Cells heterozygous for the ApcMin mutation have decreased gap junctional intercellular communication and connexin43 level, and reduced microtubule polymerization

Trine Husøy2,, Véronique Cruciani1, Helle K. Knutsen, Svein-Ole Mikalsen1, Hege B. Ølstørn and Jan Alexander

Department of Food Toxicology, Norwegian Institute of Public Health, PO Box 4404 Nydalen, NO-0403 Oslo, Norway
1 Department of Environmental and Occupational Cancer, The Norwegian Radium Hospital, NO-0310 Oslo, Norway

2 To whom correspondence should be addressed Email: trine.husoy{at}fhi.no


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutations in the tumour suppressor gene adenomatous polyposis coli (Apc) are early and critical events in the development of colon cancer. In the absence of functional Apc, ß-catenin is not degraded in the cytoplasm and can be transported to the nucleus and turn on transcription of several genes, including the gap junction protein connexin43. Apc also stabilizes microtubules and regulates microtubule polymerization. Changes in Wnt signalling and microtubule function are reported to affect the connexin level. To study the effect of heterozygous Apc mutation we examined gap junctional intercellular communication (GJIC) in IMCE (Immorto-Min colonic epithelium) cells with one mutated Apc allele and in YAMC (Young adult mouse colon) cells with normal Apc function. IMCE cells had only half the GJIC level compared with YAMC cells. RT–PCR showed that both YAMC and IMCE cells express a common complement of seven connexin genes (Cx26, Cx31, Cx39, Cx40, Cx43, Cx45 and Cx50), with an additional Cx29 gene expression in YAMC cells. We found that the Cx43 level was correspondingly lower in IMCE cells as detected by western blotting and immunofluorescence. There were no differences in the level or localization of ß-catenin and the downstream gene E-cadherin between the cells, indicating no activation of the Wnt-signalling pathway in cells with one mutated Apc allele. We also examined the microtubule polymerization rate, and IMCE cells had markedly slower microtubule polymerization than YAMC cells. Hence, it appears that mutation in one Apc allele is sufficient to affect microtubule function, while inactivation of both wild-type Apc alleles may be necessary for activation of Wnt signalling. Reduction in GJIC and Cx43 level in IMCE cells may be caused by reduced Cx43 transport as a result of alterations in microtubule function.

Abbreviations: Apc, adenomatous polyposis coli; Cx, connexin; FAP, familial adenomatous polyposis; GJIC, gap junctional intercellular communication; GSK3ß, glycogen synthase kinase 3ß; IMCE, Immorto-Min colonic epithelium; INF{gamma}, interferon-{gamma}; Min, multiple intestinal neoplasia; RT–PCR, reverse transcriptase–polymerase chain reaction; SV40, Simian virus 40; YAMC, Young adult mouse colon.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutations in the adenomatous polyposis coli (APC) gene are early and critical events in the development of colon cancer (1). Germ-line mutations in APC lead to familial adenomatous polyposis (FAP), an autosomal dominant condition characterized by the development of hundreds to thousands of colorectal adenomatous polyps in the second to third decade of life (2,3). Most of the adenomas have mutations in or loss of the remaining wild-type (wt) APC allele (4,5). Somatic APC mutations have also been observed in >80% of the sporadic adenomas and carcinomas (1,6,7).

Several important cellular functions, such as proliferation, migration, differentiation and apoptosis are affected by mutations in Apc (811). It is, however, not fully understood by which mechanisms the defect Apc protein provokes all these changes. The ability of Apc to regulate the ß-catenin level is one of the most studied functions of Apc, where Apc contributes together with axin and glycogen synthase kinase 3ß (GSK3ß) to target ß-catenin for degradation. While early studies showed that ß-catenin takes part in cell–cell adhesion (12), more recently both Apc and ß-catenin were found to be components of the Wnt-signalling pathway (13,14). In the absence of functional Apc, free ß-catenin is not degraded in the cytoplasm and can be transported into the nucleus. ß-Catenin can turn on transcription of several genes by binding to the transcription factor Tcf. To date, a handful of ß-catenin/Tcf transcriptional targets have been described. These include c-myc (15), cyclin D1 (16), matrilysin (17), E-cadherin (18) and connexin43 (19,20). Several ß-catenin/Tcf-regulated proteins are directly involved in the cellular functions, e.g. proliferation, migration, differentiation and apoptosis, which are affected by mutations in Apc.

