TECHNICAL NOTE |
Correspondence to: Assumpta Munné, Servei de Patologia, Hospital del Mar, Passeig Maritim 2529, 08003 Barcelona, Spain.
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Summary |
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ß-Catenin mediates the interaction of E-cadherin with -catenin and the actin cytoskeleton. Recent evidence indicates that when the tumor suppressor gene APC is inactivated, ß-catenin can translocate to the nucleus, where it acts as a transcriptional regulator. Because APC is inactivated in most colorectal cancers, ß-catenin nuclear localization would be expected in these tumors. In a study of adhesion molecule expression in frozen colorectal cancer tissues, we were surprised by failure to detect nuclear ß-catenin. Here we compared the reactivity of an anti-ß-catenin monoclonal antibody with 11 colorectal cancers using immunohistochemistry on sections of frozen or paraffin-embedded samples. ß-Catenin was never detected in the nuclei of normal or tumor cells in frozen tissue sections. By contrast, in 8/11 cases it was detected in the nuclei of tumor cells but not of normal cells in paraffin-embedded tissue sections. These results were confirmed with an independent rabbit polyclonal anti-ß-catenin serum. We also examined ß-catenin distribution in SW480 colon cancer cells, in which its nuclear accumulation has been reported. As in tissues, nuclear ß-catenin was detected in paraffin-embedded but not in frozen samples. These findings are relevant because of the increasing interest in the study of ß-catenin in tumors, based on its dual role in cell adhesion and transcriptional regulation. (J Histochem Cytochem 47:10891094, 1999)
Key Words: ß-catenin, colorectal cancers, frozen sections, paraffin-embedded sections, immunohistochemistry
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Introduction |
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ß-CATENIN WAS FIRST IDENTIFIED as a 92-kD member of the family of cytoplasmic proteins mediating the interaction of Ca2+-dependent transmembrane cadherin molecules with the cytoskeletal network (
ß-Catenin has recently been the object of increasing interest because of the discovery of additional functions of this protein apart from its well-known role in cell adhesion. The implication of ß-catenin in the transduction of Wingless/Wnt-dependent cellcell signaling has been demonstrated (
Most studies of the expresion of ß-catenin in tumors have focused on its membrane and cytoplasmic distribution (
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Materials and Methods |
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Tissue Specimens
Surgical specimens (n = 11) were collected from patients undergoing elective large bowel resection for colorectal carcinoma. Fresh samples of normal mucosa distant from tumor (8 cm), non-neoplastic mucosa adjacent to the tumor, and tumor were embedded in OCT, frozen in methyl-butane, and stored at -80C. An adjacent sample from each type of tissue was fixed with 4% neutral buffered formaldehyde and embedded in paraffin. Representative frozen or paraffin-embedded samples containing normal, peritumoral, and tumor tissue were used for immunohistochemistry.
Cultured Cell Line Samples
The SW480 (ATCC; Rockville, MD) and HT-29 (provided by Dr. A. Zweibaum; INSERM U178, Villejuif, France) cell lines were cultured under standard conditions until Day 15 after seeding. After trypsinization, cells were washed in PBS and centrifuged for 5 min at 1000 rpm. The resulting cell pellet was divided into two aliquots: one of them was embedded in OCT, frozen in methyl-butane, and stored at -80C, and the other aliquot was fixed and embedded in paraffin as described above.
