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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

我描述了利用不同蛋白质组技术寻找肺神经内分泌癌标志物的最新结果。1．详尽的单克隆抗体生产：我们使用肺癌细胞作为免疫原生产单克隆抗体。从一组获得的抗体中，呈现了针对活化 C 激酶 1 (RACK1) 受体的所述抗体。 RACK1在腺癌中表达显着高且频繁，但在少数鳞状细胞癌和大细胞癌中几乎检测不到。此外，RACK1表达还与腺癌患者的病理分期、肿瘤大小和淋巴结状态显着相关。这些结果表明RACK1可能是肺腺癌的新型鉴别诊断标志物。 2.癌细胞系条件培养基中肽的检测：为了鉴定肺神经内分泌肿瘤的新标志物，我们对LCNEC细胞系LCN1分泌到条件培养基中的肽进行了全面分析。通过 MALDI-TOF/TOF MS 鉴定了 40 和 19 个氨基酸残基的两个肽片段。这两个片段被证明是 VGF 的一部分。对肺癌细胞系的 RT-PCR 分析表明，vgf 仅在神经内分泌癌来源的细胞中表达。我们的数据表明 VGF 可以成为肺神经内分泌肿瘤的新型血清诊断标志物。 3.二维凝胶电泳的应用：我们利用二维凝胶电泳（2-DE）和MULDI-TOF/TOF MS研究了两种细胞系（源自小细胞肺癌（SCLC）的N231和源自肺大细胞神经内分泌癌（LCNEC）的LCN1）的细胞裂解物的蛋白质组学方法。在 25 种已鉴定的蛋白质中，与 N231 细胞相比，LCN1 细胞中的细胞角蛋白 (CK) 7、8、18 和 19 上调。对 81 例肺癌进行了免疫组织化学研究，研究了它们的表达。 LCNEC 病例中 CK7、8、18 和 19 的平均免疫染色评分显着高于 SCLC。这些数据表明LCNEC和SCLC的生物学特性可能不同，CKs的表达可能作为鉴别诊断标志物。 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法筛选来自SCLC患者的血清。为了鉴定 AA 识别的抗原，进行了二维凝胶电泳免疫印迹，并从膜和凝胶上切下目标点。通过这种方法，Villin1 被鉴定为 SCLC 患者血清中的 AA。 Villin1可能是区分LCNEC/AD与SCLC/SCC的有用标记，并且本方法可能有助于鉴定不同组织学类型的肺癌患者血清中的特定肿瘤相关分子。较少的