MECHANISMS OF LUNG CANCER PREVENTION

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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.