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[Cancer Research 61, 7978-7984, November 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Human APC2 Localization and Allelic Imbalance1

Christy Rothwell Jarrett, Jan Blancato, Tin Cao, D. S. Bressette, M. Cepeda, Paul E. Young, C. Richter King and Stephen W. Byers2

The Lombardi Cancer Research Center, Georgetown University School of Medicine, Washington, DC 20007 [C. R. J., J. B., T. C., S. W. B.]; Human Genome Sciences, Inc., Rockville, Maryland 20850 [D. S. B., M. C., P. E. Y.]; and GenVec, Rockville, Maryland 20852 [C. R. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A second adenomatous polyposis coli (APC)-like gene, APC2/APCL, was recently described and localized to chromosome 19. We have fine mapped APC2 to a small region of chromosome 19p13.3 containing markers D19S883 and WI-19632, a region commonly lost in a variety of cancers, particularly ovarian cancer. Interphase fluorescence in situ hybridization analysis revealed an APC2 allelic imbalance in 19 of 20 ovarian cancers screened and indicates that APC2 could be a potential tumor suppressor gene in ovarian cancer. When overexpressed in SKOV3 ovarian cancer cells, which express low levels of APC2, exogenous APC2 localized to the Golgi apparatus, actin-containing structures, and occasionally to microtubules. Antibodies against the NH2 terminus of human APC2 show that endogenous APC2 is diffusely distributed in the cytoplasm and colocalizes with both the Golgi apparatus and actin filaments. APC2 remained associated with actin filaments after treatment with the actin-disrupting agent, cytochalasin D. These results suggest that APC2 is involved in actin-associated events and could influence cell motility or adhesion through interaction with actin filaments, as well as functioning independently or in cooperation with APC to down-regulate ß-catenin signaling.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The APC3 tumor suppressor gene, located on chromosome 5q21, is associated with colon cancer. Possible functions include the regulation of ß-catenin protein degradation and signaling and microtubule- mediated cell migration (1, 2, 3) . ß-catenin binds to the Tcf/LEF transcription factor complex and regulates the transcription of c-myc and cyclin D1, thus indicating that this pathway may be involved in cell cycle regulation (4, 5, 6, 7, 8) . Truncating mutations in APC or mutations in certain NH2-terminal serine residues of ß-catenin result in increased ß-catenin levels and increased transcriptional activation (2 , 3 , 9 , 10) .

APCL/APC2, an APC homologue located on chromosome 19p13.3, has also been shown to interact with ß-catenin and can decrease ß-catenin levels and signaling activity in SW480 colon cancer cells (11 , 12) . hAPC2 is expressed in many different tissues and cell lines, including brain, breast, colon, and ovary. APC family members have similar NH2-terminal dimerization domains, armadillo repeats, and ß-catenin binding and regulatory domains but are less conserved at the COOH terminus, which contains the basic domain, microtubule-binding domain, and disc large binding site in hAPC. In addition, hAPC2 lacks the three 15 amino acid ß-catenin binding sites. A phylogenetic tree of the highly conserved armadillo repeat domain suggests independent Drosophila and vertebrate APC gene duplications (13) . dAPC is known to regulate wingless signaling in the eye but has not been associated with other tissues (14) . dAPC2/E-APC negatively regulates wingless signaling in the epidermis (13 , 15) . These results suggest that APC and APC2 could be tissue specific and/or have different roles in ß-catenin regulation.

Endogenous APC localizes near the ends of microtubules but does not associate with actin filaments or with microtubules in the cell body; however, when overexpressed, APC associates with microtubules throughout the cell (1 , 16 , 17) . Overexpressed hAPC2 localizes to the perinuclear region and to some microtubules in the cell body, but no studies have examined the distribution of endogenous hAPC2 (18) .

