RESUMO
In order to identify membrane proteins whose expression is induced by X-ray irradiation, we developed an antibody (Ab)-directed strategy using a phage Ab library. X-Ray-irradiated cells were screened with a phage Ab library in the presence of a large excess of polyclonal Abs prepared against membrane proteins that are commonly present at the surface of both X-ray-irradiated and nonirradiated cells. After isolation of Ab that bound only to X-ray-irradiated cells, the antigen was identified using MS. Using this approach, we found that expression of LY6D is induced in MCF10A cells by X-ray irradiation. The induction of LY6D expression is triggered through a pathway regulated by ATM, CHK2 and p53. This method is a new Ab-directed proteomic strategy for analysis of membrane proteins, and is applicable to various biological phenomena in situations in which both target molecule-expressing cells and nonexpressing cells are available.
Assuntos
Moléculas de Adesão Celular/biossíntese , Raios X , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Humanos , Mutagênicos/toxicidade , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG(1) Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG(1) revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.