MECHANISMS OF LUNG CANCER PREVENTION

肺癌患者的高死亡率和令人沮丧的5年 尽管治疗方式的进步， 迫切需要制定新的办法来防止 这种可怕的疾病。最近的临床试验显示， 作为癌症预防剂的类维生素A。这种潜力正在进一步 在这个聚焦于肺癌的项目中进行了探索。的目标 该项目旨在深入了解维甲酸对 正常、癌前和恶性支气管和肺组织和细胞 来补充临床研究据认为，维甲酸发挥 通过改变影响细胞的基因的表达， 生长和分化。类维生素A激活两种类型的核 类视色素受体（RAR和RXR），存在于三种亚型（α， β和γ），并作为转录因子。初步数据 表明RAR β水平在发育不良和恶性肺组织中降低 in vivo.这些和其他发现提出了假设，RAR β发挥作用， 在抑制癌发生中的作用，即下调 RAR β能够使癌前细胞逃脱监视， 生理水平的维甲酸，一些维甲酸可以在 药理学剂量克服了某些癌前病变的抵抗力， 细胞，也许通过增加RAR β表达。该项目乃设计 为了了解类维生素A受体特别是RAR β的参与， 在肺癌发生中的作用首先，它们的表达将在 肺癌的手术样本， 项目l患者在用13-顺式-RA和α- 生育酚，并在单层和器官型培养的正常支气管 上皮细胞、癌前细胞和肺癌细胞。的 受体的mRNA将通过原位杂交和北方 印迹为了检查类维生素A对肺细胞的调节， 天然和合成类维生素A的作用， 受体类型对类维生素A受体表达的影响，以及 正常支气管的增殖、凋亡和分化 上皮细胞、永生化支气管细胞和恶性肺细胞 在单层培养物、器官型培养物和半固体培养基中 将被确定。增殖将通过细胞计数来确定， BrdU掺入、DNA梯状条带诱导凋亡、DNA末端标记 免疫组化法进行分化 和细胞角蛋白7和13的Western印迹分析。以确定 RAR β表达或阻断是否改变了癌前病变的反应， 类维生素A对肺癌细胞生长的影响， 分化，正义和反义RAR β都将被引入 瞬时或以稳定的方式通过逆转录病毒载体进入细胞， 组成性表达或不表达该受体， 将检测细胞对类维生素A的反应。最后，确定 RAR β是否在致瘤性、肺癌细胞 表达外源性RAR β的细胞和表达外源性RAR β的细胞。 将被反义RAR β阻断的细胞皮下注射到 将测定免疫受损的小鼠的肿瘤发生率和它们的致瘤性。这些 预计研究将增加对参与的了解， RAR β在细胞对不同类维生素A的反应中的作用及其在 肺癌细胞的致瘤性。此外，新的类维生素A 可以确定在化学预防试验中的潜在应用。

The high mortality rate of lung cancer patients and the dismal 5-year survival rate despite the advancement in treatment modalities are the major reasons for the urgent need to devise novel approaches to prevent this dreadful disease. Recent clinical trials revealed the potential of retinoids as cancer preventive agents. This potential is being further explored in this program project focusing on lung cancer. The objective of this project is to provide insights into mechanisms of retinoid actions on normal, premalignant, and malignant bronchial and lung tissues and cells to complement the clinical studies. It is thought that retinoids exert their actions by changing the expression of genes that influence cell growth and differentiation. Retinoids activate two types of nuclear retinoid receptors (RARs and RXRs), which exist in three subtypes (alpha, beta, and gamma) each and act as transcription factors. Preliminary data show that RARbeta level decreases in dysplastic and malignant lung tissue in vivo. These and other findings raised the hypotheses that RARbeta plays a role in suppression of carcinogenesis, that the down regulation of RARbeta enables premalignant cells to escape the surveillance by physiological levels of retinoids, and that some retinoids can at pharmacological doses overcome the resistance of certain premalignant cells, perhaps by increasing RARbeta expression. This project is designed to understand the involvement of retinoid receptors in particular RARbeta, in lung carcinogenesis. First, their expression will be determined in surgical samples of lung carcinomas, biopsies of bronchial tissue from Project l patients before and after treatment with 13-cis-RA and alpha- tocopherol, and in monolayer and organotypic cultures of normal bronchial epithelial cells, premalignant cells, and lung carcinoma cells. The receptors' mRNAs will be analyzed by in situ hybridization and Northern blotting. To examine the modulation of lung cells by retinoids, the effects of natural and synthetic retinoids with preferences for distinct receptor types on the expression of retinoid receptors, and on proliferation, apoptosis, and differentiation of normal bronchial epithelial cells, immortalized bronchial cells, and malignant lung cells in monolayer cultures, in organotypic cultures, and in semisolid medium will be determined. Proliferation will be determined by cell counting and BrdU incorporation, apoptosis by DNA ladder formation, end labeling of DNA termini, and flow cytometry, and differentiation by immunohistochemical and Western blotting analyses of cytokeratins 7 and 13. To determine whether RARbeta expression or blockade alter the response of premalignant cells and lung carcinoma cell to effects of retinoids on cell growth and differentiation, both the sense and antisense RARbeta will be introduce transiently or in a stable fashion by retroviral vectors into cells that either express or do not express this receptor constitutively and the responses of the cells to retinoids will be examined. Lastly, to determine whether RARbeta plays a role in tumorigenicity, lung carcinoma cells expressing exogenous RARbeta and cells in which RARbeta expression was blocked by antisense RARbeta will be injected subcutaneously into immunocompromised mice and their tumorigenicity will be determined. These studies are expected to increase the understanding of the involvement of RARbeta in the cellular responses to different retinoids and its role in the tumorigenicity of lung carcinoma cells. In addition new retinoids with potential application in chemoprevention trials may be identified.