dAPC2/E-APC colocalizes with actin caps during development, and weak E-APC mutations in Drosophila females cause a loss of E-APC at the membrane of nurse cells in the ovary, which corresponds to a loss of armadillo/ß-catenin at the membrane (19 , 20) . ß-catenin localizes at the membrane to form adhesion junctions, which suggests that cell adhesion of these cells is compromised by the mutation in E-APC. In this study, we show that endogenous hAPC2 colocalizes with the Golgi apparatus and actin filaments, particularly those filaments present at the leading edge of the cell and at cell-cell contact sites but was not associated with microtubules unless overexpressed. Significantly, SKOV3 ovarian cancer cells express considerably less APC2 than other cell lines. We have fine mapped APC2 to a region of chromosome 19p13.3 containing markers D19S883 and WI-19632, a region commonly lost in a variety of cancers, particularly ovarian cancer (21, 22, 23) . Using interphase FISH, we show an allelic imbalance of APC2 in 19 of 20 sporadic ovarian tumors indicating that APC2 could be a potential tumor suppressor gene.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
FISH Analysis and Fine Mapping.
A 1-kb cDNA fragment from the NH2-terminal region of APC2 corresponding to the recombinant protein used to make antibodies was used to screen a PAC library (Human Genome FISH Mapping Resource Center at the Ontario Cancer Institute). Four genomic PAC clones were identified: 1K8, 17J21, 22K8, and 26K20. FISH to normal human lymphocyte chromosomes was used to map the genomic PAC clones to chromosome 19p13.3. Fine mapping was performed using radiation hybrid screening by PCR (Research Genetics, Inc.). Primer sequences (5'-GCTGCAGGAGCTGAAGATG; 5'-GTGGCTGGAGTTGTCCCTTA) were designed to yield a 120-bp product spanning the first exon/intron junction. Interphase FISH analysis was performed on paraffin sections of 20 sporadic ovarian and breast tumors, as well as 10 normal ovary and breast specimens.

Antibody Development.
A recombinant glutathione S-transferase-fusion protein to the NH2-terminal region of APC2 (amino acid 1–249) was produced in Escherichia coli using the pGEX-4T-2 vector, isolated, and released by protease cleavage (Pharmacia Biotech). This protein was used to inoculate both rabbit and chicken (Rockland, Inc., Gilbertsville, PA). Both the rabbit serum and IgY collected from the chicken eggs were affinity purified on an antigen-coupled CnBr column.4

Northern Analysis.
Human multiple tissue and human cancer cell line-polyadenylated RNA blots were obtained from CLONTECH and processed according to the supplied manufacturer’s protocol using a probe to the NH2-terminal region of APC2.

RT-PCR.
RNA was isolated by the RNAzol method (Tel-Test, Inc.). RT-PCR was performed using the Perkin-Elmer Gene Amp RNA PCR Core Kit. Primers to the NH2-terminal region (5'-AGGAGCTAAGGGACAACTCCA; 5'-TCCAGCAGCTCCTTGTCAAT) were designed to yield a 600-bp fragment. These primers were shown to be specific to APC2 by sequencing of the product, as well as using wild-type APC as a negative control.

Western Blot.
Cells were grown to confluence in 150-mm dishes, washed twice with PBS, and lysed for 10 min on ice in 1% HEPES lysis buffer containing 1% Triton-X and protease inhibitors (1 mM sodium vanadate, 50 mM sodium fluoride, and Boehringer Mannheim complete mini EDTA-free protease inhibitor cocktail tablet). Lysates were centrifuged at 14,000 rpm at 4°C. Protein content was determined by the bicinchoninic acid protein assay (Pierce). Western blotting was performed as described previously using either rabbit or chicken APC2 antibody at 1 µg/ml, APC Ab-1 (Oncogene) at 1 µg/ml, or ß-catenin (Transduction Laboratories) at 1:1000 (24) . The blots were developed using chemiluminescent detection (Pierce). Specificity of the antibodies was determined by incubating recombinant APC2 antigen (10 µg/ml) with the antibody for 1 h at room temperature before incubating the blot.

