Gene amplification, the generation of extra copies of a gene(s), is a common and critically important genetic defect in oral cancer (OSCC) cells. Amplification of chromosomal band 11q13, which harbors the cyclin D1 gene (CCND1) and other genes, appears to be a relatively early event in oral carcinogenesis, occurs in -45% of oral cancers, and is an indicator of poor prognosis. The mechanism underlying gene amplification has been largely unexplored in oral cancer and consequently, is not well understood. Therefore, a new approach is indicated. Based on our recent studies of oral cancer cells, we posit that 11q13 amplification in oral cancer occurs by breakage-fusion-bridge (BFB) cycles. We observed a predictable pattern of organization, an """"""""amplification fingerprint"""""""" consistent with BFB cycles, characterized by an inverted duplication chromosome pattern at 11 qI 3, with genes from the segment just distal to the proximal chromosomal breakpoint duplicated and flanking both sides of the amplified genes in the amplicon. In addition, we observed amplified CCND1 in anaphase bridges between nascent oral cancer cells. To test our hypothesis, we will carry out three Specific Aims. 1. To use our novel quantitative PCR technique (quantitative microsatellite analysis, QuMA) to prepare a high-resolution copy number map of the 11q13 amplicon in OSCC cells and primary tumors, define the minimal amplified region and the genes located therein, and determine whether breakpoints are clustered at """"""""hotspots."""""""" To identify BAC clones that span the amplicon breakpoints and validate our QuMA data using fluorescence in situ hybridization (FISH), revealing the structural organization of the amplicon and whether it fits the BFB model or an alternative model. 2. To use FISH, to show that the 11 qi 3 amplicon is localized to anaphase bridges. 3. To use Northern Blots and TaqMan quantitative RT-PCR to determine transcription patterns and the expression level of genes and novel ESTs in and around the amplicon in OSCC cells and tumors. Our data suggest that the most highly amplified segment includes expressed new genes, with CCND1 amplified to a lesser extent. The results of this study may lead to targeted methods for prevention, early detection, therapy, and/or eradication of cells harboring gene amplification.
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