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 loaded onto a PNA-agarose column equilibrated with lysis buffer A. After washing with lysis buffer A, the bound proteins were eluted with buffer containing 0.2 mol/L D-galactose. Immunoprecipitation with anti-DAF antibody. The microsome frac- tion from the nontumor region was sonicated in TBS containing 2% Triton X-100, 1 mmol/L EDTA, and protease inhibitor cocktail and then incubated at 4jC overnight. The soluble fraction was recovered by centrifugation at 100,000 Â g for 1 hour and mixed with anti-DAF antibody (4F11) at 0jC for 1 hour. Protein G–coupled Sepharose slurry (Sigma Chemical) was added to the mixture and incubated at 0jC for 30 minutes. The bound fraction was recovered with SDS-PAGE sample buffer. ELISA to quantify soluble DAF. NSCLC tissue was gently homoge- nized in a Potter-type homogenizer. The 100,000 Â g supernatants were incubated in a microtiter plate coated with anti-DAF monoclonal antibody (4F11). Plates were further incubated with biotin-conjugated anti-DAF monoclonal antibody (1C6) and horseradish peroxidase– conjugated avidin (DAKO), and the color was developed using ABTS

(Zymed, San Francisco, CA) and H2O2. Fig. 1. A, PNA lectin blotting of lung microsome fractions prepared from normal Reverse transcription-PCR of DAF, membrane cofactor protein (CD46), (N)andtumor(T) regions of NSCLC tissue. Two representative cases of patients and CD59. The expression levels of DAF, membrane cofactor protein with adenocarcinoma. B, PNA lectin blotting of the microsome fraction treated with (MCP; ref. 19), and CD59 (20) were assessed by reverse transcription- (+) and without (À)PIPLC(left)andendo-a-N-acetylgalactosaminidase (right). PCR with mRNA isolated from NSCLC tissue using Isogen (Nippon The microsome fraction used was prepared from a normal region of NSCLC tissue. C, levels of 55- to 65-kDa PNA-binding protein in lung tissue. Microsome fractions Gene Co. Ltd., Tokyo, Japan). from normal and tumor regions of 24 patients with NSCLC were subjected to PNA Real-time PCR was also done to estimate DAF and h-actin mRNA in lectin blotting followed by image analysis.The level is represented as an arbitrary an ABI Prism 7900 sequence detection system (Applied Biosystems, unit. Columns, mean; bars, SD. *, P = 0.0002.

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Table 1. Relationship between clinicopathologic variables and the level of 55- to 65-kDa PNA-binding protein and DAF in 24 patients with NSCLC

Clinicopathologic variable No. patients P 55- to 65-kDa PNA-binding protein DAF Age (y) <70 12 >70 12 0.281 0.841 Gender Male 15 Female 9 0.065 0.817 Tumor size (cm) <3 12 >3 12 0.471 0.414 Nodal involvement Negative 14 Positive 10 0.277 0.694 Pathologic stage I 10 II-IV 14 0.188 0.684 Differentiation Well differentiated 13 Not well differentiated 11 0.871 0.228 Lymphatic factor Negative 11 Positive 13 0.895 0.905 Venous factor Negative 12 Positive 12 0.928 0.879 Pleural invasion Negative 14 Positive 10 0.252 0.570 Histology Adenocarcinoma 19 Others* 5 0.267 0.659 Extent of resection Lobectomy 20 Othersc 4 0.407 0.491