Immunocytochemistry.
SKBR3, A549, MDA-MB-157, SW480, and MDCK cells were plated on 18-mm coverslips in 12 well plates at ~50,000 cells/well. SKOV3 cells were transfected with 0.2 µg of APC2 using LipofectAMINE Plus (Life Technologies, Inc.). In some experiments, cells were treated with 2 µM cytochalasin D (Sigma Chemical Co.) in media for 2 h at 37°C. Antibody blocking with the immunogen was performed as described above. Both treated and untreated cells were fixed with 2% paraformaldehyde and permeabilized with 0.2% Triton. Purified chicken APC2 antibody was used at a concentration of 1 µg/ml, and secondary antibody conjugated with fluorescein (Pierce) was used at 1:100. Other primary antibodies and reagents were used at the following concentrations: normal IgY (Rockland, Inc.) at 1 µg/ml, monoclonal ß-catenin antibody (Transduction Laboratories) at 1:100 overnight at 4°C, polyclonal anti-APC (kindly provided by P. Polakis; Ref. 3 ) at 1:100 overnight at 4°C, monoclonal antitubulin (Sigma Chemical Co.) at 1:2000, phalloidin (Molecular Probes, Inc.) at 1:200 for 15 min, and anti-PKCµ (Transduction Laboratories) at 1:200. All primary antibodies were incubated for 1 h, and all secondary antibodies were used at a 1:100 dilution for 1 h at room temperature unless otherwise noted above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chromosomal Localization and Fine Mapping.
Using a 1-kb sequence to the NH2-terminal region, four PAC clones (1K8, 17J21, 22K8, and 26K20) were isolated for APC2. FISH analysis using the four clones localized APC2 to chromosome 19p13.3, which confirms the previously published chromosomal assignment (Fig. 1ACitation ; Refs. 7 and 8 ). 19p13.3 is ~20 Mb in size. The genomic sequence of APC2 is ~40 kb, and the coding sequence is 7 kb. APC2 was then fine mapped by radiation hybrid mapping to the 800-kb region containing markers D19S883 and WI-19632 using primers designed to span the first exon/intron junction (Fig. 1B)Citation . This particular region of 19p13.3 exhibits significant LOH in many different cancers and is near the PJS-associated gene, LKB1/STK11. PJS is characterized by intestinal hamartomas and increased risk of gastrointestinal, ovarian, pancreatic, and breast cancers (25) . Although there is significant LOH in this region, there is a low frequency of mutations in the LKB1 gene in sporadic breast and colorectal cancers and adenoma malignum of PJS patients (21 , 22) . In addition, although 50% of ovarian cancers contain an LOH on 19p13.3, LKB1 is not mutated, indicating that another gene of significance in the development of cancer exists in this region (26) , e.g., marker D19S216, which is 9.5 fcM distal to marker D19S883, but not LKB1 itself, exhibits 100% LOH in sporadic adenoma malignum of the uterine cervix (23) . Therefore, APC2 could be a tumor suppressor gene affected by LOH of chromosome 19p13.3.



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  		Fig. 1. APC2 chromosomal localization and fine mapping. In A, APC2 was mapped to chromosome 19p13.3 by FISH analysis using PAC clones identified through screening with a 1-kb cDNA fragment from the NH2-terminal region of APC2. In B, fine mapping using radiation hybrid screening by PCR located APC2 to the region on chromosome 19p13.3 containing markers WI-19632 and D19S883.

 
Using PAC clone 22K8, we performed FISH analysis on the SKOV3 ovarian cancer cell line, which had reduced levels of APC2 protein, and a variety of other cancer cell lines, including those derived from breast and colon. These pilot studies showed that most had two or more copies on chromosome 19. However, although SKOV3 cells had on average three signals, none of them were on chromosome 19. To continue this study, we screened 20 sporadic breast and ovarian tumors by interphase FISH analysis. Remarkably, 19 of 20 specimens from ovarian cancer patients exhibited marked allelic imbalance (Table 1)Citation . In contrast, no significant allelic imbalance was observed in normal ovarian or breast tissue. The number of signals of the 19p telomeric marker was also significantly less than two in the ovarian cancer specimens. Even taking into account the bias toward slightly underestimating hybridization signals using interphase FISH, these data are consistent with published studies showing a marked loss of chromosome 19ptel in ovarian cancers and indicate an even more marked loss of APC2. Although the overall ratio of APC2:19p when averaged over 20 breast cancers was not markedly reduced (Table 2)Citation , 4 of 20 breast cancer specimens did actually exhibit allelic imbalance at this locus.


