Angiogenesis is considered to play an important role in the development of malignant brain tumors, especially glioblastoma multiforme (GBM). Abnormal vascular construction with a glomeruloid appearance is characteristic of GBM. fl-catenin is known as one of the adhesive molecules associated not only with cell adhesion and cell polarity, but also with carcinogenesis. We postulated the relevance of fl-catenin to vigorous endothelial proliferation in human GBM because the vascular cells (VCs) are apt to lose their cell polarity. The object of this study is to compare the immunohistochemical localization of fl-catenin in VCs between GBMs and normal brain tissues. Immunohistochemical analysis of fl-catenin for VCs in 32 GBMs and 10 normal brain tissues was performed. beta-catenin was found concentrated in the areas of vascular cellcell junction and internal surface of the vascular lumen in all normal brains. In contrast, fi-catenin, in proliferating VCs in GBMs, was stained homogeneously and intensely in the cytoplasms of 26 cases (81.3%), in which nuclear staining of beta-catenin was also recognized in four cases (12.5%). In conclusion, the intracellular localization of fl-catenin in VCs of GBMs was found to be different from that of normal brain tissues. The changes of expression of fl-catenin may be associated with the angiogenesis or transformation of the VCs in human GBM. [Neurol Res 2000; 22: 650-656]

Keywords: fl-catenin; glioblastoma multiforme; angiogenesis; endothelial proliferation; immunohistochemistry

INTRODUCTION

Angiogenesis, formation of microvasculature by capillary sprouting, is known to be crucial for development of malignant tumor. Glioblastoma multiforme (GBM) is the most common malignant brain tumor in human and often displays a glomeruloid appearance with the prominent proliferation of vascular cells (VCs). While there have been an increasing number of studies of angio enetic factors including platelet derived growth factor , basic fibroblast growth factor , vascular endothelial growth factor3'7 -9, epidermal growth factor 3'8, and transforming growth factor-beta10 few have focused on cytoskeletal or adhesive molecules of VCs in GBMs. We have reported that fl-catenin may be one of the regulators of angiogenesis through the immunohistochemical analysis of the protein in N-ethyl-Nnitrosourea (ENU) induced rat gliomas and rat normal brains" .

beta-catenin is a 92 kDa cytoplasmic protein, which forms complex with a and y-catenins and links cadherin to the actin filament network . It is also known as one of endothelial cell-cell adhesion molecules that control tube formation in endothelial cells (EC) associated with cadherin 5 (VE-cadherin), CD31 and Factin"4. The complex of vascular endothelial (VE) cadherin and catenin has been reported to play important roles in capillary tube formation Recently, the complex of cadherin and catenin has been reported to maintain the cell polarity and crucially involved in angiogenesis by making the interendothelial junction to be dissociated and reorganized2. In addition, fl-catenin that interacted with the adenomatous polyposis coli (APC) suppressor gene product, has been regarded as an element in Wnt signal transduction pathway of the cell, and a disturbance of its expression is known to be associated with the development of colorectal carcinoma

In this study, immunohistochemical analysis of betacatenin was performed to understand the molecular characteristics in VCs between GBM and normal brain tissue.

MATERIALS AND METHODS

Tissues

Thirty-two tumors including GBMs were obtained from patients by surgical operations prior to chemotherapy and radiotherapy. The diagnosis of each case was morpho logically confined. Ten normal brain tissues as controls were obtained by autopsies. For the immunohistochemical study, all tumor tissue and normal brain tissues were fixed in 10% formalin and then embedded in paraffin wax. Serial sections were cut at 4 um thickness and placed on the slides. One slide stained with hematoxylin and eosin was used for histological classification according to guidelines of the World Health Organization for histological typing of tumors of the central nervous system24 , and others were applied for the immunohistochemical analysis.

For the immunoblotting study, two cases of surgical specimens were selected from the cases for immunohistochemistry, and normal white matter was obtained by biopsy from the patient with epilepsy. The tumor tissues and normal brain tissues were immersed in 0.9%

NaCI solution to remove blood and immediately frozen in liquid nitrogen. The frozen tissues were thawed and homogenized at 4C (using a teflon homogenizer) in RIPA buffer [10 mM Tris-HCI (pH 7.4), 1% Triton-X, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCI, 1 mM ethylenediamine tetra-acetic acid (EDTA), 1 mM ethyleneglycol-bis-(betaaminoethyl ether)-N,N'-tetra-acetic acid (EGTA)]. The homogenate was centrifuged at 10,000 rpm for 5 min at 40 deg C.

