HerbolsheimerPia.pptVIP

Future Directions Identification of TSGs in breast cancer using NMD microarray technique Determine the role of EphB2 in other cancer types Search for other mechanisms for biallelic inactivation (methylation) Further prioritization by comparing candidate genes to expression data on all human genes, looking for genes with loss of expression Gene ontology First round of mutation analysis on cell lines, and if nonsense mutations found, then further validation with clinical tumor DNA Laborious, expensive, risky Initial screening using only few tumors/cell lines per each cancer type If hits are found, then screen a larger tumor material DNA methylation is an epigenetic event that alters gene expression by covalent addition of a methyl group Hypermethylation occurs at CpG sites (cytosine-phosphate-guanine sites) often found near gene promoter Hypermethylation of CpG sites inactivates the remaining wild type allele Identification of hypermethylation done by pyrosequencing Research Collaborators Georgetown University Hospital: Robert Clarke Leena Hilakivi-Clarke Nancy Dawson Translational Genomics Research Institute: Spyro Mousses VTT Technical Research Centre and Univ. Turku, Finland: Maija Wolf Olli Kallioniemi Pia Herbolsheimer (formerly Huusko), MD, PhD Georgetown University Hospital Identification of New Tumor Suppressor Genes in Breast and Prostate Cancer TSG inactivation- “Two-hit hypothesis” Biallelic inactivation is typical for tumor suppressor genes Normal Mutation Deletion Gene Amplification Normal DNA Tumor DNA Pollack et al., Nat Genet. 1999 Monni et al., PNAS. 2002 Hyman et al., Cancer Res. 2002 Comparative Genomic Hybridization (CGH) on cDNA microarray Alterations in Cancer Cell Line Genome: Alignment of Chromosomal and Microarray Based CGH Amplifications: Activated oncogenic genes Deletions: Inactivated (tumor suppressor) genes Nonsense mediated RNA decay (NMD) Premature termination codons (nonsense mutations) result in truncated transcripts NMD pa