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  		Table 1 Allelic imbalance of chromosome 19p and APC2 in ovarian tumorsa

 

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  		Table 2 Mean number of chromosome 19p telomere and APC2 signals in normal and tumor ovarian and breast tissues

 
Exogenous Expression of APC2 in SKOV3 Cells.
In pilot studies, we found that many cells expressed significant levels of APC2. However, the ovarian cancer cell line SKOV3 expressed low levels of APC2 as demonstrated by Western blot and immunocytochemistry (Fig. 2)Citation . We used SKOV3 cells to examine the distribution of exogenously expressed APC2. On APC2 transfection, the intensity of three bands was markedly increased as detected by Western blot using a chicken antibody to APC2. Immunocytochemistry showed that APC2-transfected cells could easily be detected against the background of nontransfected cells. Exogenously expressed APC2 localized around the nucleus and colocalized with PKCµ, a kinase known to associate with the Golgi apparatus (Ref. 27 ; Fig. 2A1–3Citation ). Colocalization of APC2 and PKCµ was observed for much of the Golgi stack, indicating that APC2 is associated with certain regions of the Golgi where it colocalizes with PKCµ, although the Golgi apparatus is somewhat disrupted in the transfected cells. APC2 colocalized with actin filaments at the cell membrane (Fig. 2B1–3)Citation . SKOV3 cells have less prominent actin filaments in the cytoplasm compared with the MDA-MB-157 cells shown in Fig. 5Citation . ß-catenin colocalized with exogenous APC2 in the aggregates at the Golgi apparatus, whereas in untransfected cells, ß-catenin is localized only at the membrane (Fig. 2C1–3)Citation . This could be indicative of a transport function for APC2 as suggested in recent studies (28, 29, 30) . Exogenously expressed APC2 also appears to colocalize with some perinuclear microtubules (Fig. 2D1–3)Citation .



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  		Fig. 2. Localization of exogenously expressed APC2. Immunocytochemical staining of SKOV3 cells transfected with APC2 shows perinuclear staining (A1) using the APC2 IgY antibody. Cells were stained for the Golgi apparatus marker PKCµ (A2). APC2 colocalizes with PKCµ at the Golgi apparatus (A3). Also note that the Golgi apparatus looks somewhat distorted in the transfected cells compared with the nontransfected cells. Double staining of APC2 (B1) and actin (B2) found APC2 localized with actin filaments in regions close to the membrane (B3, arrows). Cells were stained for APC2 (C1) and ß-catenin (C2). ß-catenin colocalizes with transfected APC2 in large aggregates around the nucleus (C3, arrow). In comparison, ß-catenin is only found at the membrane in the untransfected cells (arrowhead). Transfected SKOV3 cells were double stained for APC2 (D1) and tubulin (D2). Overexpressed APC2 localizes with some but not all microtubules (D3, arrows). All images are at a magnification of x630. In E, SDS-PAGE analysis was performed on a 3–8% Tris-Acetate gel. SKOV3 ovarian cancer cells express low levels of APC2 as shown by Western blot with the APC2 IgY antibody. Exogenously expressed APC2 detected four different molecular weight species.

 


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  		Fig. 5. APC2 localization at the Golgi apparatus and actin filaments. A549 cells double stained with APC2 and PKCµ or phalloidin. Cells were treated with 2 µM cytochalasin D for 2 h. In A1, PKCµ clearly stains the Golgi apparatus. In A2, APC2 localizes to the Golgi apparatus. In A3, double staining shows APC2 colocalization with PKCµ at the Golgi apparatus. B1, phalloidin staining of actin filaments in MDA-MB-157 cells. In B2, APC2 is diffusely distributed in the cytoplasm and colocalizes with actin filaments. In B3, double staining shows APC2 associated with actin filaments. C1, MDA-MB-157 cells treated with cytochalasin D and stained with phalloidin. Actin filaments are disrupted. In C2, cells treated with cytochalasin D and stained for APC2. APC2 is disrupted. In C3, double staining shows that APC2 remains associated with actin filaments after treatment with cytochalasin D.

 
APC2 Expression.
APC2 expression was determined by RT-PCR, Northern analysis, and Western analysis of both cell lines and tissue. For RT-PCR, 1 µg of total RNA from each cell line or tissue was used for amplification; for Northern analysis, 10 µg of total RNA was used; and for Western analysis, 60 µg of protein from each cell line was used. We confirmed that APC2 is expressed in a variety of tissues, including breast, colon, brain, and ovary, at both the RNA and protein level (designated as "+" in Table 3Citation ; Ref. 12 ). In some cells, APC2 levels were reduced but still detectable (+/-). APC2 expression, like APC, is greatest in the brain; however, there are differing levels in different brain regions with very little expression in the cerebellum and cerebral cortex (results not shown). Lymphoid tissues and lymphoma cell lines had no detectable APC2 at the mRNA or protein level with the exception of K-562 leukemia cells, which express low levels of APC2 (Fig. 3)Citation .