Immunohistochemistry
The streptavidinbiotinalkaline phosphatase method was applied to both tissues and cultured cells. Four-µm-thick sections of frozen and paraffin-embedded tissues were mounted on silanized slides. Cryostat sections were fixed in 10% formaldehyde for 5 min, then in acetone for 1 min, in methanol for 1 min, and washed in 50 mM Tris (pH 7.6), 150 mM NaCl. Among a variety of fixation conditions tested, the method described above was selected because it provides optimal tissue and antigen preservation. Paraffin sections were dewaxed in xylene, hydrated in ethanol, and then washed in TrisNaCl buffer. Both frozen and paraffin-embedded tissue sections were immersed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven at 700 W for 10 min. To block nonspecific binding, slides were incubated for 20 min with 5% skim milk in 20 mM Tris (pH 7.5), 0.05% Tween-20. Sections were then incubated overnight at 4C with primary antibody. After additional rinsing in TrisNaCl buffer, avidinbiotinalkaline phosphatase complex detecting mouse Igs was used (Stravigen Multilink; Biogenex, San Ramon, CA). Reactions were revealed using Fast Red TR Salt (Sigma; St Louis, MO). Sections were counterstained with Mayer's hematoxylin. A mouse monoclonal antibody detecting human ß-catenin (clone 14, catalogue reference C19220; Transduction Laboratories, Lexington, KY) was used at 1:200 dilution. Negative controls consisted of consecutive sections that were incubated with an irrelevant mouse monoclonal antibody (B12) recognizing dextran, used at a 1:2 dilution of hybridoma supernatant. As a positive control, mouse monoclonal antibody CAM 5.2 detecting cytokeratins (Becton Dickinson; San Jose, CA) was used.
Evaluation of Immunostaining
Sections were examined under a light microscope by two independent observers. Subcellular distribution (membrane, cytoplasmic, and nuclear) and intensity of immunostaining were evaluated in normal mucosa distant from the tumor, non-neoplastic mucosa adjacent to the tumor, and in the tumor itself. In the latter, the superficial, mid-, and deep regions were separately analyzed. For each sample and region, intensity was graded from 0 to 4, and the percentage of reactive cells was estimated. A staining index was calculated as the product of intensity and percentage of positive cells. A global staining index for the tumor was calculated as the average of the indices of the superficial, mid-, and deep regions.
Statistical Analysis
Fisher's exact test was used for differences between proportions. Wilcoxon's signed rank test was performed to test the difference between the distribution of staining index in the different groups. Spearman's Rho coefficient was used to test independence between membrane/cytoplasm vs nuclear staining indices.
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Results |
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In normal colon mucosa and in non-neoplastic mucosa adjacent to the tumor, ß-catenin was detected in all cases at the cell membrane and in the cytoplasm. Nuclear staining was absent from all cells in all samples analyzed. Similar results were obtained in both frozen and paraffin-embedded sections (Figure 1A and Figure 1E).
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In tumor samples, the subcellular distribution of ß-catenin was clearly different in sections from frozen and paraffin-embedded tissues (Table 1). In frozen sections, ß-catenin was always detected at both the membrane and the cytoplasmic level in superficial, mid-, and deep regions of the tumor. No nuclear staining was found in the 11 cases analyzed (Figure 1B and Figure 1C). Furthermore, ß-catenin was never found in the nuclei of colon cancer cells in frozen sections, irrespective of the fixationpermeabilization conditions used: no fixation, paraformaldehyde [2.5% in PBS for 5 min at room temperature (RT)], formaldehyde (10% in PBS for 7 min at RT), ice-cold methanol (2 min), acetone at -20C (2 min), Triton X-100 (0.2% in PBS for 10 min at RT). These treatments were performed on the frozen sections, either individually or in combination, and nuclear staining was always absent. Interestingly, in the paraffin-embedded sections, ß-catenin nuclear accumulation was observed in 8/11 cases (p<0.001) (Figure 1F and Figure 1G). Cytoplasmic and/or membrane staining was also detected in all the cases, independently of the region of the tumor. Some but not all infiltrating single tumor cells showed nuclear staining (Figure 2A). Moreover, the intensity of ß-catenin staining appeared to be higher in the invasive front of the tumors (Figure 2B), although differences in staining indices among superficial, mid-, and deep regions were not statistically significant. There was an inverse association between nuclear and cytoplasmic (p= 0.004) or membrane staining index (p=0.025).
