Microdissection-mediatedcDNA capture of human tumor genes.

Abstract

We describe a novel strategy for characterization of gene amplification in human neoplasia termed Microdisection Mediated cDNA Capture (MMcC). We demonstrated the utility of MMcC for cloning amplified genes including OSES-7, a novel potential oncogene. We further demonstrated the utility of MMcC to clone chromosome specific, single copy genes. Tumorigenesis is a multistep process that involves tumor suppressor inactivation and oncogene activation. One of several ways of oncogene activation is overexpression through gene amplification. DNA amplification in tumors is common and involves many regions not currently associated with known oncogenes. However, efforts to identify amplified genes have been very laborious because of the technical limitations of current expression mapping strategies. MMcC is based on targeting specific chromosome regions for microdissection after in situ hybridization of linkered cDNA libraries to metaphase chromosomes. This strategy allows the rapid generation of chromosome region-specific cDNAs without the need for ordered, overlapping, genomic clones of the region of interest. This makes MMcC invaluable for gene identification in amplified, rearranged DNA from tumors. We successfully used this strategy to isolate amplified 13 novel gene sequences from sarcoma and glioblastoma cell lines bearing amplified DNA. These genes significantly extended the expression map of the 12q13-15 amplicon associated with those tumors tumors. We isolated the full length cDNA for OSES-7, one of the amplified genes we identified, based on its sequence similarity to the oncogene Ost. We determined that OSES-7 was a novel member of the DBL family of oncogenes. Members of this family regulate the Rho family of GTPases involved in signal transduction cascades. These signal transduction pathways have been associated with regulation of cytokinesis, cell morphology and cell cycle progression. Thus, OSES-7 becomes an important candidate target gene for amplification with potential oncogenic properties. In addition to the unique advantage of MMcC for cloning amplified genes, we further demonstrated its value for single copy regions. We used MMcC on chromosome 6q and demonstrated, through radiation reduction hybrid panel mapping and Southern blot, successful isolation of chromosome-specific genes.