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6367.Full.Pdf Human Cancer Biology Identification of the Decay-Accelerating Factor CD55 as a Peanut Agglutinin ^ Binding Protein and Its Alteration in Non^SmallCellLungCancers Mitsunori Higuchi,1Yuichi Endo, 2 Hiroyuki Suzuki,1Fumihiko Osuka,1Yutaka Shio,1Koichi Fujiu,1 Ryuzo Kanno,1Akio Oishi,3 Teizo Fujita,2 and Mitsukazu Gotoh1 Abstract Purpose: Peanut agglutinin (PNA) recognizes tumor-associated carbohydrates. In this study, we aimed to identify the core protein harboring PNA-binding sugars in the human lung and to explore the relationship with the pathology of primary non ^ small cell lung cancers (NSCLC). Experimental Design: PNA lectin blotting was used to detect PNA-binding proteins in the microsomal fraction of lung tissue from 24 patients with NSCLC.The 55- to 65-kDa core peptide PNA-binding protein was characterized by enzymatic treatment and identified by immunopre- cipitation and affinity chromatography. The expression level and increase in size of the 55- to 65-kDa PNA-binding protein/decay-accelerating factor (DAF) were compared between normal and tumor regions of the tumor tissue by Western blotting and quantitative PCR. Results: The 55- to 65-kDa PNA-binding protein was observed in human lung. This was a glycosylphosphatidylinositol-anchored membrane protein carrying O-linked carbohydrates. This core protein was identified as DAF, one of the complementary regulatory proteins. DAF was enlarged to 65 to 75 kDa in NSCLC tumor lesions due to sialylation in the sugar moiety. At the transcription level, DAF levels were significantly lower in tumor regions, suggesting its down-regulation in NSCLC cells. Conclusions: DAF was identified as a new PNA-binding protein in the human lung. The down- regulation and heavy sialylation of DAF was associated with pathology in NSCLC, and these alterations make this protein a potential marker for NSCLC. Carbohydrates on the cell surface play an important role in lymphatic vessel invasion and a high lymph node metastatic several metastatic processes by influencing cell-cell and cell- rate in lung adenocarcinoma tissue (8). However, the precise extracellular matrix protein interactions (1). Peanut agglutinin mechanism of involvement of PNA-binding sugars in metasta- (PNA) is a plant lectin isolated from Arachis hypogaea that sis is unclear and there is no information on the core protein preferentially recognizes galactose h1-3N-acetylgalactosamine harboring PNA-binding sugars in lung cancers. To date, several linkage in O-linked glycans (2, 3), also called tumor-associated PNA-binding proteins or PNA receptors have been identified, antigen (T-antigen) or Thomsen-Friedenreich antigen (4). The including CD8, CD43, CD44, CD45, gp200, polymorphic expression levels and localization patterns of PNA-recognizing epithelial mucin, and MGC-24 (9–12). carbohydrates have been reported to correlate with the In the present study, we identified decay-accelerating factor aggressiveness of several cancers, including colorectal cancer, (DAF; CD55) as a new core protein harboring PNA-binding breast cancer, and malignant melanoma (1, 3, 5, 6). In lung sugars in the lung. DAF is present on all blood elements cancers, however, there are few reports about the relationship and most other cell types, especially in high levels on cells between T-antigen expression and clinicopathologic variables that line extravascular compartments. The protein intrinsically (7). We previously reported that the expression of PNA- functions on cell membranes to protect host cells from recognizing carbohydrates was significantly correlated to autologous complement attack by accelerating the decay of C3and C5 convertases (13–15). In addition, we report in the current study that DAF was down-regulated and processed to a high molecular weight by sialylation in non–small cell Authors’ Affiliations: Departments of 1Surgery I and 2Immunology, Fukushima lung cancer (NSCLC) tissue and the pathologic implications 3 Medical University School of Medicine; and Fukushima Red Cross Hospital, are discussed. Fukushima, Japan Received 4/5/06; revised 8/15/06; accepted 8/29/06. