fig. 1. Stable expression of functional, full-length mycAPC in SW480 cells. (A) Expression of wild-type APC mRNA in SW480APC cells by RT-PCR analysis. SW480 cells were transfected with pEF-mycAPC or pEF and neomycin-resistant clones selected. Reverse-transcribed APC cDNA was amplified over the region containing the mutation in SW480 cells and digested with PstI, which cleaves only wild-type APC (Hargest and Williamson, 1995). LIM1215 colon cancer cells contain wild-type APC. RT-PCR products from untransfected SW480 cells and control SW480 clones (.2, .7 and .9) are not cleaved by PstI. SW480APC cells (pEF-mycAPC-transfected, independent clones .23, .15, .34, .24 and .38) contain both endogenous, mutated APC mRNA that cannot be digested by PstI (436 bp RT-PCR fragment) and APC mRNA that is cleaved by PstI (294 and 142 bp RT-PCR products), demonstrating the presence of ectopically expressed APC. Some SW480APC cells (clones .14, .18 and .5) that were transfected with pEF-mycAPC and selected in neomycin did not show expression of ectopically expressed APC by RT-PCR analysis. (B) Expression of APC results in translocation of ß-catenin to the cell periphery. SW480 (parental), SW480 control (pEF vector transfected, independent clones .2, .7 and .9) and SW480APC cells (pEF-mycAPC-transfected, independent clones .23, .15, .34, .24, .14, .18, .38 and .5) were stained immunochemically with antibodies to ß-catenin and Alexa488-conjugated anti-mouse IgG. Fluorescent staining was imaged by laser-scanning confocal microscopy. ß-catenin is predominantly nuclear in SW480 cells, SW480 control clones and SW480APC clones negative for APC by RT-PCR analysis, but translocates to the cell periphery in SW480APC clones containing ectopically expressed APC (.23, .15, .34, .24 and .38). Bar, 10 µm. The cellular distribution of ß-catenin was scored as nuclear and cytoplasmic (N), nuclear, cytoplasmic and peripheral (N/P), or peripheral and no nuclear (P), and graphed. Shown is a representative of at least two independent experiments; n, total number of cells counted. (C) ß-catenin/Tcf/LEF reporter activity is downregulated in SW480APC cells. SW480 and SW480APC (independent clones .15, .34 and .24) cells were transfected in triplicate with Tcf/LEF luciferase (TOPflash) reporter gene constructs (van de Wetering et al., 1997). Shown is the ratio of TOPflash to FOPflash luciferase activity from a representative of at least three independent experiments. Error bars indicate s.e.m. from triplicate measurements. (D) Western blot analysis demonstrating expression of full-length mycAPC in SW480APC cells. SW480 and SW480APC cells were lysed, proteins separated by 4% SDS-PAGE and immunoblotted using an anti-myc (9E10) mAb. (E) Western blot analysis of total cellular levels of ß-catenin in SW480APC cells. SW480, SW480 control and SW480APC cells were lysed, 5 µg protein separated by 4-20% SDS-PAGE and immunoblotted using an anti-ß-catenin mAb and an anti-actin mAb to control for protein loading (lower panel). The level of ß-catenin in each clone was quantitated by densitometry, normalized to actin, and is represented as a ratio to ß-catenin in SW480 cells. Shown is a representative of at least three independent experiments.

View larger version (124K): [in a new window]   Fig. 2. Expression of full-length APC in SW480 cells alters cell morphology. Phase contrast micrographs of SW480 cells (A and B) and SW480APC.15 cells (C and D) plated at 104 cells/ml (A and C) or 105 cells/ml (B and D). Elongated SW480 cells (arrows) and tightly packed SW480APC colonies (arrowheads) are indicated. Bars, 20 µm.

