Nuclear β-catenin in Colorectal Tumors: To Freeze or Not To Freeze?

Abstract

β-Catenin mediates the interaction of E-cadherin with α-catenin and the actin cytoskeleton. Recent evidence indicates that when the tumor suppressor gene APC is inactivated, β-catenin can translocate to the nucleus, where it acts as a transcriptional regulator. Because APC is inactivated in most colorectal cancers, β-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 β-catenin. Here we compared the reactivity of an anti-β-catenin monoclonal antibody with 11 colorectal cancers using immunohistochemistry on sections of frozen or paraffin-embedded samples. β-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-β-catenin serum. We also examined β-catenin distribution in SW480 colon cancer cells, in which its nuclear accumulation has been reported. As in tissues, nuclear β-catenin was detected in paraffin-embedded but not in frozen samples. These findings are relevant because of the increasing interest in the study of β-catenin in tumors, based on its dual role in cell adhesion and transcriptional regulation. (J Histochem Cytochem 47:1089–1094, 1999)

Keywords

β-catenin colorectal cancers frozen sections paraffin-embedded sections immunohistochemistry

β-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 (Kemler 1993; Huber et al. 1996a). The direct interaction of β-catenin with the cytoplasmic domain of cadherins plays a crucial role in cell-cell adhesion and signal transmission between neighboring cells (Aberle et al. 1994). β-Catenin is a target for tyrosine kinases, such as src and the EGF receptor, and might transmit extracellular signals to the corresponding transduction pathways (Behrens et al. 1993; Hoschuetzky et al. 1994). A number of studies have demonstrated the association between loss of E-cadherin function, through disruption of its linkage to catenins and the cytoskeleton (Aberle et al. 1996), and the increased invasiveness and metastasis of tumors (Bracke et al. 1996).

β-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 β-catenin in the transduction of Wingless/Wnt-dependent cell-cell signaling has been demonstrated (Cadigan and Nusse 1997). Furthermore, β-catenin may also play a direct role in colorectal tumorigenesis because it binds the product of the tumor supressor gene APC (Rubinfeld et al. 1993, 1996; Kinzler and Vogelstein 1996). Glycogen synthase kinase-3β and wild-type APC regulate the level of free cytosolic β-catenin by promoting its degradation through the ubiquitin proteasome pathway (Rubinfeld et al. 1996; Aberle et al. 1997). When APC is mutated, as occurs in 85% of colorectal cancers, β-catenin accumulates in the cytosol and can translocate to the nucleus, where it binds transcription factors of the TCF/LEF gene family and activates the expression of target genes (Behrens et al. 1996; Huber et al. 1996b; Korinek et al. 1997; Morin et al. 1997; Rubinfeld et al. 1997). Moreover, it was recently reported that several colon carcinoma cell lines (Korinek et al. 1997; Morin et al. 1997) as well as colorectal tumors (Iwao et al. 1998) with wild-type APC present mutations affecting the NH₂-terminal domain of β-catenin. All these functional data are supported by the structural organization of the protein: an NH₂-terminal domain that regulates protein stability through several serine-threonine phosphorylation sites, a central domain composed of arm repeats responsible for the interactions with APC, TCF, and E-cadherin, and a COOH-terminal domain with transcriptional regulation capacity (Cadigan and Nusse 1997). Therefore, it is proposed that β-catenin plays a dual role, not only in the formation and maintenance of cell-cell interactions but also in the regulation of gene activity with a dominant oncogenic effect on tumorigenesis.

Most studies of the expresion of β-catenin in tumors have focused on its membrane and cytoplasmic distribution (Hiscox and Jiang 1997; Takayama et al. 1996, 1998; Van der Wurff et al. 1997) and few studies have reported on its nuclear localization in colorectal (Inomata et al. 1996; Hao et al. 1997; Valizadeh et al. 1997; Iwao et al. 1998) or other types of cancer (Rubinfeld et al. 1997; Palacios and Gamallo 1998). In the course of a study on the expression of cadherins and catenins in colorectal cancer tissues, we were surprised by the lack of nuclear β-catenin in frozen tumor tissue sections and we observed discrepancies between the distribution of β-catenin in frozen and paraffin-embedded samples. Because of the importance of understanding β-catenin nuclear localization in tumors, we have conducted a detailed comparison of β-catenin distribution in paired colorectal cancer samples that were either cryopreserved or paraffin-embedded.

Materials and Methods

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), nonneoplastic 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 streptavidin-biotin-alkaline 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 NaC1. 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 Tris-NaC1 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 Tris-NaC1 buffer, avidin-biotin-alkaline 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 β-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, nonneoplastic 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.

Results

In normal colon mucosa and in nonneoplastic mucosa adjacent to the tumor, β-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 (Figures 1A and 1E).

Figure 1

Reactivity of anti-β-catenin antibodies with frozen (A-D) or paraffin-embedded (E-H) sections from colorectal tissues. Mouse monoclonal anti-β-catenin: A-C,E-G; rabbit polyclonal anti-β-catenin: D,H. In normal mucosa (Case 8, A,E), β-catenin is exclusively detected in the membrane and cytoplasm in both frozen and paraffin-embedded samples. In frozen sections of colorectal cancer tissues, β-catenin is present exclusively in the membrane and cytoplasm (Case 8, B,D; Case 3, C). In paraffin-embedded sections of colorectal cancer tissues, nuclear β-catenin is detected in 8/11 cases (see text) (Case 8, F,H; Case 3, G). Bar = 34 μm.

