The pathophysiological role of transforming genes in human breast cancer remains obscure largely because of the difficulty involved in obtaining sufficient amounts of homogeneous primary epithelium necessary to undertake the appropriate genetic studies. The unique resources of the program's Clinical and Cell Biology Cores can provide this project with substantial amounts of malignant and non-malignant epithelial cells obtained from the same patient and free of heterogeneous tissue elements such as macrophages, lymphocytes, and stromal cells which can obscure any attempt at fine molecular analysis. This project will focus on oncogene action in breast cancer by isolating dominant-acting transforming genes, screening for augmented expression of known proto-oncogenes, searching for undiscovered amplified DNA sequences, and introducing activated oncogenes to examine their effect on the phenotype of primary breast epithelial cells. DNA extracted from neoplastic and benign cells from the same patient will be cotransfected into NIH 3T3 cells and bioassayed for tumorigenicity in nude mice. The tumor and germline DNA will also be analyzed by an in-gel renaturation technique to detect any amplified DNA sequences present only in the patients' tumor specimens. RNA extracted from short-term cultures of benign and malignant breast epithelial cells will be screened by dot-blot and Northern analysis for differential oncogene expression using a panel of nearly 20 oncogene probes, and the DNA from those tumor samples showing increased oncogene RNA expression will be probed by Southern analysis to detect amplification of these specific oncogenes. Lastly, constructs containing activated oncogenes will be introduced into malignant and non-malignant primary breast epithelial cells looking for differential induction of phenotypic changes and cell immortalization. Hopefully these efforts will provide evidence for transforming gene expression in the pathogenesis and progression of breast cancer, and in so doing assist in the development of new genetic markers useful in the diagnosis and evaluation of this disease.
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