Additionally to, or simultaneously with, regulation of ß-catenin stability, Apc binds to microtubules and participates in regulation of microtubule dynamics. Cells homozygous for the multiple intestinal neoplasia (Min) mutation in Apc (ApcMin) show aberrant chromosome segregation, which leads to chromosomal instability (21,22). There are, however, very few mechanistic studies on the effect of one mutated Apc allele. Truncated Apc proteins were reported to bind wt Apc and it has been suggested that truncated Apc could function in a dominant negative manner (23,24).

Gap junction channels mediate the direct exchange of ions and small molecules between the cells. The gap junction channel proteins are called connexins, a family with as many as 20 distinct isoforms in mammals. The connexin family members are named by their molecular mass in kilodaltons (kDa), e.g. connexin43 (Cx43). Impairment of gap junctional intercellular communication (GJIC), caused by mutations or loss of function of connexins, is involved in a number of diseases including the development of cancer (2529). Although gap junctions are found in the intestine (30,31), there is limited knowledge of their significance in this tissue. ß-Catenin can regulate the expression of Cx43. Changes in Wnt signalling could therefore affect GJIC and/or Cx expression. Wnt-1 and Wnt-8 increase GJIC in Xenopus embryos (32,33). Wnt signalling has also been shown to induce Cx43 expression directly (19) and to be an important modulator of Cx43-dependent intercellular coupling in the heart (20). Changes in microtubule function are also found to affect GJIC in some cells (34,35). More recently the C-terminal tail of Cx43 was found to bind microtubules directly (36), which indicates an even closer connection between microtubule function and GJIC than anticipated previously.

The objective of this study was to examine whether GJIC is changed in cells heterozygous for mutation in Apc (Immorto-Min colonic epithelium, IMCE) compared with cells with two wt Apc alleles (Young adult mouse colon, YAMC). We also investigated the Cx expression of the cells, and possible changes in Wnt signalling and microtubule function.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibodies
A polyclonal rabbit anti-Cx43 antiserum was raised against the 20 C-terminal amino acids of Cx43. A tyrosine was added N-terminally to this peptide for coupling purposes. The antiserum recognizes Cx43 from many cell types both in western blots and in immunofluorescence experiments (3739). Goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) were obtained from Bio-Rad (Hercules, CA). Goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to biotin and fluorescein avidin were from Vector Laboratories (Burlingame, CA). Antibodies against E-cadherin (C20820) and ß-catenin (C19220) were purchased from Transduction Laboratories (Lexington, KY), and monoclonal anti-{alpha}-tubulin was from Zymed Laboratories (San Francisco, CA). APC was detected by polyclonal antibodies directed against the N- or C-terminal tails of Apc from Santa Cruz Biotechnology (Santa Cruz, CA) and by a monoclonal antibody against the N-terminal, Ab-1, from Oncogene (Cambridge, MA).

Cell cultures
The epithelial cell lines derived from colon mucosa of Immorto mouse (YAMC) and from an Immorto-Min mouse hybrid (IMCE) were a generous gift from Robert H.Whitehead (Ludwig Institute for Cancer Research, Melbourne, Australia). Both YAMC and IMCE cells are immortalized by heat-labile Simian virus 40 (SV40) large T antigen, which is inactivated at 39°C, and is under control of an interferon-{gamma} (INF{gamma}) sensitive promoter (40,41). YAMC and IMCE cells were grown with INF{gamma} (5 U/ml) at permissive temperature (33°C) in 5% CO2, in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin. The cells were transferred to non-permissive temperature (39°C), with or without INF{gamma} for 2 or 4 days before experiments were performed. The results from cells grown at non-permissive conditions where SV40 large T antigen is inactive (39°C, absence of INF{gamma}) are assumed to be closer to a normal in vivo situation, and were therefore chosen as the main experimental condition unless otherwise is mentioned.

GJIC assay
GJIC was measured by microinjection of Lucifer Yellow (Sigma) into single cells, and the dye-containing cells were counted 5–8 min after microinjection (37). A two-tailed t-test was used for the statistical calculations.