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  		Table 3 APC2 expression

 


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  		Fig. 3. Expression of APC2 and Western blot of cell lysates comparing APC2 and APC. In A, SW480 cell lysates, containing a truncated form of APC (T-APC), were used to compare affinity-purified APC2 rabbit antibody (1 µg/ml) to APC antibody-1 (1 µg/ml). SDS-PAGE analysis was performed on a 4–12% Tris-Glycine gel. No cross-reactivity could be found. The affinity-purified APC2 IgY antibody gave similar results (results not shown). In B, varying protein patterns of APC2 are observed by Western blot analysis of five different cell lysates using the IgY antibody (1 µg/ml). Equal amounts of protein (60 µg) were loaded in each lane. SDS-PAGE analysis was performed on a 3–8% Tris-Acetate gel. Note that HL-60 and K-562 lymphoma cell lines have no detectable APC2 as shown on a 4–12% Tris-Glycine gel. In C, APC2 was immunoprecipitated from SW480 and HBL-100 cell lysates using the affinity-purified rabbit antibody. Rabbit IgG was used as a control. Complexes and a control lysate were analyzed for APC by Western blot using APC Ab-1 (1 µg/ml). The truncated form of APC found in SW480 cells and full-length APC found in HBL-100 cells exist in a complex with APC2.

 
Both rabbit and chicken hAPC2 antibodies were affinity purified on an antigen-coupled CnBr column. Western blot analysis determined that both antibodies were specific to hAPC2 with no cross-reactivity to APC (Fig. 3A)Citation . This was confirmed using the SW480 colon cancer cell line that contains a COOH-terminally truncated form of APC. Neither rabbit nor chicken hAPC2 antibodies detect this truncated APC protein, although it contains the conserved NH2 terminus. The predicted molecular weight of full-length APC is Mr ~310,000 and that of APC2 is Mr ~245,000. In addition, APC2 is present in T84 cells, which have a homozygous deletion of the APC gene. To additionally determine specificity, we blocked the antibody with recombinant antigen before Western blot analysis and found that all bands are specific to APC2 (results not shown). Similarly, when preimmune IgY was used to probe Western blots, no staining was observed (data not shown). Western blot analysis showed that APC2 is expressed in many cells lines, including SKBR3, SW480, MDCK, MDA-MB-157, and 436 (Fig. 3B)Citation . HL-60 lymphoma cells have no detectable APC2 protein, which correlates with the mRNA data. A characteristic pattern of immunoreactive species was observed. Three major bands larger than Mr 200,000 and three smaller molecular weight species of Mr ~121,000; 81,000; and 51,000 (data not shown on this blot) were present consistently. Other cell lines, e.g., MDA-MB-157 and SKOV3, have significantly less of all bands. The presence of multiple bands by Western blotting with NH2-terminal APC2 antibodies is similar to that observed for APC using NH2-terminal APC antibodies (31) . In the case of APC, the multiple banding pattern has been ascribed to a combination of multiple splice variants and to degradation products. Because several lower molecular weight species are present in cells that are transfected with full-length APC2 (Fig. 2)Citation , it is likely that these bands represent degradation products. Some differences in the pattern of immunoreactive species were observed when endogenous APC2 was compared in different cell lines and when compared with exogenously expressed APC2. These variations are likely to represent variable degrees of proteolytic degradation in the different situations. However, it is possible that like APC, APC2 may also have many splice variants.

The NH2-terminal region of APC2 contains the highly conserved dimerization domain. This region in APC has been shown to dimerize in vitro (32 , 33) . To determine whether APC associates with APC2, APC2 was immunoprecipitated with the purified rabbit antibody, and APC was detected using APC Ab-1 in SW480 and HBL-100 cells (Fig. 3C)Citation . Full-length APC was detected in HBL-100 cells, and the Mr 150,000 truncated form of APC was detected in SW480 cells. This result demonstrates that APC and APC2 can either dimerize or associate in a complex in a detergent soluble lysate.

