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Exhaustive proteome analyses of pulmonary neuroendocrine carcinomas

肺神经内分泌癌的详尽蛋白质组分析

基本信息

批准号：
19590365
负责人：
SATO Yuichi
金额：
$ 2.91万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2007
资助国家：
日本
起止时间：
2007 至 2009
项目状态：
已结题

项目摘要

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

我描述了用不同的蛋白质组技术寻找肺神经内分泌癌标志物的最新结果。穷举单克隆抗体生产：我们使用来自肺癌的细胞作为免疫原产生单克隆抗体。从一组获得的抗体中，本发明描述了活化C激酶1（RACK 1）受体的抗体。RACK 1在腺癌中的表达显著增高，但在少数鳞状细胞癌和大细胞癌中几乎未检测到。RACK 1的表达与腺癌患者的病理分期、肿瘤大小及淋巴结转移状态有关。提示RACK 1可能是肺腺癌的一个新的鉴别诊断标志物。2.癌细胞系条件培养液中多肽的检测：为了鉴定肺神经内分泌肿瘤的新标志物，我们综合分析了与肺神经内分泌肿瘤相关的多肽， ...更多信息由LCNEC系LCN 1分泌到条件培养基中。经MALDI-TOF/TOF MS鉴定，得到了40和19个氨基酸残基的两个肽段，这两个肽段是VGF的一部分。肺癌细胞系的RT-PCR分析显示，vgf仅在神经内分泌癌源性细胞中表达。我们的数据表明，VGF可以成为一种新的血清学诊断标记物的肺神经内分泌肿瘤。3.双向凝胶电泳的应用：我们研究了蛋白质组学的方法，使用细胞裂解液从两个细胞系（N231来源于小细胞肺癌（SCLC）和LCN 1来源于肺大细胞神经内分泌癌（LCNEC））。在25个鉴定的蛋白质中，细胞角蛋白（CK）7，8，与N231细胞相比，LCN 1细胞中18和19的表达上调。应用免疫组化方法检测81例肺癌组织中的表达。CK 7、8、18和19的平均免疫染色评分在LCNEC病例中显著高于SCLC。提示LCNEC和SCLC的生物学特性可能不同，CKs的表达可作为鉴别诊断的标志物。4.肺癌患者血清中自身抗体的检测：为了检测自身抗体（AA），使用四种细胞系的细胞裂解物通过免疫印迹筛选来自SCLC患者的血清。为了鉴定AA识别的抗原，免疫印迹二维凝胶电泳，并从膜和凝胶上切出靶点。用该方法对小细胞肺癌患者血清中的villin 1进行了鉴定。Villin 1可能是区分LCNEC/AD和SCLC/SCC的有用标志物，本方法可能用于鉴定不同组织学类型肺癌患者血清中的特异性肿瘤相关分子。少