RT–PCR and sequencing of PCR products
RT–PCR was performed on RNA extracted from IMCE and YAMC cells using the GenElute Mammalian Total RNA kit (Sigma, St Louis, MO). Three independent extractions were made from each cell type. Potential contaminating DNA was removed from the RNA using DNA-free kit (Ambion, Austin, TX). RNA was reverse transcribed by Superscript II (Invitrogen, Carlsbad, CA) and the cDNA was amplified by standard PCR procedures using the primer sets shown in Table I. Typically, PCR reactions consisted of 32–35 cycles (denaturing: 95°C for 45 s; annealing: 46–60°C for 30 s; elongation: 72°C for 30 s). After cleaning and treatment with exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT, USB, Cleveland, OH), a dye terminator cycle sequencing reaction was performed using the DYEnamic ET dye terminator kit (Amersham Biosciences, Little Chalfont, UK). The products were thereafter analysed on a MegaBACE (Amersham Biosciences) to ensure the specificity of the primer sets. All kits were used according to the suppliers' recommendations.


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Table I. Sequences of the primer sets used for amplification of connexin mRNA

 
Western blotting
Cells were washed in ice-cold PBS before adding electrophoresis sample buffer (6.25 mM Tris-base, pH 6.8, 5% glycerol, 2% sodium dodecyl sulphate, 13% 2-mercaptoethanol and bromophenolblue). The cells were further homogenized by sonication for 3 x 5 s, followed by heating of the samples at 96–98°C for 5 min. Protein concentrations were measured by the Bradford procedure (Bio-Rad). Similar amounts of protein (10–30 µg) were loaded, and separated on a discontinuous SDS–PAGE gel. The proteins were electroblotted onto nitrocellulose membranes for 1 h (overnight at 4°C for APC detection), and blocked in 5% skimmed milk for 1 h. The membranes were first probed with the primary antibodies in suitable dilutions for 1 h at room temperature or overnight at 4°C, washed and treated with the HRP-conjugated secondary antibody for another hour. The proteins were detected by chemiluminescence (Pierce, Rockford, IL). Equal loading was verified by Ponceau S (Sigma, St Louis, MO) staining of the filters. Quantification of band intensities was done in MatLab, and protein levels were calculated from the area under the curves.

Microtubule polymerization
The cells were incubated on ice for 1 h to depolymerize microtubules (42). Then the cold medium was removed and fresh medium at 39°C was added. The cells were incubated at 39°C for 0, 1, 2, 4 and 6 min and fixed with ethanol. Immunofluorescence staining of the cells is described below. The proportion of cells with microtubule asters in the preparations was counted. Results from a representative experiment in triplicate are shown.

Immunofluorescence
The cells were fixed in cold absolute ethanol for 10 min and permeabilized in PBS with 0.75% Triton X-100 for 30 min. Avidin–biotin blocking kit (Vector Laboratories) was used to quench the endogenous biotin–avidin, and mouse on mouse blocking from Vector MOM immunodetection kit (Vector Laboratories) was used for the monoclonal antibodies. After 1 h in normal donkey serum or mouse IgG, the cells were incubated for 30 min with primary rabbit or mouse antibodies, respectively. The cells were then incubated with biotin-conjugated secondary antibody for 10 min followed by fluorescein–avidin DCS for 5 min. Between each step, the cells were washed in PBS. The cells were finally mounted in Vectashield mounting medium (Vector Laboratories).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
GJIC in cells with different Apc status
Changes in GJIC, as a secondary effect of Apc mutation, was measured in YAMC (Apc+/+) and IMCE (ApcMin/+) cells grown at 33°C (permissive temperature) and after 2 and 4 days at 39°C (non-permissive temperature). The cells did not differ significantly in GJIC at 33°C without INF{gamma}, with a level of about 20 communicating cells (Figure 1). When the cells were transferred from 33 to 39°C for 2 and 4 days the GJIC level in IMCE cells was gradually reduced to half compared with YAMC cells (P < 0.0001), in which GJIC was unaffected by the shift in temperature (Figure 1). INF {gamma} reduced GJIC in YAMC cells at 39°C and in IMCE cells at 33°C (not shown).



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Fig. 1. GJIC measurements in YAMC and IMCE cells. GJIC was measured after 2 days at 33°C and after 2 and 4 days at 39°C, without INF{gamma}. The error bars show SEM for each measurement with n = 24-51.

 
Characterization of Cx expression profile, level and localization
To further investigate the basis for the difference in GJIC, we examined Cx expression in YAMC and IMCE cells. The Cx isoforms expressed at the mRNA level were characterized by RT–PCR. Primers to eleven different Cx were constructed (Table I). Both cell lines expressed Cx26, Cx31, Cx39, Cx40, Cx43, Cx45 and Cx50 mRNAs (Table II). Only YAMC cells expressed the recently cloned Cx29 (Table II). Sequence analysis of the Cx29 RT–PCR product from YAMC cells revealed a sequence identical to that reported previously (not shown) (43). The cell lines did not express Cx30, Cx32 or Cx36 (Table II).


