Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I

Journal of Experimental & Clinical Cancer Research, Feb 2011

Background This study aimed to investigate the molecular structural relationship between cell adhesive molecule CD44 and Lewis y antigen, and determine the effects of Lewis y antigen on CD44-mediated adhesion and spreading of ovarian cancer cell line RMG-I and the Lewis y antigen-overexpressed cell line RMG-I-H. Methods The expression of CD44 in RMG-I and RMG-I-H cells before and after treatment of Lewis y monoclonal antibody was detected by immunocytochemistry; the expression of Lewis y antigen and CD44 was detected by Western Blot. The structural relationship between Lewis y antigen and CD44 was determined by immunoprecipitation and confocal laser scanning microscopy. The adhesion and spreading of RMG-I and RMG-I-H cells on hyaluronic acid (HA) were observed. The expression of CD44 mRNA in RMG-I and RMG-I-H cells was detected by real-time RT-PCR. Results Immunocytochemistry revealed that the expression of CD44 was significantly higher in RMG-I-H cells than in RMG-I cells (P < 0.01), and its expression in both cell lines was significantly decreased after treatment of Lewis y monoclonal antibody (both P < 0.01). Western Blot confirmed that the content of CD44 in RMG-I-H cells was 1.46 times of that in RMG-I cells. The co-location of Lewis y antigen and CD44 was confirmed by co-immunoprecipitation. The co-expression of CD44 and Lewis y antigen in RMG-I-H cells was 2.24 times of that in RMG-I cells. The adhesion and spreading of RMG-I-H cells on HA were significantly enhanced as compared to those of RMG-I cells (P < 0.01), and this enhancement was inhibited by Lewis y monoclonal antibody (P < 0.01). The mRNA level of CD44 in both cell lines was similar (P > 0.05). Conclusion Lewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells.

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Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I

Journal of Experimental & Clinical Cancer Research Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I Lili Gao 0 Limei Yan 0 Bei Lin 0 Jian Gao 0 Xiuyun Liang 0 Yanyan Wang 0 Juanjuan Liu 0 Shulan Zhang 0 Iwamori Masao 1 0 Department of Obstetrics and Gynecology, Shengjing Hospital Affiliated to China Medical University , Shenyang, 110004, P R of China 1 Departments of Biochemistry, Faculty of Science and Technology, Kinki University , Osaka, 577-8502 , Japan Background: This study aimed to investigate the molecular structural relationship between cell adhesive molecule CD44 and Lewis y antigen, and determine the effects of Lewis y antigen on CD44-mediated adhesion and spreading of ovarian cancer cell line RMG-I and the Lewis y antigen-overexpressed cell line RMG-I-H. Methods: The expression of CD44 in RMG-I and RMG-I-H cells before and after treatment of Lewis y monoclonal antibody was detected by immunocytochemistry; the expression of Lewis y antigen and CD44 was detected by Western Blot. The structural relationship between Lewis y antigen and CD44 was determined by immunoprecipitation and confocal laser scanning microscopy. The adhesion and spreading of RMG-I and RMG-I-H cells on hyaluronic acid (HA) were observed. The expression of CD44 mRNA in RMG-I and RMG-I-H cells was detected by real-time RT-PCR. Results: Immunocytochemistry revealed that the expression of CD44 was significantly higher in RMG-I-H cells than in RMG-I cells (P < 0.01), and its expression in both cell lines was significantly decreased after treatment of Lewis y monoclonal antibody (both P < 0.01). Western Blot confirmed that the content of CD44 in RMG-I-H cells was 1.46 times of that in RMG-I cells. The co-location of Lewis y antigen and CD44 was confirmed by coimmunoprecipitation. The co-expression of CD44 and Lewis y antigen in RMG-I-H cells was 2.24 times of that in RMG-I cells. The adhesion and spreading of RMG-I-H cells on HA were significantly enhanced as compared to those of RMG-I cells (P < 0.01), and this enhancement was inhibited by Lewis y monoclonal antibody (P < 0.01). The mRNA level of CD44 in both cell lines was similar (P > 0.05). Conclusion: Lewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells. - Background Glycosylated antigens, important components of glycolipids and glycoproteins, are widely expressed on cell membrane and are involved in cell adhesion, recognition, and signal transduction [1]. The alterations of type II sugar chains, such as Lewis and Lewis y, are common in ovarian cancer: 75% of epithelial ovarian cancers have overexpression of Lewis y antigen which shows obvious relationship with prognosis; tumor marker CA125 in epithelial ovarian cancer also contains Lewis y structure [2,3]. Alpha1, 2-fucosyltransferase (a1, 2-FT) is a key enzyme for synthesizing Lewis y antigen. In our previous study, we successfully transferred a1, 2-FT gene into ovarian cancer cell line RMG-I and established a cell line RMG-I-H with stable high expression of Lewis y antigen, which showed obviously enhanced malignant behaviors [4-6]. CD44, one of important adhesive molecules on cells, is involved in the adhesion and metastasis of tumor cells and plays an important role in tumor development [7-10], but the regulatory mechanism is unclear yet. The molecule CD44 is abundant of a-L-fucose, and is an important a1, 2-fucose antigen-containing protein on the surface of cells [11]. CD44 is expressed on several tissue cells, binds to receptors in extracellular matrix such as hyaluronic acid (HA) and laminin, and mediates cell-cell and cell-matrix adhesion [12,13]. The present study aimed to determine the impact of a1, 2-FT gene transfection on the expression of CD44 on cells and the effects of Lewis y antigen on CD44-mediated cell adhesion and spreading. Methods Materials Lewis y monoclonal antibody was purchased from Abcam Co.; CD44 monoclonal antibody from Santa Cruz Co. and Wuhan Boster Co.; Protein A-agarose, ECL chromogenic agent, and 5 SDS-PAGE loading buffer from Shanghai Beyotime Institute of Biotechnology; SABC kit from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd; HA from Hefei Bomei Biotechnology Co., Ltd; DMEM culture medium from Gibco Co.; fetal bovine serum (FBS) from Shenyang Boermei Reagent Co.; Coomassie brilliant blue from Beijing Solarbio Science & Technology Co., Ltd; Trizol reagent, PrimeScriptRT reagent kit, and SYBR Premix Ex Taqfrom Dalian TaKaRa Biotechnology Co. The sequences of primers were synthesized by Shanghai Invitrogen Co. Cell line and cell culture The cell line RMG-I was originated from ovarian clear cell cancer tissues. The cell line RMG-I-H with high expression of a1, 2-FT and Lewis y antigen was established in our lab [14]. RMG-I and RMG-I-H cells were cultured in DMEM medium containing 10% FBS at 37C in 5% CO2 and saturated humidity. Cells are grouped in immunocytochemistry, c (...truncated)


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Lili Gao, Limei Yan, Bei Lin, Jian Gao, Xiuyun Liang, Yanyan Wang, Juanjuan Liu, Shulan Zhang, Iwamori Masao. Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I, Journal of Experimental & Clinical Cancer Research, 2011, pp. 15, 30, DOI: 10.1186/1756-9966-30-15