SPRL--A MOLECULAR MARKER FOR BRONCHIAL PRENEOPLASIA

描述(申请者描述)：该计划旨在 确立申请人为生物标记物研究的独立调查者 在肺癌的发生、早期发现和化学预防中的作用 上呼吸道癌症。申请人以前有过 在分子和细胞药理学方面的研究经验，最近 成为一名成熟的内科肿瘤学家，专门治疗 肺癌。赞助商是一位有成就的肺部生理学家， 发育和分化领域的分子生物学家 支气管上皮，以及在调节生物标志物的领域 支气管上皮对环境毒素如臭氧和 烟草烟雾。发起人的计划是监督 申请人在分子技术、设计和技术方面获得专门知识 进行实验以阐明调节A蛋白的机制 与支气管化生相关的分子标志物SPR1，以及在 该生物标志物在肺癌早期检测中的应用。 赞助商已经克隆了一个富含脯氨酸的小蛋白(spr-1)，它是 化生过程中气管、支气管壁上皮的过度表达 从正常的粘液纤毛表型到鳞状外观。SPR1 维生素A下调表达，肿瘤上调表达 推动者。因此，SPR1可能是一个潜在的早期生物标志物。 用于支气管上皮的癌前转化。在他们的 实验室里，有一系列的人支气管上皮细胞展示 不同程度的SPR1表达和致瘤性。这 因此，该提案旨在利用这种细胞培养模型来阐明 SPR1在多步致癌中的表达调控机制 并使用档案标本来绘制SPR1图 肺癌患者肺组织癌变过程中的表达 肺癌。 第一个目标是检验这一假设，即 SPR1在癌变过程中的表达 支气管鳞癌向周围不典型增生、化生和 免疫组织化学染色和免疫组织化学方法检测正常支气管上皮 人肺鳞状细胞癌的原位杂交研究。第二 目的是使用人类气管、支气管细胞模型来检验这一假说 SPR1的细胞类型特异性表达在 通过使用核连续转录分析和一个 SPR1启动子区域嵌合构建的转基因研究 依附于一种记者基因。第三个目标是确定顺式要素 以及参与SPR1基因调控的转录因子 利用DNA结合迁移率变化凝胶分析技术， 足迹和表达克隆。 这些研究是为将SPR1用作生物标志物奠定基础 识别多步致癌的早期步骤，以便早期发现 治疗肺癌，并测试其疗效和机制。 化学预防药物逆转卵巢癌恶变的研究 支气管上皮细胞。

DESCRIPTION (Applicant's Description): This program is designed to establish the applicant as an independent investigator of biomarker research in bronchial carcinogenesis, and in early detection and chemoprevention of cancers of the upper aerodigestive tract. The applicant has previous research experience in molecular and cellular pharmacology and has recently become an established medical oncologist specializing in the treatment of lung cancer. The sponsor is an accomplished pulmonary physiologist and molecular biologist in the field of development and differentiation of bronchial epithelium, and in the area of regulation of biomarkers of the bronchial epithelium in response to environmental toxins such as ozone and tobacco smoke. The plans of the sponsor are to provide supervision to the applicant to acquire expertise in molecular techniques, in the design and execution of experiments to elucidate the mechanisms of regulation of a molecular marker, spr1, associated with bronchial metaplasia, and in the application of this biomarker for early detection of lung cancer. The sponsor has cloned a small proline-rich protein (spr-1) which is overexpressed in tracheobronchial epithelium undergoing metaplastic change from a normal mucociliary phenotype to a squamous appearance. The spr1 expression is down-regulated by vitamin A and up-regulated by tumor promoters. Therefore, it appears that spr1 is a potential early biomarker for preneoplastic transformation of the bronchial epithelium. In their laboratory, there is a series of human bronchial epithelial cells displaying varied degree of spr1 expression and tumorigenicity potential. This proposal is, thus, intended to use this cell-culture model to elucidate the mechanism of regulation of spr1 expression in the multistep carcinogenesis of the bronchial epithelium, and to use archival specimens to map spr1 expression in the field of cancerization of lung tissues from patients with lung cancer. The first aim is to test the hypothesis that there is an increasing expression of spr1 in the field of cancerization extending from a focus of bronchial squamous carcinoma to the surrounding dysplastic, metaplastic and normal bronchial epithelia as determined by immunohistochemical staining and in situ hybridization of archival human squamous lung carcinoma. The second aim is to use a human tracheobronchial cell model to test the hypothesis that the cell type-specific expression of spr1 is regulated at the level of transcription by using a nuclear run-on transcriptional assay and a transfection study with chimeric constructs of the spr1 promoter region attached to a reporter gene. The third aim is to identify the cis-elements and the transcriptional factors involved in the regulation of spr1 gene by employing the techniques of DNA-binding mobility shift gel assay, DNA footprinting and expression cloning. These studies are to set the stage for using spr1 as a biomarker for identifying the early steps of multistep carcinogenesis, for early detection of lung cancer, and for testing the efficacy and mechanisms of chemopreventive agents in reversing malignant transformation of the bronchial epithelium.