Figure 2

Reactivity of anti-β-catenin antibody with the deep region of tumor (paraffin-embedded sections). (A) Some infiltrating single cells show nuclear staining. (B) Invasive front of the tumor, with strong nuclear staining. Bar = 34 μm.

Figure 3

Reactivity of anti-β-catenin antibody with SW480 (A,C) and HT-29 (B,D) cells. In sections of frozen cell pellets, no nuclear staining is observed (A,B). In sections of paraffin-embedded cell pellets, strong nuclear staining reactivity is observed in SW480 cells (C) but not in HT-29 cells (D). Bar = 34 μm.

In tumor samples, the subcellular distribution of β-catenin was clearly different in sections from frozen and paraffin-embedded tissues (Table 1). In frozen sections, β-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 (Figures 1B and 1C). Furthermore, β-catenin was never found in the nuclei of colon cancer cells in frozen sections, irrespective of the fixation-permeabilization 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, β-catenin nuclear accumulation was observed in 8/11 cases (p < 0.001) (Figures 1F and 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 β-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).

Table 1

Global staining indices of B-catenin in 11 colorectal tumors (superficial, mid-, and deep regions) a

			Nuclear		Cytoplasm/Membrane
Case	HG	S	F	P	F	P
1	G2	II	0	1.76	4.00	1.00
2	G2	II	0	1.06	4.00	1.50
3	G1	II	0	0	3.20	2.25
4	G2	I	0	1.00	2.90	1.60
5	G2	I	0	0.88	2.40	1.55
6	G3	I	0	2.20	3.60	1.25
7	G2	II	0	0.30	2.00	2.70
8	G2	IV	0	0.60	2.80	1.90
9	G3	III	0	0	2.20	2.20
10	G2	III	0	0	3.00	2.40
11	G2	III	0	1.20	3.00	1.50
Mean ± SD			0	0.82 ± 0.73	3.01 ± 0.67	1.80 ± 0.53

HG, histophatologic grade (G1, well-differentiated; G2, moderately differentiated; G3, poorly differentiated); S, stage grouping; F, frozen; P, paraffin.

To examine the basis of this discrepancy related to the tissue preservation method (frozen vs paraffin), we performed β-catenin immunostaining experiments on sections from pellets of colorectal cancer-derived cultured cells presenting well-characterized mutations in the APC gene. Nuclear (3-catenin has been consistently detected in paraformaldehyde-fixed and Triton X-100-permeabilized SW480 cells (Behrens et al. 1998: and unpublished observations). In sections of paraffin-embedded SW480 cell pellets, β-catenin was detected in both nucleus and cytoplasm (Figure 3C). However, when the same experiment was performed on sections of frozen cell pellets, β-catenin was detected only in the cytoplasm (Figure 3A). As an additional control, we used HT-29 cells because (3-catenin is exclusively detected in the membrane and cytoplasm in fixed and permeabilized cells (unpublished observations). In these cells, β-catenin was never detected in the nucleus, independent of the cell preparation used: paraformaldehyde-fixed (2.5% in PBS for 5 min) and Triton X-100-permeabilized (0.2% in PBS for 10 min), frozen or paraffin-embedded sections (Figures 3B and 3D).

Discussion

Cadherins and catenins have attracted much interest because of their role in cell-cell adhesion during carcinogenesis. The recent evidence supporting an additional role for β-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 β-catenin in human tumors without reference to its nuclear localization (Hiscox and Jiang 1997: Van der Wurff et al. 1997: Takayama et al. 1996, 1998). Only four studies have reported β-catenin nuclear localization in colorectal tumors (Inomata et al. 1996: Hao et al. 1997: Valizadeh et al. 1997: Iwao et al. 1998). Interestingly, all of them were performed on paraffin-embedded tissues sections with the antibody used in our study.

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 β-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, β-catenin was never found in the nuclei of colon cancer cells in frozen sections, irrespective of the fixation-permeabilization 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-β-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 β-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 (Figures 1D and 1H). It is important to emphasize that, until now, most immunohistochemical studies of β-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 β-catenin localization and the invasive characteristics of the tumor. However, and in agreement with other reports (Hao et al. 1997), our data support the notion that nuclear β-catenin accumulation is associated with decreased localization at cell-cell contact sites.

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 β-catenin-independent pathway such as microsatellite instability owing to defects in mismatch repair genes (Miyaki et al. 1994; Kinzler and Vogelstein 1996).

The findings reported here are important because antibodies to the COOH-terminus of β-catenin are very commonly used to examine the distribution of this molecule, not only in colorectal cancer but in other tumor types as well (Palacios and Gamallo 1998). Furthermore, they may be relevant to the interpretation of studies in transgenic mice, including the Min model of familial adenomatous polyposis (Su et al. 1992; Wong et al. 1998).

Footnotes

Acknowledgments

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-β-catenin antiserum.

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