*Squamous cell carcinoma and large cell lung carcinoma. cBilobectomy and pneumonectomy. localization on the membranes. Treatment of the lung micro- detected a 55- to 65-kDa band in the eluate. Furthermore, anti- some fraction with endo-a-N-acetylgalactosaminidase resulted DAF antibody pulled down a 55- to 65-kDa band in the in a complete loss of the 55- to 65-kDa PNA-binding protein immune precipitate from the solubilized microsome fraction (Fig. 1B), indicating that this protein possesses O-linked glycans (Fig. 2B). These results clearly indicate that one of the core recognized by PNA. Treatment with PIPLC significantly reduced proteins of the 55- to 65-kDa PNA-binding protein is DAF. the amount of 55- to 65-kDa PNA-binding protein in the Increased molecular size and down-regulation of DAF in microsome fraction (Fig. 1B). These results indicate that the NSCLC tissue. In almost all cases of primary NSCLC, DAF core protein is a glycosylphosphatidylinositol-anchored mem- was expressed as a 55- to 65-kDa protein in the nontumor tissue, brane protein with O-linked carbohydrates. The 55- to 65-kDa whereas it was expressed at a larger molecular weight of 65 to 75 PNA-binding protein was not detected in peripheral blood or kDa in the tumor lesions (Fig. 3A). Treatment of the microsome in peripheral hemocytes, indicating that this protein is not a fraction with neuraminidase resulted in reduction of the component of peripheral blood that contaminated the lung molecular weight of DAF to 55 kDa, both in the nontumor tissue specimen (data not shown). No 55- to 65-kDa PNA- and tumor tissue (Fig. 3B), suggesting the involvement of sialic binding protein was detected in nontumor tissue or tumor acid residue. DAF was observed at a larger size of 65 to 75 kDa in tissue from colorectal and stomach cancer (data not shown). a tumor environment using DAF-overexpressed PC14 cells As shown in Fig. 1C, the levels of the 55- to 65-kDa PNA- (Fig. 3C). Treatment of the transformants with neuraminidase binding protein were significantly lower in NSCLC tumor tissue resulted in a reduction in size to 55 kDa, like native DAF. than in nontumor tissue from the same specimens (P = 0.0002; In 20 of the 24 cases of NSCLC, the amount of DAF was n = 24). No clear correlation was found between the level of significantly reduced in tumor tissue compared with nontumor 55- to 65-kDa PNA-binding protein and clinicopathologic tissue (Fig. 4A). The soluble form of DAF, which is detached variables, such as tumor size, International Union Against from the membrane or secreted from lung cells as a soluble Cancer staging, and histology of NSCLC (Table 1). isoform (14, 22, 23), also decreased in tumor tissue (Fig. 4B). Identification of DAF as a core protein of the 55- to 65-kDa The level of membrane DAF showed no clear correlation with PNA-binding protein. To identify the core peptide of the the clinicopathologic variables (Table 1). These results were in 55- to 65-kDa PNA-binding protein, the microsome fraction close agreement to the PNA lectin blotting findings. As shown from lung tissue was solubilized and subjected to affinity in Fig. 4C, a correlation was observed between the level of chromatography with a PNA-agarose column. The eluate with 55- to 65-kDa PNA-binding protein and DAF (r = 0.582; P < D-galactose from the column revealed a major band of 55- to 0.001). Eighteen of the 24 cases that showed a decrease of 65-kDa by PNA lectin blotting (Fig. 2A). Based on the structural 55- to 65-kDa PNA-binding protein showed a similar decrease features of the 55- to 65-kDa PNA-binding protein described of DAF in tumor tissue. DAF mRNA, assessed by a real-time above, we suspected DAF (CD55) as a candidate protein. As PCR, was lower in the tumor tissue compared with normal shown in Fig. 2A, Western blotting with anti-DAF antibody tissue (Fig. 4D), suggesting that DAF is down-regulated in

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Fig. 2. Identification of DAF as the core protein of 55- to 65-kDa PNA-binding protein. A, PNA lectin blotting and Western blotting of bound (B) and unbound (UB) fractions of the solubilized lung microsome to PNA-agarose. B, PNA lectin blotting and Western blotting of immunoprecipitates of the solubilized microsome pulled down with (Ab+) and without (Ab À) anti-DAF antibody.

NSCLC tissue at the transcription level. When expression levels of the other complement regulatory proteins were assessed by reverse transcription-PCR, it was found that MCP mRNA was

Fig. 4. A, DAF levels in normal and tumor regions of NSCLC tissue. DAF in the microsome fraction from 24 patients with NSCLC was quantified by Western blotting followed by image analysis.The level is expressed as an arbitrary unit. Columns, mean; bars, SD. *, P =0.0056.B, levels of soluble DAF in the soluble fractions of NSCLC tissue that were determined by ELISA. **, P =0.018(n =20). C, correlation between the levels of 55- to 65-kDa PNA-binding protein and DAF in the microsome fraction. Open, closed, and gray circles, levels in the normal tissue regions from patients with adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, respectively. Open, closed, and gray squares, corresponding tumor regions. D, DAF mRNA levels estimated by real-time PCR in the normal and tumor regions of NSCLC tissue. DAF mRNA levels are presented as a ratio of DAF mRNA to h-actin mRNA. Columns, mean; bars, SD. ***, P = 0.003. E, expression of DAF, MCP, and CD59 estimated by reverse transcription-PCR in normal and tumor regions of NSCLC tissue.The levels of DAF, MCP, and CD59 mRNA were evaluated by reverse transcription-PCR. Multiple minor bands were observed for DAF and MCP, which have been identified as splicing variants generated from the DAF and MCP genes, respectively (19, 22).Typical case of a patient with adenocarcinoma.