View larger version (36K): [in a new window]   Fig. 3. Expression of full-length APC in SW480 cells inhibits cell growth, soft agar colony formation and tumour growth. (A) Cell growth. SW480 and SW480APC.15 cells, seeded in triplicate, were harvested at the time points indicated and counted using a haemocytometer (four fields counted in duplicate). Shown is the mean (±s.d.) number of cells counted from a representative of at least three independent experiments. (i) SW480APC cells (filled symbols) or parental SW480 cells (open symbols) were seeded at either 5x104 cells/ml (squares) or 1x104 cells/ml (diamonds). (ii) SW480 (triangles) and SW480APC (squares) cells were seeded in triplicate at 105 cells/ml. (B) Soft agar colony assay. SW480 or SW480APC.15 cells were seeded in triplicate into 0.3% agar overlayed on 0.6% agar in 35 mm dishes at the densities indicated. Phase contrast micrographs were taken of colonies stained with Crystal Violet formed by SW480 [(i) and (iii)] or SW480APC [(ii) and (iv)] cells after 10 days growth in soft agar. Arrows and arrowheads indicate colonies classified as large and small, respectively. (C) Xenograft tumour growth rate. 5x106 SW480 control cells (clone .7) and 5x106 SW480APC (clone .15) cells were injected into nude Balb/c mice (n=8/group) and tumour volumes of xenografts measured. Shown is the mean (±s.d.) tumour volume (n=8) for SW480 control cells (squares) and SW480APC cells (triangles).

View larger version (47K): [in a new window]   Fig. 4. Alterations in the localization, expression levels and charge of E-cadherin in SW480APC cells. (A) Redistrubution of E-cadherin in SW480APC cells. Cells were immunostained using an antibody against E-cadherin and Alexa488-conjugated anti-mouse IgG. Confocal microscopy reveals a punctate distribution for E-cadherin in SW480 and SW480 control cells (indicated by arrowheads) that is redistributed to sites of cell-cell contact in SW480APC cells (arrows). Bars, 10 µm. (B) Increased expression of E-cadherin in SW480APC cells by western blot analysis. Cells were lysed, 20 µg protein separated by 4-20% SDS-PAGE and immunoblotted using anti-E-cadherin and anti-actin (lower panel) mAbs. The level of E-cadherin in each clone was quantitated by densitometry, normalized to actin, and is represented as a ratio to E-cadherin in SW480 cells. Shown is a representative of at least three independent experiments. (C) 2DE gel analysis. SW480 or SW480APC.15 cells were lysed in 2DE gel extraction buffer containing 9 M urea, 4% (v/v) Chaps, 50 mM DTT, separated in two dimensions (IEF and SDS-PAGE), and analysed by immunoblotting with an anti-E-cadherin antibody. A paired sample of a representative of at least three independent experiments is shown.

View larger version (111K): [in a new window]   Fig. 5. Reorganization of the actin cytoskeleton in SW480APC cells. Vehicle control-treated SW480 (A-C) or SW480APC.15 (panels D-F), and cytochalasin D-treated SW480 (G-I) or SW480APC.15 (J-L) cells, were co-stained with Rhodamine-Phalloidin to visualize F-actin (A,D,G,J) and antibodies to ß-catenin and Alexa488-conjugated anti-mouse IgG to visualize ß-catenin (B,E,H,K). Areas with overlapping actin (Rhodamine, red) and ß-catenin (Alexa488, green) staining are seen as yellow in C, F, I and L (composite). Fluorescent staining was imaged by laser-scanning confocal microscopy. The images shown are single-representative focal planes. Bar, 10 µm.

View larger version (50K): [in a new window]   Fig. 6. Junctional ß-catenin and E-cadherin staining in SW480APC cells is disrupted by EGTA treatment and restored with excess Ca2+. MDCK (panels i-iii), SW480APC.15 (panels iv-vi) or SW480 (panels vii-ix) cells were stained with antibodies to ß-catenin (A), E-cadherin (B) or with Rhodamine-Phalloidin to visualize actin (C). Prior to staining, cells were serum starved overnight and left untreated (i, iv and vii), treated with 4 mM EGTA and 1 mM MgCl2 for 1 our (ii, v, viii) or with 4 mM EGTA and 1 mM MgCl2 for 1 hour followed by treatment with 10 mM Ca2+ for 2 hours (iii, vi, ix). Arrows indicate intact adherens junctions, and arrowheads indicate relocalized staining. Fluorescent staining was imaged by laser-scanning confocal microscopy. The images shown are for single-representative focal planes. Bar, 10 µm.

View larger version (70K): [in a new window]   Fig. 7. SW480APC cells migrate differently to SW480 cells in a wound assay. SW480 (A-F) or SW480APC.15 (G-L) cells were grown as confluent monolayers on a plastic surface and a wound applied with a plastic pipette tip. Cells were photographed at 0, 24 and 48 hours after wounding at 4x (A,C,E,G,I,K) and 10x (B,D,F,H,J,L) magnification. The width of the wound at time (T)=0 is indicated by black lines on the 4x magnification micrographs. The data shown are representative of at least three independent experiments performed in triplicate. Bars, 50 µm.

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