Inactivation of genes normally involved in growth suppression may lead to malignant transformation. Some of these tumor suppressor genes have been identified but a large variety of cancer-associated genetic abnormalities suggests the existence of many presently unknown tumor suppressors. Identification and functional analysis of new tumor suppressors would be greatly enhanced by the ability to select genetic elements that induce a transformed phenotype through suppression of specific genes. A general methodology for production of efficient genetic suppressor elements (GSEs) is based on the isolation of biologically active random fragments of the target genes from expression libraries. The isolated GSEs encode either antisense RNA or dominant negative truncated proteins. This approach was first used in mammalian system to isolate a set of GSEs for human topoisomerase II; such GSEs cause resistance to cytotoxic drugs that interact with topoisomerase II. In an extension of this methodology, a large retroviral library (3 X 10 7 recombinant clones) carrying random fragments of normalized (uniform abundance) cDNA form mouse NIH 3T3 cells has been generated. This library has been already used to isolate several biologically active GSEs, derived from previously unknown genes and inducing drug resistance or transformation-associated phenotypic changes. The latter GSEs were derived from known and unknown genes that were not previously associated with negative growth control. Under the present proposal, the GSEs capable of inducing transformation or immortalization of mouse fibroblasts in vitro, or tumor formation in nude mice will be isolated from cell populations transduced with the same library or with a similar library prepared from primary corresponding mouse genes. To study the function of these putative tumor suppressors, phenotypes associated with their inhibition or overexpression will be analyzed in cell culture and in GSE-carrying transgenic mice. Expression of human homologs of the cloned mouse genes will be also analyzed in different tumors and normal tissues. Their chromosomal location will be determined to investigate their possible association with tumor-specific chromosomal rearrangements.
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