It is proposed to exploit a powerful and novel new method of selecting genes whose expression is altered in breast cancer. This method, invented by Dr. Arthur Pardee is called Differential Display (DD). DD and the even newer method, called Subtractive Display (SD) to be developed will be applied in my laboratory to the identification and selection of both TSGs (tumor suppressor genes) and oncogenes, on the basis of differential expression at the mRNA level. With this method virtually every expressed gene in a cell population can be displayed on a sequencing gel after PCR amplification. By comparing the banding patterns of mRNAs from normal and tumor cells, displayed on adjacent lanes of the gel, it is evident by eye which bands show differential expression. Two, three, or four cell lines can readily be compared simultaneously. Over-expressed bands can be recovered directly from the gel, further amplified by PCR, verified by Northern analysis, and sequenced to provide a partial sequence for identification in GenBank. Selected genes will be fully sequenced to obtain full-length cDNAs for analysis by transfection of candidate TSGs into tumor cells and candidate oncogenes into normal cells. This methodology will initially be applied to normal and tumor-derived mammary epithelial cells developed in my laboratory. The differentially expressed genes from these comparisons will provide a library for further investigation. A subset will be selected by criteria including consistent expression changes in a small test series of cell lines and primary invasive tumors detected at the mRNA level, and utilized for antibody production to examine expression at the protein level in an expanded set of neoplastic and pre-neoplastic tissues. Subsequently, with the development of a miniaturized procedure, tumor cells from staged surgical or fine needle biopsy specimens (Smith/Thor ROI) will be utilized directly to identify selected differentially expressed genes in primary tissues, and in micro metastases. Thus one focus of this application is to utilize the Pardee methodology to recover, clone, and sequence genes whose expression is altered in the early stages of breast cancer, using tissue-derived cells directly. Since some, presumably most of these genes will already be present in our library derived from normal and tumor cells grown in culture, the tissue studies will in addition validate use of cell cultures for functional studies of these genes. Initial screening of candidate TSGs and oncogenes will be carriend out by partial sequence analysis to identify known genes, relatives of known genes, as well as novel unknown genes. Selected genes will be screened by treatment of tumor cells with antisense oligonucleotides to look for growth inhibition in the set of potential oncogenes, overexpressed in tumor cells, and by treatment of normal cells with antisense to look for loss of the serum requirement for growth or by other growth proliferation assays.
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