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Mitsukazu Gotoh, Department of Surgery I, Fukushima Tissue specimens. Tumor lung tissue specimens were obtained from Medical University School of Medicine, 1-Hikariga-oka, Fukushima 960-1295, 24 patients with NSCLC who underwent surgery at Fukushima Medical Japan. Phone: 81-24-547-1254; Fax: 81-24-548-2735; E-mail: [email protected]. University Hospital (Fukushima, Japan). Specimens included adeno- F 2006 American Association for Cancer Research. carcinoma, squamous cell carcinoma, and large cell lung carcinoma. doi:10.1158/1078-0432.CCR-06-0836 Tumors were classified according to the WHO classification of lung www.aacrjournals.org 6367 Clin Cancer Res 2006;12(21)November 1, 2006 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Human Cancer Biology tumor (16). All tissue samples were obtained within 30 minutes after Foster City, CA). The relative gene expression was calculated as a fold surgical resection and stored in liquid nitrogen until further use. induction compared with h-actin. Normal, nontumor tissue was obtained from regions sufficiently far Preparation of DAF-overexpressed PC14 cells. Full-length DAF away from the cancerous lesion in the same specimen. A signed consent cDNA, which was kindly provided by Dr. D.M. Lublin (Washington form was obtained from each patient. University School of Medicine, St. Louis, MO), was cloned in the PNA lectin blotting. Lung tissue was homogenized in PBS containing pcDNA3plasmid (Invitrogen, Carlsbad, CA). The plasmid was trans- protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). fected into PC14 cells (21), a human lung carcinoma cell line, by Microsome fraction was recovered by differential centrifugation of the electroporation with Nucleofector (Wako Pure Chemicals). Positive homogenate and solubilized by sonication in 0.1 mol/L Tris-HCl clones were screened by 400 Ag/mL neomycin (Geneticin, Life (pH 6.8) containing 4% SDS and 20% glycerol (SDS-PAGE sample Technologies, Gaithersburg, MD). buffer). Total protein concentration was determined with bicinchoninic DAF immunostaining. Formalin-fixed, paraffin-embedded sections acid protein assay reagent (Pierce, Rockford, IL). Samples of 20 to 30 Ag of NSCLC tissue were prepared following standard procedures. of protein were subjected to SDS-PAGE on a 7.5% to 10% poly- Deparaffinized sections were incubated with anti-DAF antibody acrylamide gel under nonreducing conditions as described previously (4F11) followed by biotinylated rabbit anti-mouse IgG. Color was (17). Briefly, the blotted membrane was probed with a biotin- developed by incubation with 3,3¶-diaminobenzidine-H2O2.The conjugated PNA (E.Y. Laboratories, San Mateo, CA) and the color was sections were counterstained with hematoxylin. developed with the Vectastain avidin-biotin complex method kit (Vector Statistics. Differences in levels of the 55- to 65-kDa PNA-binding Laboratories, Burlingame, CA) and nitroblue tetrazolium (Wako Pure protein and DAF between normal and tumor tissues were evaluated by Chemicals, Osaka, Japan). Wilcoxon signed ranks test, and the correlation between both levels was DAF Western blotting. Western blotting for the microsome DAF estimated by Pearson’s correlation coefficient. The correlation between fraction was carried out by a protocol similar to the aforementioned clinicopathologic feature and the normal/tumor expression ratios of the PNA lectin blots, except for sequential incubation with anti-DAF 55- to 65-kDa PNA-binding protein and DAF was also evaluated with antibody (4F11; ref. 18) and then with biotinylated anti-mouse IgG the Student’s t test. antibody (DAKO, Carpinteria, CA), instead of biotinylated PNA. To quantify the bands in PNA lectin blots and Western blots, the signal Results intensity of each band was estimated using NIH image software (version 1.56; Wane Rasband; NIH, Bethesda, MD). PNA-binding protein (55 to 65 kDa)is present in human Treatment with neuraminidase, endo-a-N-acetylgalactosaminidase lung. PNA lectin blots showed that normal human lung tissue (O-glycanase), and phosphatidylinositol-specific phospholipase C. Selective expressed PNA-binding protein with a size of 55 to 65 kDa, removal of the terminal sialic acid and all O-linked glycans was termed 55- to 65-kDa PNA-binding protein (Fig. 1A). The achieved by treatment of the microsome fraction with 0.1 unit 55- to 65-kDa PNA-binding protein was observed in the neuraminidase (Wako Pure Chemicals) alone and with neuraminidase microsome fraction of lung tissue homogenate, suggesting its plus 20 milliunits endo-a-N-acetylgalactosaminidase (Seikagaku Co., Tokyo, Japan), respectively, at 37jC for 16 hours. Treatment of the microsome fraction with phosphatidylinositol-specific phospholipase C (PIPLC; Molecular Probes, Inc., Eugene, OR) was done in PBS contain- ing 0.5 units/mL enzyme at 37jC for 60 minutes. Affinity chromatography with PNA-agarose column. The microsome fraction from normal tissue was solubilized in TBS containing 1% Triton X-100 and protease inhibitor cocktail (lysis buffer A) at 4jC overnight and subsequently centrifuged at 100,000 Â g for 1 hour. The supernatant was
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