Immunohistochemical procedure

The sections were deparaffinized in xylene, dehydrated in a graded alcohol series and rinsed in 0.05 M Tris-HCI, pH 7.6 for 20 min. Before application of primary antibody, sections were subjected to antigen retrieval by boiling for 15 min in sodium citrate buffer (0.01 M, pH 6.0) in a microwave oven. Subsequently, endogenous peroxidase activity was blocked with 5% hydrogen peroxide in methanol for 10 min. After washing in 0.05 M Tris-HCI buffer, pH 7.6 for 20 min, non-specific staining was blocked by 2% normal bovine serum for 45 min. Anti-fl-catenin mouse monoclonal antibody 1 : 1000 (Transduction Laboratories, Lexington, MA, USA) was applied to specimens. LSAB Kit (DAKO, Denmark) was used as biotinylated secondary antibody and Streptavidine conjugated to horseradish peroxidase (HRP). Washing between steps was with Tris-HCI buffer. 3-3' Diaminobenzidine (DAB; Sigma Chemicals, St. Louis, MO, USA) was used as the chromogen. Subsequently, tumor specimens were stained with periodic acid-Schiff (PAS) to identify the basement membrane of the VCs after immunohistochemical process for betacatenin. The cells surrounded by PAS-positive basement membrane were defined as 'VCs'. These VCs included ECs, pericytes and vascular smooth muscles. Finally, all specimens were counterstained with Mayer's hematoxylin. Normal choroid plexuses in rats were used as positive controls (Figure 1), while negative controls were obtained by using mouse serum instead of the primary antibody. The cytoplasmic staining of VCs for fl-catenin was graded according to the following criteria: +, immunopositive cells were observed in 10% to 50% of all VCs; ++, in more than 50% of all VCs. The VCs with immunopositive nucleus were graded as follows: +, less than 10%; ++, more than 10% of all VCs. The localized staining in cell to cell junction and internal surface of VCs was graded as '+' when they were observed in more than 10% per slide. The immunopositive tumor cells were graded as follows: +, from 20% to 50% of all tumor cells; ++, more than 50% of all tumor cells.

Electrophoresis and immunoblot analysis

Twenty micrograms of supernatant fractions prepared as described above and 6 ug of HeLa lysate (positive control) (Transduction Laboratories, Lexington, USA) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli25. Immunoblotting was performed after transfer of proteins onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, USA) from 10% SDS-PAGE. Blots were blocked for 1 h in phosphatebuffered saline (PBS)/0.05% Tween 20 (PBS-T) containing 5% skimmed milk and then probed with primary antibody against fl-catenin (1 : 3000) for 30 min at room temperature. After washing with PBS-T, the membrane was incubated with HRP-labeled secondary antibody (DAKO, Denmark) (1 :4000) for 30 min at room temperature, and rinsed in PBS-T, and the bands of betacatenin were detected with ECL system (Amersham, Buckinghamshire, UK). In all experiments, protein was determined with the protein assay reagent (Bio-Rad, Munich, Germany) using bovine albumin as a standard.

RESULTS

Immunohistochemistry

The immunohistochemical findings are summarized in Tables 1 and 2. The normal choroid plexus had staining activity for fl-catenin at cell-cell junction (Figure IAB). In the normal brain vessels, fl-catenin was basically speckled at vascular cell-cell junction (Figure 2A--C and thinly expressed along the internal surface of vascular lumen (Figure 2B-D) in all of 10 normal brain tissues (100%) (Table 1). In contrast, VCs in GBMs were homogeneously and intensely stained for fl-catenin in the cytoplasm in 26 cases (81.3%) out of 32 GBMs (Figure 3A-E and Table 1). The cytoplasmic staining was also observed in glomerular like formation (Figure 3D,E). Furthermore, nuclear staining was recognized in four cases (12.5%) (Figure 3G-fi. All negative controls were not stained for /-catenin (Figure 3F. Such intense cytoplasmic staining and nuclear staining of fl-catenin were never seen in VCs in normal brains. A few of these VCs in GBMs retained to express fl-catenin in cell-cell junction, however, these VCs also had fl-catenin expression in the cytoplasm with or without expression in the nucleus. The tumor cells also expressed /-catenin in cell-cell junction with weak or intense cytoplasmic staining in 31 cases (96.9%) (Figure 4 and Table 2). In contrast, the glias in normal brain tissues were not stained for fi-catenin, while the neuropil was weakly positive for fl-catenin (Figure 2 and Table 2).

