Applications of novel monoclonal antibodies specific for synuclein-γ in evaluating its levels in sera and cancer tissues from colorectal cancer patients
Introduction
Synucleins are a family of small highly charged neuronal proteins consisting of three known members, synuclein-α (SNCA), synuclein-β (SNCB), and synuclein-γ (SNCG). Synuclein genes map to 4q21.3-q22 (SNCA), 5q35 (SNCB), and 10q23 (SNCG), respectively. The structural differences are likely to mediate functional variations by its unique C-terminal tail [1]. Synuclein-α has been extensively characterized due to its role in neurodegenerative diseases, including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. In these diseases, the normally soluble synuclein-α assembles into abnormal filaments, which is inhibited by synuclein-β and -γ [2]. synuclein-γ gene, also called BCSG1 [3] or persyn [4], is composed of five exons, which encode a 127 amino acid protein and shares 54% and 56% amino acid sequence identity with SNCA and SNCB, respectively. SNCG expression is restricted to the brain and periphery neuronal tissues [5]. However, its aberrant expression beyond neuronal system is highly associated with human malignancies [6], [7], particularly breast cancer and ovarian cancer. Several studies have shown that SNCG was abnormally expressed in a high percentage of advanced and metastatic breast tumors but not in normal or benign breast tissues [6], [7]. Overexpressed SNCG stimulates proliferation and induces metastasis of breast cancer cells [8], [9]. Further investigations have shown that SNCG overexpression in breast cancer cells resulted in a compromised mitotic checkpoint [10], accelerated rate of chromosomal instability [11], increased resistance of tumor cells to certain chemotherapeutic drugs [13] or anti-microtubule agents [12]. Down-regulation of SNCG expression sensitized breast cancer cells to anti-microtubule drug-induced cytotoxicity [12]. The inhibitory effects of SNCG on mitotic checkpoint function may play an important oncogenic role in human cancers besides breast or ovarian carcinomas [11].
Previous studies have shown that SNCG is aberrantly expressed in diversified cancer types, including liver, esophagus, colon, gastric, lung, prostate, cervical, and breast cancer, but rarely expressed in tumor-matched non-neoplastic adjacent tissues, and expressions of SNCG protein in different cancer types positively correlated with stages of diseases [3], [6], [14], [15]. Recent studies demonstrate that SNCG is an independent predictive marker for recurrence and metastasis in breast cancer progression by detecting SNCG mRNA levels [16] or protein levels of primary tumor tissues [17]. However, SNCG protein expressions in other human cancers except breast cancer have not been systematically examined. The previous studies on SNCG expression are based on mRNA [16], [18], [19], [20] or/and protein levels using anti-SNCG polyclonal antibodies [10], [11], [13], [15], [16], [18], [19], [20], [21], [22], [23]. Although SNCG mAbs made previously by our laboratory can be used in immunohistochemical analysis for breast cancer [17], these SNCG mAbs can not specifically recognize endogenous SNCG in other immunoassays. Detection of SNCG protein in cell lysates and supernatant of CRC cell lines has not been verified. In the present study, we generated a panel of SNCG-specific mAbs and developed a sandwich ELISA for SNCG. With these novel SNCG mAbs, we found elevated levels of SNCG in CRC cell lines, serum samples and tumor tissues from CRC patients. Our results validate the clinical applications of these mAbs and suggest that SNCG is a potential biomarker for CRC.
Section snippets
Antigens and chemical reagents
Recombinant human GST-SNCG, GST-SNCA, and GST-SNCB were purified and characterized as previously described [17]. Molecular weight standards were purchased from Bio-Rad Canada (Mississauga, Ontario, Canada). Protein-A Sepharose, protein-G Sepharose, HRP-conjugated anti-mouse IgG, and the enhanced chemiluminescence (ECL) Western blot system were obtained from Amersham Pharmacia Biotech Inc.
Cell lines and culture conditions
SP2/0 myeloma cells, breast cancer cell lines MCF-7 and T47D, colorectal cancer cell lines HCT-116, RKO,
Generation of anti-SNCG mAbs and characterization of their specificities
After fusing spleenocytes from GST-SNCG immunized mouse with SP2/0 myeloma cells, 2880 clones were initially tested for SNCG binding by indirect ELISA. Forty-five clones showed good reactions with SNCG. Among them, 10 hybridoma clones stably secreting specific anti-SNCG mAbs were established for further characterization, and the mAbs from these hybridomas were designated as 1# (IgG2a), 9# (IgG1), 11# (IgG1), 14# (IgG2b), 18# (IgG1), 22# (IgG2b), 31# (IgG1), 36# (IgG2a), 42# (IgG1), and 211#
Discussion
Study in SNCG had been hampered by the lack of monoclonal antibodies with high specificity and affinity against endogenous SNCG. In this study, we generated a panel of SNCG mAbs recognizing both exogenous and endogenous SNCG. These mAbs can be utilized in ELISA, Western blot, immunoprecipitation, immunocytochemistry, and immunohistochemistry. By using these antibodies, we show for the first time that SNCG proteins can be secreted extracellularly in SNCG transfected cell (MCF7-SNCG) line, which
Conflict of interest statement
Authors report no conflict of interest.
Acknowledgements
This work was supported by Foundation of Capital Medicine Development of Beijing (2002-2023, 2005-3051), “985 program” of Peking university and 863 High Technology Program (2006AA02A249). The authors thank Dr. Jiafu Ji of Cancer Tissue Bank and Dr. Aiping Lu of Pathology Department of Beijing Cancer Hospital for providing the serum and tissue specimens.
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