Subcellular distribution of Wnt pathway proteins in normal and neoplastic colon - PubMed (original) (raw)

Subcellular distribution of Wnt pathway proteins in normal and neoplastic colon

Christine B Anderson et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Mutations in the APC tumor suppressor gene are present in approximately 85% of colorectal tumors and are thought to contribute early in the process of tumorigenesis. The truncated protein resulting from most APC mutations can lead to elevated beta-catenin levels in colon tumor cells. APC and associated proteins thus form a beta-catenin regulatory complex, with axin playing a key role. Although cell culture studies have revealed intriguing aspects of this complex, little characterization has been done in human colonocytes, the target tissue of colon carcinogenesis. The present study of intact human colon crypts, adenomatous polyps, and adenocarcinomas focuses on subcellular localization of some key elements of the complex: beta-catenin, APC, axin, and axin2. We examined endogenous protein localization within the framework of three-dimensional tissue architecture by using laser scanning confocal microscopy, and immunofluorescence staining of whole-mount fixed tissue from more than 50 patients. Expression patterns suggest that APC and axin colocalize in the nucleus and at lateral cell borders, and show that axin2 is limited to the nucleus. Altered nuclear expression of axin seen in colon polyps and carcinomas may be a consequence of the loss of full-length APC and the advent of nuclear beta-catenin. The observation of nuclear beta-catenin in fewer than half of carcinoma images and only rarely in polyps indicates that nuclear translocation of beta-catenin may not be an immediate consequence of the loss of APC.

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Figures

Figure 1

Figure 1

(a) Serial optical sections of mouse colon crypts labeled with the nuclear counterstain To-Pro 3. Sequential images along the _Z_-axis were collected using laser scanning confocal microscopy. Optical section 2 gives the clearest longitudinal section of the center crypt. To compare tissue preparation methods, colon tissue from a single patient was either fixed and the crypts manually microdissected (b), or treated with chelating agents to release the crypts, which were then fixed (c). Crypts were immunostained for APC and the protein localization compared. Crypts manipulated before fixation (c) exhibited altered protein localization.

Figure 2

Figure 2

Expression of APC, β-catenin, axin, and axin2 in optical sections of whole-mount human colon tissue. The basic units of normal colonic mucosa, the crypts, are vase-shaped structures comprised of rectangular epithelial cells, stacked lengthwise. These cells have an apical side that faces the crypt lumen and a basal side that houses the nucleus. A grayscale image shows the primary antibody alone. Three-color merged images of APC or β-catenin show primary antibody in green, a nuclear counterstain in blue, and phalloidin-labeled actin in red, delineating a ring of adhesion proteins just below the apical cell membrane. The two-color merged images show axin or axin2 in red with a nuclear counter stain in blue. Arrows indicate apical surfaces and arrowheads mark nuclei. In normal crypts (a), APC and axin are found diffusely in the nucleus and near the borders between cells. APC is especially prominent near the apical junctions. β-catenin is found at lateral cell junctions and axin2 is nuclear. Adenomatous polyps (b) show strong nuclear N-terminal (truncated) APC, whereas C-terminal (full-length) APC and axin are missing or reduced in the nucleus. C-terminal APC is variably retained at cell–cell borders, and near the face of the apical membrane. Axin is strongly cytoplasmic. The β-catenin pattern is similar to that in normal crypts and axin2 remains nuclear. In carcinoma (c), expression patterns of the four proteins are similar to those in polyps except that β-catenin and axin are found in the nucleus. (Scale bar, 10 μm.)

Figure 3

Figure 3

Graphic representation of data from Table 1, grouped by site of expression, cytoplasmic, cell border, or nuclear. In a, cytoplasmic expression of axin is seen in normal cells, but is found with greater frequency in polyp and carcinoma, as are cytoplasmic N-terminal (truncated) APC and axin2. Incidence of cytoplasmic β-catenin also rises slightly in carcinoma. In b, cell border expression of N-terminal APC is diminished in polyp and carcinoma. In c, nuclear expression of APC, axin, and axin2 is seen in normal cells. Nuclear C-terminal APC and axin are diminished in polyp, but nuclear axin is found again in carcinoma in conjunction with the expression of nuclear β-catenin.

Figure 4

Figure 4

Double staining of β-catenin and axin in adenomatous polyp and adenocarcinoma. The polyp image (a) shows β-catenin and axin near cell–cell junctions, and axin strongly in the cytoplasm. Neither protein is obvious in the nucleus. The carcinoma image (b) shows both β-catenin and axin in the nucleus. Primary antibodies in each double-stained image are presented singly in grayscale, and in a three-color merged image showing β-catenin in green, axin in red, and nuclei in blue. (Scale bar, 10 μm.)

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