Immunoblots

Western blots were used to confirm the expression of fl-catenin in GBM specimens. The observed bands were revealed at 92 kDa corresponding to fl-catenin at the same level of a positive control (Figure 5).

DISCUSSION

This study was performed to study the immunological localization of beta-catenin, a cytoplasmic protein with two biological roles, as a regulator of the adhesion of the cells and the maintenance of polarity in vascular formation19,20. Cadherin 5 and CD31, involved in tube formation of EC, exert the vascular construction by binding of fl-catenin 13. At formation of EC monolayer, it has been reported that the content of fl-catenin gradually decreases while that of y-catenin increases'9. Therefore, it is suggested that f-catenin is one of the key regulators for the cadherin-mediated cell-cell adhesion system26. In this study, fl-catenin was localized in the areas of vascular cell-cell junction and internal surface of vascular lumen in the normal brain vessels. In contrast, fi-catenin was homogeneously and intensely expressed in the cytoplasm of VCs in GBMs. Nuclear staining of VCs was also revealed in some GBM cases. As the capillary endothelial cells are regarded as the vital component with possible proliferative activity in GBM27, it is especially noteworthy that VCs in GBMs apparently express fl-catenin as compared with those in normal brain tissues. We have reported the immunohistochemical analysis of fl-catenin in ENU-induced rat gliomas and rat normal brain tissues. In that experiment, the cytoplasmic/nuclear distribution of fl-catenin in tumor vessels was more frequently observed with the degree of malignancy of the tumor, while fl-catenin was found concentrated in vascular cell-cell junction and internal surface of the vascular lumen in normal brains. These findings supported the presenting results in human GBM tissues.

Another role of beta-catenin is to interact with APC suppressor gene product2l 23, and the mutation of the APC gene initiates the majority of colorectal cancers associated with the dysfunction of fi-catenin 22. fl-catenin is also considered to associate with cell polarity based on previous evidence that fl-catenin has a high sequence similarity with a Drosophila segment polarity gene product Armadillo (71 % amino acid identity)12,1 which plays a role in the formation of the anterior-posterior polarity of the fly segments28'29 and that over-expression of fl-catenin is known to induce the formation of a complete secondary body axi S29. From our results, it is speculated that the homogeneously stained VCs may lose the cell polarity and involve formation of vascular construction like a glomeruloid appearance. In this study, junctional staining of fl-catenin was recognized in normal VCs, while cytoplasmic/nuclear staining of betacatenin was proved in GBMs. This suggested that cellular distribution of beta-catenin activity is closely associated with the polarity of VCs.

Our findings that increased expression of cytoplasmic beta-catenin is related to the morphological changes in GBM seem to be in agreement with the observation that transformation from an epithelial to a mesenchymal phenotype is associated with increased levels of cytoplasmic fl-catenin in melanoma cel IS30. Strongly positive staining of fl-catenin in VCs in GBMs may indicate the proliferative potentials in the cells, as shown in a previous report that the proliferative potential of VCs in GBM has been achieved in response to the degree of malignancy in the human glioma

CONCLUSION

The differential expression of beta-catenin in VCs of GBMs and of normal brain tissues was characterized in this study. The altered expression of betacatenin may be involved in the angiogenesis or transformation of the VCs in GBM.

ACKNOWLEDGEMENTS

This study was partly supported by the Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, and Culture of Japan. The authors would like to acknowledge the technical assistance of Miss Kyoko Takahashi, Miss Chikako Usui, and Miss Tomoko Kajita.

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Hirohito Yano, Akira Hara*, Katsunobu Takenaka, Kei Nakatani:, Jun Shinoda, Kuniyasu Shimokawat, Naoki Yoshimi*, Hideki Mori* and Noboru Sakai

Department of Neurosurgery, *Department of Pathology, Department of Laboratory Medicine Gifu University School of Medicine, Gifu

*Department of Neurosurgery, Gifu Prefectural Hospital, Gifu, Japan

Correspondence and reprint requests to: Hirohito Yano, MD, Department of Neurosurgery, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu City 500-8705, Japan. [hirohito@cc.gifu-u.ac.jp] Accepted for publication June 2000.

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