MOLECULAR ANALYSIS OF THE REGULATION PERTURBATION OF CELL CYCLE IN LUNG CANCER

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This proposal is part of a joint effort (SPORE) to provide better methods of clinical diagnosis, prognosis and treatments for lung cancer patients by studying the molecular basis of the disease. This project interacts with many of the other projects of this SPORE by sharing results and similar technologies to produce a synergistic effort. The project utilizes the human tissue procurement, tissue bank and animal cores. The focus of this project is on the role of cell cycle regulation in the etiology of lung cancer. The hypothesis is that precise, biochemical knowledge of the alterations of cell cycle regulatory molecules in cancer cells is essential for understanding the etiology of all cancers. This knowledge can then be exploited for solving the clinical problems of lung cancer patients, who have very little hope treatment. Molecular analysis focuses on the family of cyclin-dependent kinase (CDKs) and inhibitors (CDIs) and important CDK substrates such as the Rb (Retinoblastoma) tumor suppressor. A biochemical analysis of cyclin D1- CDK6 complexes and two CDIs, the MTS1 and mTS2 (multiple tumor suppressor) genes, is being performed in order to investigate the mechanism of perturbed cell cycle regulation in NSCLC cells. This molecular analysis can also reveal additional molecules altered in NSCLC and provide the basis for potential prognosis and therapy. Potential gene therapies and a test of the hypothesis that overexpressed cyclin D1 and loss of MTS1-2 is important for aberrant proliferation in NSCLC is accomplished by the use of recombinant DNA technologies, which employ antisense cyclin D1 retroviruses, plasmids and synthetic oligodeoxynucleotides, as well as MTS1-2 expression plasmids. Liposome technologies are used to deliver the plasmids and synthetic oligodeoxynucleotides to NSCLC cells in vitro and in vivo. Nude rodents with xenografts of human tumor cells are used to test the efficacy of these genetic manipulations and therapies in vivo. Molecular analysis of human tumors, dysplastic tissues and sputum samples are screened with a large panel of molecular markers, including but is not limited, to, cyclin D1, CDKs, CDIs and Rb using PCR, PCR-SSCP, immunoblots and immunohistochemical analyses. In this manner, a systematic study of any genetic changes that occur during tumor progression is done. These genetic changes can then be correlated with pathological changes in the cells to produce a molecular model for tumor progression. Dysplastic human cells that have cell cycle alterations are transformed with a variety of oncogenes as an assay of their state of progression to provide direct experimental evidence for such a molecular model. This molecular model has the potential to be used as a monitor of tumor progression in patients by providing oncologists with both prognostic and diagnostic information.