SPRL--A MOLECULAR MARKER FOR BRONCHIAL PRENEOPLASIA

描述（申请人的描述）：该计划旨在 使申请人成为生物标志物研究的独立研究者 在支气管癌发生中，以及在早期发现和化学预防中， 上呼吸消化道的癌症。 申请人以前 在分子和细胞药理学方面的研究经验， 成为一名成熟的医学肿瘤学家，专门治疗 肺癌 申办者是一位有成就的肺生理学家， 发展和分化领域的分子生物学家 支气管上皮，以及在调节支气管上皮细胞的生物标志物的领域中， 支气管上皮细胞对环境毒素如臭氧和 烟草烟雾。 赞助商的计划是为 申请人获得分子技术，在设计和 执行实验，以阐明调节的机制， 与支气管化生相关的分子标志物spr 1， 该生物标志物用于肺癌的早期检测。 申办者克隆了一种富含脯氨酸的小蛋白（spr-1）， 在发生化生的气管支气管上皮中过度表达 从正常的粘膜纤毛表型变为鳞状外观。 spr1 表达受维生素A的下调和肿瘤的上调 发起人。 因此，spr 1似乎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 在实验室中，有一系列人类支气管上皮细胞显示 不同程度的spr 1表达和致瘤潜力。 这 因此，该建议旨在使用这种细胞培养模型来阐明 spr 1在多步骤癌变过程中的表达调控机制 的支气管上皮细胞，并使用档案标本映射spr 1 在来自患有肺腺癌的患者的肺组织的癌变领域中的表达 肺癌 第一个目的是检验一个假设，即 spr 1在癌灶延伸的癌变区域中的表达 支气管鳞状细胞癌向周围异型增生、化生和 通过免疫组织化学染色确定的正常支气管上皮， 人肺鳞癌组织原位杂交研究 第二 目的是使用人气管支气管细胞模型来验证这一假设 spr 1的细胞类型特异性表达是在 通过使用核连续转录测定和 用spr 1启动子区嵌合构建体的转染研究 连接到一个报告基因上 第三个目的是确定顺式元件 以及参与spr 1基因调控的转录因子， 采用DNA结合迁移率变动凝胶分析技术， 足迹和表达克隆。 这些研究将为使用spr 1作为生物标志物奠定基础， 识别多步骤致癌的早期步骤，以便早期检测 肺癌，并测试的疗效和机制， 化学预防剂在逆转恶性转化中的作用 支气管上皮

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.