1. Walter Piper Dr. Andrew Buermeyer Department of Environmental and Molecular Toxicology Oregon State University 2009 HHMI Undergraduate Research Program Blue DNA – by Christopher Escabarte http://yethzart.deviantart.com/art/Blue-DNA-75781660 ©2008-2009 ~yethzartyethzart

2. Figure 1 – Colon Cancer and Polyp http://images.medicinenet.com/images/ILLUSTRATIONS/colon_ cancer.jpg MedicineNet.com  Alcohol (+80%-250%)  Smoking (+18%)  BMI (Body Mass Index)  (+50% in men when BMI>25. Less risk in women)  Diet  Genetic influence (+200%)  A 1 st degree relative with the cancer is one of the biggest risk factors.  The exact causes of this genetic risk are unknown.

3.  Hereditary  Sporadic 5% 95% Figure 2 – Percentage of total cases of CRC by type.

4.  Current CRC risk evaluation  family history  Cytotoxicity assay  physical basis to evaluate risk  Uses:  Large scale studies  Individual patient evaluation

5.  DNA mismatch repair (MMR) is a complex pathway involved in the maintenance of DNA integrity.  In the event that this repair process fails, a mutation will occur, which may lead to cancer.  MMR deficiency is linked to hereditary colorectal cancer. Figure 3 – DNA Mismatch Repair http://www.rndsystems.com/mini_review_detail_objectname_MR03_DNADamageResponse.a spx R&D Systems

6.  Moderate deficiencies present in the normal range of the population contribute to the genetic risk for sporadic CRC.  How can we accurately assess MMR proficiency?  Genetically  Biochemically  Cell-based cytotoxicity assay

7. Figure 4 MMR proteins in DNA-repair and DNA-damage-response pathways Figure 4 MMR proteins in DNA-repair and DNA-damage-response pathways Biochemical Society Transactions www.biochemsoctrans.org Biochem. Soc. Trans. (2005) 33, 689-693 Biochemical Society Transactions www.biochemsoctrans.org Biochem. Soc. Trans. (2005) 33, 689-693

8. Figure 5 – MNNG induced methylation and attempted mismatch repair leading to apoptosis.

9.  Use MTS to measure the relative surviving cell fraction following exposure to MNNG.  Relative cell survival should correlate with the level of MMR proficiency.  Does MMR proficiency correlate with the genetic risk for sporadic CRC?

10.  Cell survival is then measured using an MTS Assay.  Cells biochemically reduce the tetrazolium compound, MTS, into a colored formazan product that has an absorbance maximum at 490 nm.  The amount of formazan dye is determined by absorption of 490 nm UV light in a plate reader. Figure 6 – General reaction for the reduction of a tetrazolium to a formazan. http://en.wikipedia.org/wiki/F ile:Tetrazolium_reduction.png

11.  We are using immortalized lymphoblasts from blood samples to measure varying degrees of MMR proficiency in the general population.  Differing degrees of MMR proficiency can be characterized by consistent results showing a distinct level of cell survival.  Is the assay sensitive and precise enough to measure the slight differences in MMR proficiency?

12.  Culture human lymphoblastoid cells  Growth concentration: 50,000 – 1,000,000 cells/ml  Doubling time: ~21 hours  Treat cells in 96-well plates with the harmful mutagen MNNG  Cell cultures that are more MMR proficient undergo higher rates of apoptosis because they have a more effective DNA damage signaling pathway.  MTS assay is used to measure relative cell survival.

13.  Currently establishing conditions for cytotoxicity measurement using MTS assay, so an accurate cell count can be determined based on signal reading.  What kind of media is best? Standard RPMI media, media without phenol red, or media buffered with 25 mM HEPES? Figure 7 – Signal in plate reader vs. number of cells in a well.

14.  Validate assay with initial set of clinical samples.  Study the normal range of MMR proficiency in the general population.  Large-scale studies could also be done to definitively link MMR to sporadic CRC risk.

15.  Howard Hughes Medical Institute  Dr. Andrew Buermeyer  Vidya Schalk  Sarah Ferrer  Anneka Wickramanayake  Dr. Kevin Ahern