This proposal uses a new methodology to identify human genes that are required for tumor cell growth. Such genes, which provide potential targets for cancer treatment, will be identified through expression selection of genetic suppressor elements (GSEs). GSEs are biologically active sense- or antisense-oriented cDNA fragments that inhibit the function of the gene from which they are derived. Genes that are essential for cell proliferation are expected to give rise to GSEs that inhibit cell growth. Such GSEs can be isolated by bromodeoxyuridine (BrdU) suicide selection from a normalized (reduced-redundance) library of human cDNA fragments in an inducible retroviral vector. In preliminary studies, selection for growth-inhibitory GSEs has been carried out in breast carcinoma cells, yielding growth-inhibitory GSEs from about 60 genes. Many of the genes identified by GSE selection are known oncogenes or positive regulators of cell growth, while other genes have no known function or had not been previously implicated in cell proliferation. This analysis will now be extended to several other types of tumor and normal cells. A normalized cDNA fragment library in an inducible retroviral vector will be generated from a mixture of RNA preparations from multiple human tumor cell lines. This library will be transduced into recipient cell lines derived from several major types of human cancer and into telomerase-immortalized lines of normal human cells. Prior to transduction, the recipient cell lines will be derivatized to provide for high efficiency of retroviral infection and for the ability to regulate gene expression from retroviral vectors. The transduced cells will be subjected to BrdU suicide selection for growth-inhibitory GSEs, and GSE-enriched population of cDNA fragments will be recovered from the selected cells. Genes enriched by GSE selection will be identified by sequencing, and representative GSEs from each gene will be tested by several functional assays. The role of GSE-cognate genes in cell proliferation will be confirmed via siRNA inhibition. Genes identified through GSE selection will be prioritized as potential targets by comparing the ability of their cognate GSEs to inhibit cell growth in different types of tumor and normal cells and by analyzing the ability of the GSEs to induce tumor cell death through mitotic catastrophe. This analysis will provide a database of potential new targets for the development of anticancer drugs.