Subcellular Localization of APC2.
To investigate the localization of endogenous APC2 in the cell, we performed immunocytochemistry on 10 cell lines, including SKBR3 breast cancer cells, MDCK normal canine kidney cells, SW480 colon cancer cells, and A549 lung carcinoma cells. Although both rabbit and chicken antibodies exhibited a similar staining pattern by immunocytochemistry, the chicken antibody was exceptional and was used for these studies. Preimmune chicken IgY and antigen-blocked antibody, as well as IgY before antigen affinity purification, were completely negative (Fig. 4, A and B)Citation . Specific APC2 staining was similar in all cell lines and was observed diffusely in the cytoplasm, as well as being associated with tubular structures adjacent to the nucleus that resembled the Golgi apparatus (Fig. 4)Citation . Staining was also concentrated along filamentous structures and in what appeared to be lamellipodia membranes.



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  		Fig. 4. Immunocytochemical staining for endogenous APC2. In A, SKBR3 cells were stained using preimmune IgY (3 µg/ml). Little staining could be seen even in this overexposed image. B, MDA-MB-157 cells stained using IgY antibody blocked with APC2 protein. C, MDA-MB-157 cells stained for APC2 using APC2 IgY antibody (1 µg/ml). APC2 can be seen concentrated along filamentous structures, as well as concentrated along the membrane (arrows). In D, A549 cells were stained with APC2 affinity-purified IgY antibody (1 µg/ml). Arrow, staining resembling the Golgi apparatus surrounding the nucleus. E, A549 cells stained for APC2 as above. Box, a region of small vesicles/particles concentrated in a lamellipodia membrane. F, A549 cells again stained for APC2. Arrows, staining resembling the Golgi apparatus surrounding the nucleus and staining along actin filaments.

 
APC2 Association with Actin Filaments.
As was observed with exogenously expressed APC2 in SKOV3 cells, endogenous APC2 colocalized with PKCµ at the Golgi apparatus (Fig. 5A1–3)Citation . A relationship between APC2 and actin filaments was observed in cells stained with phalloidin (Fig. 5B1–3)Citation . However, not all actin filaments stained, and APC2 appeared to be concentrated near actin-associated membrane ruffles and lamellipodia, as well as cell-cell contact sites. Treatment with cytochalasin D, an actin-disrupting agent, causes actin filaments to collapse and coalesce mostly at the cell periphery but also throughout the cell. After treatment with cytochalasin D, APC2 remained associated with the actin filaments in MDA-MB-157 cells (Fig. 5C1–3)Citation .

Endogenous APC is localized at the tips of microtubules in MDCK cells and is not associated with actin filaments (1) . However, overexpression of APC results in the decoration of microtubules throughout the cell (16 , 17) . Consistent with this, cytochalasin D treatment does not affect APC staining, but disruption of microtubules with nocodazole does (1) .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Subcellular Localization of hAPC2.
hAPC2 is diffusely distributed in the cytoplasm, is localized to the Golgi apparatus, and is associated with actin filaments. In some instances, such as lamellipodia or membrane ruffles, APC2 exhibits a punctate staining at the ends of actin filaments. Unlike APC, APC2 remains associated with the disrupted actin filaments after treatment with cytochalasin D. Recent studies show that dAPC2/E-APC colocalizes with actin caps during Drosophila development and negatively regulates wingless signaling in the epidermis (13 , 20 , 34) . These data suggest that although sequence similarity is low, hAPC2 and dAPC2/E-APC may be functional homologues and that both may be involved in actin-associated events, such as motility, as well as in ß-catenin signaling.

In contrast, endogenous APC localizes near the ends of microtubules in a punctate pattern but does not colocalize with actin (1) ; however, when overexpressed, APC associates with microtubules throughout the cell (16 , 17) . Overexpressed APC2 forms aggregates at the Golgi apparatus and colocalizes with actin filaments as well as some microtubules. Similarly, Nakagawa et al. (18) reported that overexpressed, epitope-tagged APCL/APC2 localizes in the perinuclear region and microtubule network. Our results indicate that as is the case with APC, APC2 localization with microtubules in the cell body may only occur when APC2 is present at very high levels. It has been suggested that APC might be involved in microtubule-regulated membrane protrusion and cell migration, as well as inhibition of ß-catenin signaling (35) . Like APC, APC2 also can inhibit ß-catenin signaling (11 , 12) . Although APC2 and APC do not colocalize at the membrane or cytoskeletal structures, they are both present in the cytoplasm. Immunoprecipitation results show that they can exist in the same complex in this environment. Taken together, these findings suggest an intriguing scenario in which cytoplasmic APC and APC2 regulate related microtubule and actin-based functions and ß-catenin signaling either independently or in cooperation. APC and APC2 could cooperate in the cytoplasm or in association with microtubules and actin filaments, respectively, to control such processes as ß-catenin signaling and cell motility as suggested by Barth et al. (35) . In addition, interactions between microtubules and actin filaments occur during cell motility (36) . The cellular location and many binding domains of APC2 suggest that it has multiple and perhaps dynamic functions, e.g., recent studies located a nuclear export signal that allows APC, APC2, and E-APC to shuttle between the nucleus and the cytoplasm, thus suggesting a transport mechanism (28, 29, 30) .