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Table II. Connexins expressed in YAMC and IMCE cells as detected by RT–PCR

 
We studied Cx43 and Cx29 in more detail, as Cx43 is affected by Wnt signalling and Cx29 is found in YAMC cells but not in IMCE cells. Both cell lines were found to produce Cx43 protein (Figure 2A). IMCE cells produced the same amount of Cx43 as YAMC cells at 33°C (not shown). However, IMCE cells contained considerably less Cx43 than YAMC cells when grown at 39°C, particularly after 4 days at non-permissive conditions (Figure 2A and B). INF{gamma} reduced the protein level of Cx43 in both cell lines, but seemed to increase the relative band intensity of the middle band of Cx43 (Figure 2A). Immunofluorescence experiments revealed similar cellular localization of Cx43 in YAMC and IMCE cells, with Cx43 present in the cell membrane, Golgi apparatus and vesicles in cytoplasm, probably lysosomes or endosomes (Figure 3). There was no difference in Cx43 intensity between the cells at 33°C (not shown). IMCE cells, however, contained considerably less Cx43 in the cell membrane than YAMC cells after 4 days at 39°C (Figure 3). The difference in GJIC between YAMC and IMCE cells correlated well with the observed Cx43 level.



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Fig. 2. Levels of ß-catenin, E-cadherin and Cx43. (A) YAMC (Y) and IMCE (I) cells were incubated 2 and 4 days at 39°C with (+) and without (-) INF{gamma} and subjected to western blotting. (B) Quantification of Cx43 level from western blots in YAMC and IMCE cells without INF{gamma}.

 


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Fig. 3. Immunofluorescence of Cx43. YAMC and IMCE cells were incubated 4 days at 39°C without INF{gamma}. Cells without primary anti-Cx43 antibody (control) gave no signal.

 
The observed difference in Cx29 expression between YAMC and IMCE cells could also contribute to the difference in GJIC (Table II). However, further studies on Cx29 proteins could not be performed due to the lack of suitable antibodies. At present, only one commercial antibody is available. However, this antibody gave the same band pattern in YAMC and IMCE on western blots with bands only at ~55 and 80 kDa as reported previously (44), but no band at 29 kDa (not shown). Immunofluorescence studies of Cx29 revealed a weak diffuse staining of the cytoplasm, and no staining of the cell membrane as would be expected for gap junction proteins (not shown).

Apc and ß-catenin level and localization, and activation of Wnt signalling
IMCE (ApcMin/+) cells were found to express less full length Apc than the YAMC (Apc+/+) cells (Figure 4), as could be expected since IMCE cells contain a mutated Apc allele. In addition, prolonged treatment of YAMC and IMCE from 2 to 4 days at 39°C clearly increased the wt Apc level in both cell lines, but IMCE cells still expressed less wt Apc than YAMC cells after 4 days at 39°C (Figure 4). The ApcMin mutation in IMCE cells results in a truncated Apc protein, which appeared on western blots at ~100 kDa (Figure 4). Apc had a ‘punctate’ localization throughout the cytoplasm, with no difference between the cell lines (not shown).



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Fig. 4. Western blotting of Apc. YAMC (Y) and IMCE (I) cells were incubated 2 and 4 days at 39°C, without INF{gamma} (-). Wt Apc (wt) and truncated Apc (Min) are marked with arrows.

 
As activation of Wnt signalling by mutation in Apc could possibly affect ß-catenin/ Tcf-mediated transcription of Cx43, we examined the level of ß-catenin in the cells by western blotting (Figure 2A) followed by quantification of the bands (not shown). The amount of ß-catenin was neither affected by the reduced level of wt Apc and the presence of a truncated Apc protein in IMCE compared with YAMC, nor did it change by increased wt Apc caused by the shift in temperature (Figure 2A). INF{gamma} did not influence the protein level of ß-catenin in either cell type.

Possible activation of Wnt signalling caused by re-localization of ß-catenin from the cytoplasm to the nucleus was studied by immunofluorescence. ß-Catenin was clearly localized to the cell membrane of most cells and in the nucleus of some cells, with only minor differences between the cell types (not shown). The presence of ß-catenin in the nucleus seemed to be dependent on the cell density. Cells in exponential growth contained more ß-catenin in the nucleus than confluent cells, where ß-catenin was more localized to the cell membrane.