Fig. 3. A, Western blotting of DAF in microsome fractions of normal and tumor regions of lung tissue from patients with adenocarcinoma (adeno), squamous cell carcinoma (Sq), and large cell carcinoma (Large). B, reduced molecular mass of decreased in tumor tissue, but CD59 mRNA in tumor tissue was DAF by selective removal of sialic acid. The microsome fraction prepared from the not significantly different from normal tissue (Fig. 4E). normal region of NSCLC tissue was treated with (Neu +) and without (Neu À) As shown in Fig. 5, after immunohistochemical staining, DAF neuraminidase. C, Western blotting of DAF in the cell lysate of DAF-overexpressed PC14 cells (Tx+) before (Neu À)andafter(Neu +) treatment with neuraminidase. was localized to the alveolar epithelial cells in the normal lung, TxÀ, native PC14 cells without transfection. whereas tumor cells were not or very weakly stained. In the

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Discussion

The present study showed that PNA recognized a structure of O-linked carbohydrates on DAF. It is likely that PNA recognizes the limited molecular species of DAF because DAF band detected by Western blot was broader than that seen by PNA lectin blot (Figs. 1-3), although these results might be also explained by the different sensitivities of the two methods. In almost all NSCLC tissues, the expression level of DAF was Fig. 5. Immunostaining of DAF in the normal and tumor regions of NSCLC tissue. reduced compared with normal lung tissue. A similar down- Typical result from a patient with adenocarcinoma. DAF staining is observed in alveolar epithelial cells in normal tissue (A), whereas blood cells and connective regulation was observed in ovarian cancer (24, 25), whereas an tissue cells are positive in tumor tissue (B). up-regulation was reported in other malignancies, such as colorectal cancer and gastric cancer (26–30). From the suggested that the molecular mass of DAF of 65 kDa in normal viewpoint of immunologic surveillance, it is reasonable to tissue consists of 42% total sugars, including 15% sialic acids, suggest that, in a tumor environment, cells are exposed to an and that the molecular mass of DAF of 75 kDa in tumor lesions augmented complement attack and therefore generate high consists of 49% total sugars, including 27% sialic acids. This levels of DAF to protect themselves. The dynamics of DAF in clearly indicates that sialylation in the O-linked sugar is NSCLC tissue require a very different explanation. This down- responsible for the enlarged molecular weight of DAF in regulation did not correlate with any specific clinicopathologic NSCLC tissue. It is unknown how the activity of DAF is variables, suggesting that it is a basal feature of NSCLC. A clue modified by sialylation. for a possible explanation might lie in the characteristics of Using histochemical PNA staining, we previously reported sialylated DAF, which should be able to execute its function for that the expression of PNA-recognizing carbohydrates correlat- longer periods than the regular form because of its high ed with lymph node metastasis in human lung adenocarcino- resistance against proteolysis (31). The highly sialylated DAF is ma tissue (8). This observation implies that the net amount of retained on the cell surface for a long period, and further PNA-binding carbohydrates increased in tumor lesions and biosynthesis of DAF might be suppressed in these tumor cells. correlated with the metastasis of tumor cells. At present, we Another explanation could lie in the architecture of NSCLC have no evidence about whether the increased level of 55- to tissue. Unlike normal lung cells, the tumor cells formed a bulky 65-kDa PNA-binding protein and/or DAF is responsible for the cluster of tissue where the inside was not faced to alveolar cavity high frequency of metastasis in NSCLC because we observed a (Fig. 5), therefore possibly do not need a high expression of decrease of DAF in NSCLC and no correlation between PNA- complement regulatory proteins. binding protein/DAF level and clinicopathologic variables, Interestingly, a similar down-regulation was observed for including nodal involvement. In addition, in in vitro chemo- MCP, another complement regulatory protein of the regulators invasion assays, we observed that the metastatic activity of DAF- of the complement activation family. MCP has a transmem- overexpressed PC14 cells was not statistically different from brane protein with a domain structure very similar to DAF. It is that of nontransfected cells (P = 0.058; n = 6). Thus, although likely that DAF and MCP are regulated in a similar manner in DAF is one of the PNA-binding proteins on the surface of lung NSCLC tissue. However, it is unclear about whether MCP is cells, its expression alone seems to have no significant effect on another 55- to 65-kDa PNA-binding protein, although a small the metastatic property of NSCLC. amount of PNA-binding protein was left on the cell membrane In conclusion, we report a new 55- to 65-kDa PNA-binding after PIPLC treatment (Fig. 1B). protein in normal lung tissue where the core protein was iden- It is of special interest that a larger size DAF was present in tified as DAF, CD55. In NSCLC tissue, DAF was down-regulated tumor tissue of NSCLC and that the larger molecular weight at the transcription level and processed to a larger molecule was due to sialylation of the sugar moiety. In general, larger through sialylation of the sugar moiety. Our results indicate that molecular weight glycoproteins are known as Warren-Glick the reduced expression and heavy sialylation of DAF was asso- phenomenon in cancer (32–35), a finding considered due to ciated with basal pathology of NSCLC and that these altera- increased amounts of N-linked glycans by N-acetylglucosami- tions of DAF make this protein a potential marker for NSCLC. nyltransferase (36). DAF has an N-linked sugar that is essential for its function (14), but it contributes little to the molecular Acknowledgments weight. In the present study, neuraminidase treatment resulted in reduction of the molecular weight to 55 kDa. Based on the We thank Professor Tsukasa Seya (Hokkaido University, Sapporo, Japan) for calculated mass of the peptide portion of DAF of 38 kDa, it is valuable suggestions and discussions.

