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PhIP-induced Mutation in Mlh1 -deficient Mice

PhIP-induced Mutation in Mlh1 -deficient Mice

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PhIP-induced Mutation in Mlh1 -deficient Mice

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  1. PhIP-induced Mutation in Mlh1-deficient Mice Stacy Hill Stacy Hill Buermeyer Lab Group

  2. Significance • 2-5% of all colorectal cancer cases in humans result from a genetic predisposition called hereditary nonpolyposis colorectal cancer (HNPCC). • HNPCC is the most commonly inherited form of colorectal cancer. • Those who have inherited HNPCC have an 80% risk of developing an early onset internal cancer, such as colorectal cancer. Stacy Hill Buermeyer Lab Group

  3. HNPCC Syndrome & Mlh1 • HNPCC is a syndrome caused by inherited defects in DNA Mismatch Repair (MMR). • There are 4 different MMR genes linked to HNPCC, Mlh1 and Msh2 being the most common. • Most HNPCC individuals have normal MMR activity in their cells, due to the fact that they are heterozygous for their MMR mutation. • In the rare event that an individual is born with no proficient MMR activity, the individual usually dies by the age of 5 years. Stacy Hill Buermeyer Lab Group

  4. HNPCC Syndrome • Individual cells that become homozygous deficient for Mlh1 are at high risk for becoming cancers. Stacy Hill Buermeyer Lab Group

  5. Risk Factors Of HNPCC • Little is known about the modulation of cancer risk by environmental factors, such as heterocyclic amines (HCA). • Mlh1-/- mice can be used to identify modulators of cancer risk associated with MMR deficiency. • Research Question: Does HCA exposure actually increase the risk of cancer development in an individual with MMR deficiency? Stacy Hill Buermeyer Lab Group

  6. What Are Heterocyclic Amines? • Heterocyclic amines (HCA) are the carcinogenic chemicals formed from the cooking of muscle meats such as beef, pork, fowl, and fish. • HCA form when amino acids and creatine (a chemical found in muscles) react at high cooking temperatures. • Frying, broiling, and barbecuing produce the largest amounts of HCA because the meats are cooked at very high temperatures. Stacy Hill Buermeyer Lab Group

  7. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) PhIP is the most abundant HCA, which we are currently using for analysis. MMR deficient cells have been shown to be hypermutable by exposure to PhIP in cell culture. What Are We Afraid Of? Stacy Hill Buermeyer Lab Group

  8. Wildtype Mice • PhIP • Vehicle • Mlh1-/- Mice • PhIP • Vehicle Experimental Model • We use genetically engineered mice to model cancer predisposition in individuals with MMR deficiency. Stacy Hill Buermeyer Lab Group

  9. PhIP vs. Vehicle Mutagenesis Tumorigenesis Cell Turnover Study Design Stacy Hill Buermeyer Lab Group

  10. Research Focus • Mutagenesis • The level of mutations, induced by PhIP, in both wildtype (normal) mice and in Mlh1-/- mice. • Mlh1-/- or “knockout” mice are Mlh1-deficient. • Mutations resulting from PhIP exposure are believed to be a result of the formation of PhIP-DNA adducts, primarily to guanine residues. Stacy Hill Buermeyer Lab Group

  11. Characterizing PhIP-induced Mutations • A recoverable lambda shuttle vector, previously integrated into the mouse genome, will be used to characterize mutations. • Illustration of the mouse chromosome. Stacy Hill Buermeyer Lab Group

  12. Isolating Genomic DNA Mutation Selection Phage Packaging G1250 E.coli infection Recovering Lambda Phage Stacy Hill Buermeyer Lab Group

  13. The Selection Process Stacy Hill Buermeyer Lab Group

  14. Data Analysis • All of the plates are then collected and the number of visible plaques, or plaque forming units (pfu) are counted, to determine mutation frequency (MF). • MF = # of pfu 24ºC ‘# of pfu 37ºC x Dilution Factor • Plaques are then purified and the cII gene is sequenced. Then mutations are determined. Stacy Hill Buermeyer Lab Group

  15. Results (Mutation Frequency) • PhIP-induced mutations: • Wildtype 6 x 10-5 • Knockout 47 x 10-5 Stacy Hill Buermeyer Lab Group

  16. Results (cII Gene Map) Stacy Hill Buermeyer Lab Group

  17. Results (Mutation Spectrum) Stacy Hill Buermeyer Lab Group

  18. PhIP, and perhaps other HCA, exposure may increase cancer risk in people with MMR deficiencies. Summary • Mlh1-/- mice were hypersensitive to PhIP-induced mutations in the colon. • Most PhIP-induced mutations in both wildtype and knockout mice involved G/C base pairs. • Preliminary data suggest Mlh1-/- mice are hypersensitive to PhIP-induced carcinogenesis. Stacy Hill Buermeyer Lab Group

  19. Acknowledgements • Howard Huges Medical Institute (HHMI) • Undergraduate Research Innovation Scholarship Creativity (URISC) • Stephanie Smith-Roe • Andrew Buermeyer • Kevin Ahern Stacy Hill Buermeyer Lab Group