Another target of the ß-catenin/Tcf transcription factor, E-cadherin, was not affected by the changes in wt Apc. The same amount (Figure 2A) and localization (not shown) of E-cadherin were observed in both YAMC and IMCE cells. Altogether, the results for ß-catenin and E-cadherin indicate a lack of activation of Wnt signalling in IMCE cells as a result of reduced amount of wt Apc compared with YAMC cells.

Microtubule polymerization
Transport of Cx43 to the cell membrane could be dependent on functional microtubules and we tested whether reduced Cx43 level in the membrane could be caused by changes in microtubule dynamics. Western blots did not reveal any difference in the tubulin content between YAMC and IMCE cells (not shown). However, after microtubule depolymerization on ice, we found that IMCE cells polymerized microtubules at a slower rate than YAMC cells (Figure 5 and Table III). The microtubule polymerization rate was visualized by the size and intensity of the asters (see legend to Figure 5). IMCE cells had smaller and less intense asters at all time points. This was especially evident at 1 and 2 min after temperature change, but was still evident after 6 min (Figure 5). The numbers of cells with microtubule asters were counted and correlated to the total numbers of cells present (Table III). It was evident that a much larger fraction of YAMC cells had visible microtubule asters than that of IMCE cells at all measured times after ice treatment (Table III).



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Fig. 5. Immunofluorescence of microtubules in YAMC and IMCE cells after depolymerization. Polymerization rate of microtubules after ice treatment for 1 h was studied by adding medium at 39°C for 1, 2, 4 and 6 min followed by staining with anti-tubulin antibody. The control cells were stained without ice treatment, and maximally depolymerized microtubules are shown after 1 h ice treatment (0 min). The new microtubules start to grow from the centrosome in the cell to form a small star-like structure, called aster (arrow). One aster was observed in each cell. Note smaller and less intense asters in IMCE cells.

 

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Table III. The number of cells with visible microtubule astersa as percent of total cell number

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several important observations have been made in this study. Cells heterozygous for the ApcMin mutation had decreased GJIC and reduced Cx43 levels, especially in the cell membrane. However, no increase in the Wnt signalling was found, as determined by ß-catenin level and localization. Most interestingly, we found a reduced microtubule polymerization rate as a result of one mutated Apc allele. This shows for the first time that mutation in one Apc allele influences microtubule function.

Neither YAMC nor IMCE cells are tumorigenic in nude mice or grow in soft agar (45). However, mice with the Min mutation in one Apc allele (Min-mice) spontaneously develop adenomas in the intestine similar to those found in FAP patients upon loss or inactivation of the remaining wt Apc allele (4648). In sporadic cancer, two hits in the Apc genes are necessary for tumour development. Accordingly, the Min-derived cell line, IMCE, is supposed to reflect cells predisposed to intestinal carcinogenesis. The results present in this paper suggest that reduced GJIC in the intestinal crypts may contribute to the development of cancer in the intestine, secondary to a first hit in Apc.

The observed increase in wt Apc level in IMCE cells from 2 to 4 days at 39°C was apparently correlated with a corresponding reduction in GJIC and Cx43 level, whereas no such correlation was found for YAMC cells where GJIC and Cx43 levels were unaffected. In contrast, YAMC cells with consistently higher levels of wt Apc than IMCE cells at 39°C had higher GJIC and more Cx43 in the membrane. Thus, there is no correlation between the level of wt Apc on one side, and GJIC and Cx43 level on the other side. However, there is a good correlation between GJIC and Cx43 level in both cell lines. We suggest that the presence of the Min mutation is more important for GJIC and the Cx43 level than the amount of wt Apc. This is supported by studies in Min-mice, where we observed changes in the Cx level both in the normal intestine and in the tumours (Husøy et al., unpublished). A difference in cell growth between the cell lines is not supposed to influence GJIC or Cx43 levels at 39°C, as YAMC and IMCE cells do not grow at non-permissive conditions and the cell density was similar during experiments (not shown) (45). At 33°C the difference in cell growth was minimal (not shown) (46). INF{gamma} is shown previously to give a persistent reduction in GJIC (49). The Cx43 level was clearly reduced by INF{gamma} in both YAMC and IMCE cells at 39°C, with a simultaneous increase in the band intensity of the middle Cx43 band probably caused by increased phosphorylation. However, INF{gamma} only reduced GJIC in YAMC cells at 39°C. INF-{gamma} has been reported previously to activate PKC (50), which is known to phosphorylate Cx43 with a following reduction in GJIC (38).