References 1. Zebda N, Bailly M, Brown S, Dore JF, Berthier- 2. NovogrodskyA, Lotan R, Ravid A, Sharon N. Peanut associated cytostructural antigenic alterations. Cancer Vergnes O. Expression of PNA-binding sites on agglutinin, a new mitogen that binds to galactosyl 1981;47:2872 ^ 7. specific glycoproteins by human melanoma cells is sites exposed after neuraminidase treatment. J Immu- 4. Pereira ME, Kabat EA, Lotan R, Sharon N. Immuno- associated with a high metastatic potential. J Cell nol 1975;115:1243 ^ 8. chemical studies on the specificity of the peanut Biochem1994;54:161^73. 3. Howard DR, Ferguson P, Batsakis JG. Carcinoma- agglutinin. Carbohydr Res 1976;51:107 ^18.

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5. Cochran AJ, Wen DR, Behthier-Vergnes O, et al. 15. Nicholson-WellerA, MarchJP,Rosen CE, Spicer DB, 26. Mizuno M, Nakagawa M, Uesu T, et al. Detection Cytoplasmic accumulation of peanut agglutinin- Austen KF. Surface membrane expression by human of decay-accelerating factor in stool specimens of binding glycoconjugates in the cells of primary mela- blood leukocytes and platelets of decay-accelerating patients with colorectal cancer. Gastroenterology noma correlates with clinical outcome. Hum Pathol factor, a regulatory protein of the complement system. 1995;109:826^ 31. 1999;30:556^61. Blood 1985;65:1237 ^ 44. 27. Koretz K, Bruderlein S, Henne C, Moller P. Decay- 6. Kellokumpu I, Kellokumpu S, Andersson LC. Identifi- 16. WHO. Histological typing of lung and pleural accelerating factor (DAF, CD55) in normal colorectal cation of glycoproteins expressing tumor-associated tumours. WHO international histological classification mucosa, adenomas and carcinomas. Br J Cancer PNA-binding sites in colorectal carcinomas by SDS- of tumours. 3rd ed. Geneva: WHO; 1999. 1992;66:810 ^ 4. GEL electrophoresis and PNA-labelling. Br J Cancer 17. Laemmli UK. Cleavage of structural proteins during 28. Niehans GA, Cherwitz DL, Staley NA, Knapp DJ, 19 87;55 :3 61 ^ 5. the assembly of the head of bacteriophageT4. Nature Dalmasso AP. Human carcinomas variably express the 7. Takanami I. Expression of T homsen-Friedenreich 1970;227:680 ^ 5. complement inhibitory proteins CD46 (membrane antigen as a marker of poor prognosis in pulmonary 18. Ohya S, Mizuno M, Kawada M, et al. Improvements cofactor protein), CD55 (decay-accelerating factor), adenocarcinoma. Oncol Rep 1999;6:341 ^ 4. in the measurement of stool decay-accelerating factor and CD59 (protectin).AmJPathol1996;149:129^ 42. 8. Suzuki H, Kawaguchi T, Higuchi M, et al. Expression in the detection of colorectal cancer. Acta Med 29. Inoue T, Yamakawa M, Takahashi T. Expression of of peanut agglutinin (PNA lectin) binding carbohy- Okayama 2002;56:171 ^ 6. complement regulating factors in gastric cancer cells. drates correlates with nodal involvement in human 19. Lublin DM, Liszewski MK, Post TW, et al. Molecular MolPathol2002;55:193^9. lung adenocarcinoma. Cancer Lett 2002;187:215^ 21. cloning and chromosomal localization of human 30.Varsano S, Rashkovsky L, Shapiro H, Radnay J. 9. Wu W, Harley PH, Punt JA, Sharrow SO, Kearse KP. membrane cofactor protein (MCP). Evidence for inclu- Cytokines modulate expression of cell-membrane Identification of CD8 as a peanut agglutinin (PNA) sion in the multigene family of complement-regulatory complement inhibitory proteins in human lung cancer receptor molecule on immature thymocytes. J Exp proteins. J Exp Med 1988;168:181 ^ 94. cell lines. Am J Respir Cell Mol Biol 1998;19:522^ 9. Med 1996;184:759 ^ 64. 