Exhaustive proteome analyses of pulmonary neuroendocrine carcinomas

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I describe recent results of the search for pulmonary neuroendocrine carcinoma markers with diverse proteome techniques.1. Exhaustive monoclonal antibodies production: We generated monoclonal antibodies using cells derived from pulmonary carcinomas as an immunogen. From a group of obtained antibodies, present described antibody for the receptor of activated C kinase 1 (RACK1). The expression RACK1 was significantly high and frequent in adenocarcinomas but was barely detected in a few squamous cell carcinomas and large cell carcinomas. Moreover, RACK1 expression was also significantly associated with the pathological stage, tumor size and lymph node status of adenocarcinoma patients. These results suggest that RACK1 may be a novel differential diagnostic marker for pulmonary adenocarcinomas. 2. Detection of peptides in the conditioned medium from carcinoma cell lines: Aiming to identify new markers of pulmonary neuroendocrine tumors, we analyzed comprehensively analyzed peptides which w … More ere secreted into the conditioned medium by LCN1, an LCNEC line. Two peptide fragments of 40 and 19 amino acid residues were identified by MALDI-TOF/TOF MS. These two fragments were demonstrated to be parts of VGF. RT-PCR analysis of lung cancer cell lines showed that vgf was expressed only in neuroendocrine carcinoma-derived cells. Our data suggest that VGF can be a novel sero-diagnostic marker of pulmonary neuroendocrine tumors. 3. Application of two-dimensional gel electrophoresis: We studied the proteomic approach using cell lysate from two cell lines (N231 derived from small cell lung carcinoma (SCLC) and LCN1 derived from pulmonary large cell neuroendocrine carcinoma (LCNEC)) with two-dimensional gel electrophoresis (2-DE) and MULDI-TOF/TOF MS. Within the 25 identified proteins, cytokeratins (CK) 7, 8, 18 and 19 were up-regulated in LCN1 cells compared with N231 cells. Their expressions were studied immunohistochemically with 81 pulmonary carcinomas. The mean immunostaining scores of CK7, 8, 18, and 19 were significantly higher in LCNEC cases than in SCLC. These data suggest that the biological characteristics of LCNEC and SCLC may be different and the expressions of CKs may serve as differential diagnostic markers. 4. Detection of autoantibodies in sera of patients with lung cancer: To detect autoantibodies (AAs), sera from SCLC patient was screened by immunoblotting using cell lysate of four cell lines. To identify the antigens recognized by AAs, two-dimensional gel electrophoresis was immunoblotted and target spots were cut out from the membrane and gel. By this method, villin1 was identified with AA in sera from patients with SCLC. Villin1 may be useful markers to distinguish LCNEC/AD from SCLC/SCC, and the present method might be useful to identify specific tumor-associated molecules in sera from pulmonary carcinoma patients with different histologic types. Less