The overall goal of this proposal is to increase knowledge of the early molecular events underlying the development of lung cancer. This will entail identifying molecular and immunoassays that can elucidate genetic and epigenetic alterations in lung tumor cells acquired by sputum sampling or bronchial brushing. Studies will then ascertain whether these genetic alterations can be detected in lung cells removed from human donors by these minimally-invasive methods before evidence of cancer. This latter information will be obtained by screening for genetic alterations in lung archival samples and tumor tissues obtained from Colorado Plateau uranium miners from 1960 to the present. Finally, preliminary studies will be initiated to determine which genetic alterations detected in human lung tumors are also present in a rodent model system where one can match molecular events with discrete stages of lung cancer evolution. The gene dysfunction assays will be centered around immunocytochemical, in situ hybridization, and polymerase chain reaction-based techniques. Expression of gene products for c-raf-1, c-myc, c-myb, ErbB, ErbB-2, p53, Rb, TGF- alpha, PDGF, MHC-1, and CEA will be discerned. The frequency for mutation of the K-ras and p53 genes and for loss of heterozygosity at chromosomal loci 13q14, 17p13, 3p21, 11p13-15, and trisomy 7 will also be examined. Initially, human lung cells with one or more of the genetic alterations described above will be mixed at varying concentrations with cells that are normal for the parameter being assessed. Cells will be prepared under conditions identical to those used for collection and preservation of specimens acquired by sputum sampling or bronchial brushing for cytological examination. Conditions established in these reconstruction experiments will then be applied toward examining gene dysfunctions in sputum samples, bronchial brushings obtained from donors at the Johns Hopkins University and University of New Mexico Hospitals, and in archival samples collected from the uranium miners of the Colorado Plateau. These latter studies will determine whether aberrant gene functions can be detected in sputum samples prior to the onset of rank disease. This research provides a basis for defining the significant genetic and epigenetic alterations that arise prior to any obvious change in cell morphology and may also advance our understanding of the sequence that aberrations arise during the evolution of a normal lung epithelial cell into its malignant counterpart.
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