20. Davis A, Simmons DL, Hale G, et al. CD59, an 31. Reddy P, Caras I, Krieger M. Effect of O-linked 10. Schopperle WM, Armant DR, Dewolf WC. Purifica- LY-6-like protein expressed in human lymphoid cells, glycosylation on the cell surface expression and sta- tion of a tumor-specific PNA-binding glycoprotein, regulates the action of the complement membrane bility of decay-accelerating factor, a glycophospholi- gp200, from a human embryonal carcinoma cell line. attack complex on homologous cells. J Exp Med pid-anchored membrane protein. J Biol Chem 1989; Arch Biochem Biophys 1992;298:538 ^ 43. 1989;170:637 ^ 54. 264:17329^ 36. 11. MasuzawaY, MiyauchiT, Hamanoue M, et al. A novel 21. Shindo-Okada N, Takeuchi K, Nagamachi Y. Estab- 32. Buck CA, Glick MC, Warren L. Effect of growth on core protein as well as polymorphic epithelial mucin lishment of cell lines with high- and low-metastatic the glycoproteins from the surface of control and Rous carry peanut agglutinin binding sites in human gastric potential from PC-14 human lung adenocarcinoma. sarcoma virus transformed hamster cells. Biochemis- carcinoma cells: sequence analysis and examination Jpn J Cancer Res 2001;92:174 ^ 83. try 1971;10:2176 ^ 80. of gene expression. J Biochem 1992;112:609 ^ 15. 22. Osuka F, Endo Y, Higuchi M, et al. Molecular clon- 33. Buck CA, Glick MC,Warren L. A comparative study 12. HudsonDL,SleemanJ,WattFM.CD44isthe ing and characterization of novel splicing variants of glycoproteins from the surface of control and Rous peanut lectin-binding glycoprotein of human epider- of human decay-accelerating factor (DAF, CD55). sarcoma virus transformed hamster cells. Biochemis- mal keratinocytes and plays a role in intercellular Genomics 2006;88:316^ 22. try 1970;9:4567 ^ 76. adhesion. J Cell Sci 1995;108:1959 ^ 70. 23. Caras IW, Weddell GN. Signal peptide for protein 34. Buck CA, Glick MC, Warren L. Glycopeptides from 13. Coyne KE, Hall SE,Thompson ES, et al. Mapping of secretion directing glycophospholipid membrane an- the surface of control and virus-transformed cells. epitopes, glycosylation sites, and complement regula- chor attachment. Science 1989;243:1196 ^ 8. Science 1971;172:169 ^ 71. tory domains in human decay accelerating factor. 24. Bjorge L, Hauklinen J, Wahlstrom T, et al. Comple- 35.Warren L, Critchley D, Macpherson I. Surface glyco- J Immunol 1992;149:2906 ^ 13. ment-regulatory proteins in ovarian malignancies. Int proteins and glycolipids of chicken embryo cells trans- 14. Medof ME, Walter EI, Rutgers JL, Knowles DM, J Cancer 1997;70:14^ 25. formed by a temperature-sensitive mutant of Rous Nussenzweig V. Identification of complement decay- 25. Macor P, Mezzanzanica D, Cossetti C, et al. Com- sarcoma virus. Nature 1972;235:275 ^8. accelerating factor (DAF) on epithelium and glandular plement activated by chimeric anti-folate receptor 36. Kobata A. Altered glycosylation of surface glyco- cells and in body fluids. J Exp Med 1987;165: antibodies is an efficient effector system to control proteins in tumor cells and its clinical application. 848^64. ovarian carcinoma. Cancer Res 2006;66:3876 ^ 83. Pigment Cell Res 1989;2:304 ^ 8.

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Mitsunori Higuchi, Yuichi Endo, Hiroyuki Suzuki, et al.

Clin Cancer Res 2006;12:6367-6372.

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