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To examine the basis of this discrepancy related to the tissue preservation method (frozen vs paraffin), we performed ß-catenin immunostaining experiments on sections from pellets of colorectal cancer-derived cultured cells presenting well-characterized mutations in the APC gene. Nuclear ß-catenin has been consistently detected in paraformaldehyde-fixed and Triton X-100-permeabilized SW480 cells (
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Discussion |
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Cadherins and catenins have attracted much interest because of their role in cellcell adhesion during carcinogenesis. The recent evidence supporting an additional role for ß-catenin in transcriptional regulation has renewed interest in this molecule as a potential dominant oncogene. In the past few years, many studies have described the membrane and cytoplasmic distribution of ß-catenin in human tumors without reference to its nuclear localization (
While analyzing adhesion molecule expression in a series of more than 100 colorectal cancers by immunohistochemistry on frozen tissue sections, we were surprised by the lack of detection of nuclear ß-catenin, a finding that is not in agreement with current hypotheses about the accumulation of this molecule in APC-mutated colorectal cancers. The possibility that a technical artifact could explain this discrepancy led us to perform a detailed immunohistochemical study using paired samples of fresh and paraffin-embedded tissues. As described above, ß-catenin was never found in the nuclei of colon cancer cells in frozen sections, irrespective of the fixationpermeabilization conditions used. By contrast, it was frequently detected in the nucleus when paraffin-embedded tissues were used. Several sources of evidence indicate that the nuclear reactivity observed after such processing is not due to a technical artifact. (a) Nuclear staining was never observed in normal cells, as expected because they harbor wild-type APC protein. (b) When SW480 cells were used, the reactivity of the anti-ß-catenin antibody with sections of paraffin-embedded, but not frozen, cell pellets precisely reflected the findings with formaldehyde-fixed cultured cells. Finally (c), a rabbit polyclonal antiserum raised against a synthetic peptide from the ß-catenin sequence (PGDSNQLAWFDTDL; kindly provided by J.W. Nelson, Stanford University, Palo Alto, CA) yielded the same pattern of reactivity with both frozen and paraffin-embedded tissues as did the monoclonal antibody (Figure 1D and Figure 1H). It is important to emphasize that, until now, most immunohistochemical studies of ß-catenin have employed either the monoclonal antibody used here or antisera raised against the peptide used by the Nelson laboratory. Interestingly, both antibodies recognize the COOH-terminus, a domain endowed with transcriptional activation capacity. This suggests that, in frozen tissues, this domain is cryptic, possibly due to its interaction with other molecules. In paraffin-embedded tissues this domain would be available for antibody binding. However, this hypothesis does not explain the results with cultured cells fixed and permeabilized in situ.
The very small number of cases studied in this series does not allow conclusions about the association between nuclear ß-catenin localization and the invasive characteristics of the tumor. However, and in agreement with other reports (
Regarding the three tumors lacking nuclear staining in paraffin-embedded section samples (Table 1; Figure 1G), they may have arisen through alterations in an APC- and ß-catenin-independent pathway such as microsatellite instability owing to defects in mismatch repair genes (
The findings reported here are important because antibodies to the COOH-terminus of ß-catenin are very commonly used to examine the distribution of this molecule, not only in colorectal cancer but in other tumor types as well (
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Footnotes |
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1 AM, MF, and MLM have made equivalent contributions to this work.
2 Members of the Colon Cancer Team at IMAS (CCTI): F. Alameda, J. Baulida, E. Batlle, D. Dominguez, M. Fabre, M. Gallén, A. García de Herreros, J. Lloreta, M. L. Mariñoso, A. Munné, F. X. Real, S. Serrano, and M. C. Torns.
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Acknowledgments |
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Supported by Fondo de Investigación Sanitaria (Grant 97-1216) and by la Maratò de TV3.
We would like to thank Julian García and Josep M. Estañol for valuable contributions and James W. Nelson for providing anti-ß-catenin antiserum.
Received for publication January 25, 1999; accepted March 9, 1999.
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