Significance of the Chromosomal Location of APC2.
The chromosomal localization of APC2 to chromosome 19p13.3 within 12 fcM of the telomere is significant because this region is associated with PJS and exhibits significant LOH in sporadic cancers. Patients with PJS are more susceptible to breast, testis, gastrointestinal, and ovarian cancers (25) . Loss of 19p13.3 occurs in many sporadic cancers, including those of the breast, and is remarkably common in sporadic ovarian carcinomas (~50%; Ref. 26 ). Ovarian cancers are also characterized by a high rate (~16%) of stabilizing ß-catenin mutations (37) . However, mutations in the PJS gene, LKB1, are not present in most of these sporadic cancers, suggesting the existence of other tumor suppressor loci in this region of chromosome 19 (21 , 26) . Our fine-mapping analysis shows that APC2 is located in the region of markers D19S883 and WI-19632 between the LKB1 gene and the site of 100% LOH found in adenoma malignum of the uterine cervix (23) . Importantly, we found significant allelic imbalance of APC2 in sporadic ovarian cancers. Therefore, like APC, APC2 could be a tumor suppressor gene important in several cancers, particularly ovarian cancer.

A recent study by Townsley and Bienz (19) showed that the majority of offspring of homozygous Drosophila females carrying a weak dAPC2/E-APC mutation die as embryos with defects similar to wingless stimulation. Interestingly, E-APC was lost at the membrane of nurse cells in the ovary. This loss coincided with a loss of armadillo/ß-catenin at the membrane and suggests that cell adhesion might be affected by the weak mutation in E-APC. Our data indicate that if similar mutations occur in hAPC2, they may be relevant in ovarian cancer. At this time, no single nucleotide polymorphisms have been discovered in the APC2 gene. Additional studies searching for single nucleotide polymorphisms and a more formal LOH study will better determine whether APC2 is a tumor suppressor gene.


   ACKNOWLEDGMENTS
 
We thank Y. Nakamura for the full-length APC2/APCL cDNA and P. Polakis for the polyclonal APC antibody. We also thank Michele Dougherty for help with the RT-PCR experiments, Emma Bowden and Susette Mueller for advice on PKCµ antibody, P. Burbello for advice on tubulin, and B. Haddad for information and help with FISH analysis and radiation hybrid screening. Finally, we thank the support of the Lombardi Cancer Center SPORE Imaging and Microscopy Core Facility and the Cytogenetics facility.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Department of Defense (DAMD 17-98-8089) and NIH (R21 CA87749). C. R. J. was supported by a Department of Defense Predoctoral Fellowship Grant DAMD 17-99-1-9197. Back

2 To whom requests for reprints should be addressed, at Georgetown University Medical Center, Research Building E415, 3970 Reservoir Road Northwest, Washington, DC 20007. Phone: (202) 687-1813; Fax: (202) 687-7505; Back

3 The abbreviations used are: APC, adenomatous polyposis coli; APC2, APC-like gene; dAPC, Drosophila APC; dAPC2/E-APC, Drosophila APC2; FISH, fluorescence in situ hybridization; hAPC2, human APC2; PAC, P-1-derived artificial chromosome; PJS, Peutz-Jeghers syndrome; RT-PCR, reverse transcription-PCR; LOH, loss of heterozygosity. Back

4 Internet address: http://viz.e222.zo.utexas.edu/Marker_pages/methods_pages/affinity_col.html. Back

Received 12/11/00. Accepted 8/27/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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