Apc is an important participant in the Wnt-signalling pathway. Wnt binds to the Frizzled receptor, which leads to inhibition of GSK3ß activity, and stabilization of cytoplasmic ß-catenin (reviewed in ref. 51). A similar increase in ß-catenin can result from mutations in Apc. Cell lines with homozygous mutations in Apc are reported to have increased ß-catenin level and constitutive transcriptional activation by ß-catenin (14,52). Thus, loss of Apc function and activation of the Wnt pathway would be expected to have similar consequences with regard to levels of ß-catenin in the cell. Several publications have shown that Wnt signalling increases the production of Cx43 protein (19,20,53). In addition, Cx43 is a direct target for ß-catenin-mediated gene transcription (19) and is co-localized with ß-catenin in cardiomyocytes (20). A mutation in Apc could therefore be expected to increase the level of Cx43 in a similar way. On the contrary, we observed a reduction in GJIC and Cx43 protein level in IMCE cells compared with YAMC cells. In addition, we found no change in the level or localization of E-cadherin, which also is a target for ß-catenin transcription. Furthermore, no difference in ß-catenin content or localization was found between IMCE and YAMC cells. Hence, the observed reduction in GJIC and Cx43 protein in IMCE cells are probably not caused by alterations in the Wnt-signalling pathway. This may indicate that both Apc alleles must be affected in order to change ß-catenin signalling and thereby of ß-catenin responsive genes like Cx43. In adenomas from Min-mice the second Apc allele was either lost or mutated (5,47,48,54). Consequently, ß-catenin was upregulated in the adenomas in Min-mice (55,56).

Another function of Apc is its binding to tubulin (57). Recent publications address the biological significance of this property. Apc stabilized microtubules (58), and Apc in interaction with the microtubule binding protein EB1 participated in the regulation of microtubule polymerization (42). Consequently, mouse embryonic stem cells homozygous for the ApcMin mutation had alterations in the chromosome segregation, which lead to chromosomal instability (21,22). Our finding that microtubule function is affected also in cells with one truncated and one full length Apc protein is intriguing, indicating that already at this stage several cellular functions could be changed.

During the study of microtubule polymerization, YAMC and IMCE cells were incubated on ice for 1 h. Reactivation of SV40 large T antigen, which is able to bind microtubule and to reduce GJIC (59,60), would probably occur during ice treatment. We find it less likely that this explains the observed differences because both cell lines express the same level of SV40 large T antigen (unpublished results), and would therefore be affected to the same degree.

Very few mechanistic studies have been done in cells heterozygous for Apc mutation. The recent report on reduced number of microtubules in the cochlea of Min-mice compared with wild-type littermates (61), support our findings on altered microtubule function as a result of one mutated Apc allele. Cx43 may bind to microtubule directly (36) and it is suggested that the Cx43 transport to the cell membrane is mediated by vesicular transport along microtubules, as the transport is reduced by the microtubule disrupter nocodazole (62,63). Nocodazole reduces GJIC in some cell types (34,35), although contrasting results also are reported (63,64). We suggest that the observed reduction in membrane-associated Cx43 and GJIC in IMCE cells could alternatively be caused by alterations in these cells' ability to polymerize and de-polymerize microtubules, and thereby affect the transport of Cx43 to the membrane. Another membrane protein, E-cadherin, was not affected in IMCE cells. To our knowledge there is no report on transport of E-cadherin along microtubules. In addition, disruption of microtubules with nocodazole or colcemid induced an increase in E-cadherin in the membrane accompanied by actin polymerization (65).

Hence, it appears that mutation in one Apc allele is sufficient to affect microtubule function, whereas inactivation of both wt Apc alleles may be necessary for activation of Wnt signalling. Further studies are needed to clarify the nature and role of changed microtubule polymerization caused by ApcMin mutation and how this is connected to the reduced expression of Cx43 in the cell membrane.


    Acknowledgments
 
We thank Dr Robert Whitehead (Vanderbilt University, Nashville, TN) for the IMCE and YAMC cells. We also greatly appreciate the excellent technical assistance by Hege Haugen on the experiments. Karen-Marie Heintz is thanked for running the MegaBACE. We thank Dr E.Rivedal for making the anti-Cx43 antiserum available to us. Véronique Cruciani is supported by the Research Council of Norway. This study was supported with grants from the Research Council of Norway and the Norwegian Cancer Society.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 11, 2002; revised January 13, 2003; accepted January 15, 2003.