Identification and characterization of the CD226 gene promoter.

CD226 is one of the main activating receptors on natural killer cells, and it can induce cytotoxicity to target cells through interaction with its ligands CD155 or CD112. CD226 is also involved in T cell differentiation, activation, and cytotoxicity. The expression of CD226 on natural killer cells and T cells can be regulated by cytokines and chemical stimuli; however, the mechanism of the regulation of the CD226 gene is still unknown. In this study, we have identified two promoters in the human CD226 gene named P1 and P2, which are located at -810 to -287 bp and +33 to +213 bp, respectively, and a negative regulation element between P1 and P2. Both P1 and P2 can be regulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and calcium ionophore (A23187). Bioinformatics analysis shows that, within this CD226 gene region, there are putative binding sites for transcription factors AP-1, Sp1, PEA3, and Ets-1. We have found that transcription factor activating protein-1 (AP-1) can up-regulate CD226 promoters P1 and P2 in human hepatocarcinoma cells, a hepatocarcinoma cell line with low expression of endogenous AP-1 and Ets-1. Interestingly, the transcription factor Ets-1 promotes AP-1-induced P2 activity but inhibits AP-1-induced P1 activity for which a 10-bp AP-1/Ets-1 composite site (CCTTCCTTCC) in P1 may be responsible.

Generation of monoclonal antibodies to cancer/testis (CT) antigen CT10/MAGE-C2.

CT10/MAGE-C2 is a recently identified antigen that, typically of cancer/testis (CT) antigens, can be found in various malignant tumors and in normal adult testis. As with many other CT antigens, our knowledge is based mainly on mRNA expression data. In the present study, we describe the generation of mAbs to CT10/MAGE-C2 for the analysis of its protein expression. Newly generated clones were chosen based on their reactivity in ELISA, immunoblotting, and immunohistochemistry (IHC). Emphasis was put on the reactivity of newly generated reagents on formalin-fixed, paraffin-embedded tissue to ensure their applicability to archival material. Eventually we selected two clones, LX-CT10.5 and LX-CT10.9, that showed intense reactivity to CT10/MAGE-C2 protein and CT10/MAGE-C2 mRNA-positive cell lines, but no cross-reactivity with other CT antigens. Both mAbs show superior staining characteristics in IHC and are applicable to frozen and paraffin sections. In testis, CT10/MAGE-C2 displays the typical CT pattern with regard to staining of germ cells, which is intense during the early maturation stages. In tumors, we analyzed a limited number of cases displaying the typical heterogeneous CT expression pattern. Interestingly, immunoreactivity was seen solely in the nucleus: No staining was seen in the cytoplasm of tumor cells.

[Identification of cell lines expressing the putative ligand(s) for LAIR-1(CD305) and LAIR-2(CD306)].

AIM: To identify cell lines expressing the putative ligand(s) for LAIR-1 and LAIR-2. METHODS: CHO cell lines secreting LAIR-1-Fc or LAIR-2-Fc fusion protein were prepared and the supernatants from the CHO cell lines were collected and purified by protein A affinity chromatography column. Several cell lines were stained with the purified LAIR-1-Fc and LAIR-2-Fc fusion proteins and then analyzed by flow cytometry for detecting the expression of their putative ligand(s). To exclude non-specific binding between the cells and the fusion proteins, the cell lines expressing their putative ligand(s) were pre-incubated with the mAbs against LAIR-1 or LAIR-2, stained with the fusion proteins, and analyzed by flow cytometry. RESULTS: Both LAIR-1 and LAIR-2 fusion proteins bound strongly with human amnion-derived epithelial cell line WISH, and moderately with human melanoma cell line C32, human embryo kidney epithelial cell line 293T, and human umbilical vein endothelial cell line ECV304. Furthermore, this binding could be blocked by the mAb FMU-LAIR-2.2 (recognizing both LAIR-1 and LAIR-2) or mAb FMU-LAIR-2.1(recognizing LAIR-2). CONCLUSION: The putative ligand(s) for LAIR-1 and LAIR-2 were expressed on WISH, C32, 293T and ECV304 cell lines. LAIR-1 and LAIR-2 may share the same ligand(s). These findings lay the foundation for the molecular cloning of the putative ligand(s) for LAIR-1 and LAIR-2.