The objectives are to analyze the cellular, biochemical and genetic changes associated with progression of the transformed phenotype in cloned populations of type 5 adenovirus (Ad5) transformed rodent cells and in lectin resistant (""""""""glycosylation mutant"""""""") human melanoma cells stably expressing an increased metastatic potential in vivo. Based on experiments indicating that the progression phenotype in Ad5-transformed rat embryo cells in reversible and no systematic changes occur in either the transcription of Ad5-early genes or the levels of Ad5-transforming proteins in cells displaying various stages of progression, experiments will focus on determining the role of cellular genes (including oncogenes) in regulating the progression phenotype. Several approaches will be used to study this process, including: (a) somatic cell hybridization between cells displaying a progressed phenotype and normal, unprogressed or subclones of progressed cells treated with azacytidine and stably displaying an unprogressed phenotype; (b) DNA transfection experiments to determine if various oncogene products localized in the cytoplasm can complement the nuclear localized Ad5 E1a gene products resulting in progression; (c) high resolution 2-D protein gel electrophoresis to investigate the spectrum of protein changes associated with the acquisition and the reversion of the progressed phenotype; and (d) molecular cloning techniques to identify and clone potential suppressor genes which when expressed prevent progression of the transformed phenotype. Using the human melanoma model system, investigations will be conducted to determine the potential mechanism by which metastatic melanoma cells display the progressed phenotype. These studies will include: (a) evaluating the potential role of oncogene activation in determining the metastatic phenotype; (b) identifying specific protein changes by 2-D protein gel electrophoresis which occur concomitant with expression of the metastatic phenotype; and (c) DNA-transfection studies to transfer and identify human metastasis genes. The present investigations should permit a better molecular definition of the process of progression of the transformed phenotype in both rodent and human cells. In addition, since the carcinogenic process is often a multistep process the present studies should also provide insights into the molecular basis for carcinogenesis and potential mechanism for suppressing oncogenic transformation.
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