Suppression Subtractive Hybridization for Identification and Functional Analysis of Tumor Suppressor

Alterations leading to inactivation of tumor suppressor genes are often associated with reduced levels of tumor suppressor gene expression. Specific tumors or cell lines may exhibit deletion of one or both gene copies, promoter methylation, splice-site mutations, nonsense mutations that induce premature translational termination and destabilize mRNA transcripts, or a combination of these. Such mutations result in complete absence or partial reduction in levels of tumor suppressor mRNA. Therefore, cDNA subtraction has been frequently used for identification of candidate tumor suppressor genes (1 –7 ). As multiple tumor suppressor genes function in signaling pathways that regulate expression of oncogene transcription factors (8 ), subtraction methods are also useful for identification of downstream targets of these pathways. For example, embryo cells can be derived from mice engineered to be deficient in both alleles of a tumor suppressor. Then primary cell cultures can be analyzed by comparison with littermate control cells for identification of candidate target genes.