Pathogenic Mechanisms of Cancer Causing hMLH1 Mutations Functional Relationship between DNA Mismatch Repair and Tumor Formation Eddie O’Donnell Laboratory of Dr. Andrew B. Buermeyer Department of Environmental and Molecular Toxicology

2003 Estimated US Cancer Deaths ONS=Other nervous system. *Excludes basal and squamous cell skin cancers and in situ carcinomas except urinary bladder. Source: American Cancer Society, Men 285,900 Women 270,600 25%Lung & bronchus 15%Breast 11%Colon & rectum 6%Pancreas 5%Ovary 4%Non-Hodgkin lymphoma 4%Leukemia 3%Uterine corpus 2%Brain/ONS 2%Multiple myeloma 23%All other sites Lung & bronchus31% Prostate10% Colon & rectum10% Pancreas5% Non-Hodgkin4% lymphoma Leukemia4% Esophagus4% Liver/intrahepatic3% bile duct Urinary bladder3% Kidney3% All other sites22%

Colon Cancer Causes of Cancer Mutations within cells cause uncontrolled cell growth Cancer may be inherited or sporadic Colorectal Cancer 15 % - Mismatch Repair (MMR) deficiency observed 90 % of Sporadic cases linked to MMR deficiency are mlh1 deficient (loss of expression) 2-5 % - Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Recent Discoveries involving HNPCC 1993 – MSH2 mutations linked to 35% of HNPCC 1994 – hMHL1 mutations linked to 35 % of HNPCC Non Hereditary HNPCC, FAP

DNA Mismatch Repair DNA Mismatches arise from errors during DNA Replication MMR is an essential process for maintaining genomic integrity Basic Mechanism: Mismatch recognition Strand choice Excision Resynthesis G T T T A G T *

DNA Synthesis ErrorMutation Base Mismatches  Base Substitution Mutations Insertion / Deletion Loops  Microsatellite Instability (MSI) Microsatellite loci are common and unique to a persons genome Looking specifically at dinucleotide repeated sequences Deficient MMR can lead to increased probability of replication errors Mutation Types GTGT No Repair, Continued Replication ATAT ATAT GCGC Incorrect DNA sequence, mutation in genome copy Incorrect Placement of Base Successful Repair Dinucleotide Loop Insertion AC TG No Repair, Continued Replication Incorrect DNA sequence, mutation in genome copy

hMLH1 significance hMLH1 is an essential protein for the prevention of mutations. Exact function is unknown. Treatments MMR deficient cancers may respond differently to chemotherapeutic drugs. Detection MMR deficient cancers are commonly detected through screening for MSI, however… Several hMLH1 Mutations have been implicated in HNPCC cases showing only high levels of base substitution mutations: not initially identified as MMR deficient cancers. Clinical relevance of the hMLH1 gene E578G K618A D132H V716M hMLH1 amino acid site 578 changed from E (glutamic acid) to G (Glycine)

Preliminary Research with hMLH1 E578G Data from analysis of cells expressing E578G demonstrate: No MSI, consistent with observed cancers Increased levels of base substitution mutations E578G mutation affects repair of base base mismatches but not dinucleotide loops Increased base substitutions may explain pathogenicity of mutations suggests a possible novel role for MMR repair genes, containing MLH1, in substrate recognition and / or commitment to initiate repair

Research Questions & Goals The goal of the research is to determine the mutation prevention capabilities of the MLH1 mutants E578G, K618A, V716M, and D132H using biochemical assays Are mutations of hMLH1 responsible for the observed molecular phenotypes?

Project Overview Stable transfections create extracts containing repair factors including mutant hMLH1 protein Generation of substrates to model DNA mismatches Biochemical Assays will elucidate the repair efficiency of the mutant hMLH1 protein

Biochemical Assay Procedure Length of repair products will be measured with Analytical gel electrophoresis.

Cytoplasmic Extracts Mutant hMLH1 gene introduced via electroporation A specific sequence, unrelated to the hMLH1 gene, which enables resistance to the drug G418 is also introduced to allow for selection of cells that were succesfuly transfected Cells expressing the desired MLH1 mutation provide the source for MLH1 mutant protein and other repair proteins Selection of cell lines expressing sufficient levels of MLH1 mutants relative to wildtype MHL1 using western blotting Stable Transfection E578G Cell Line Selection Selected Cell Lines Positive Control Negative Control Extracts 250 kD 150 kD 100 kD 75 kD 50 kD MSH6 (140.1 kD) PMS2 MLH1 ß-Tubulin (50.9 kD)

Substrate Formation Research will involve in-vitro MMR reactions and different plasmid substrates, that model presumed replication errors, with either -single base pair mismatches model base substitutions -dinucleotide insertion loops model MSI mutations TGTG + - Contaminating Plasmid Purified Substrate

Substrate Formation A nicking enzyme, which cuts one strand of a double stranded sequence, cuts at two sites on the plasmid DNA. The Plasmid is heated to remove the cut fragment, and the complementary strand is added to create a pure gap molecule A circular piece of DNA, known as a plasmid, serves as the starting material for the mismatch repair substrate. A DNA oligo, similar to removed fragment, is introduced to the gap molecule, and DNA Ligase is used to anneal the mismatch oligo to form the mismatch substrate.

Does the starting plasmid cut correctly? How efficient was the nicking of the DNA Purity of gap molecule ? Is the gap molecule resistant to cutting ? Is the mismatch substrate resistant to cutting ? Substrate Formation Diagnostics used at each step to ensure quality of substrate Restriction Endonuclease Cut Starting Plasmid Uncut Plasmid Gap Molecule Mismatch Substrate Linearized DNA Supercoiled DNA Linear DNA from double digest Nicked Plasmid Nicked DNA

Preliminary Repair Assays Cellular extracts without MLH1 protein and extract with functional (wildtype) MLH1 protein will serve as experimental controls Extracts: A – HeLa cells (positive control) B – WT22 cells (positive control) C – E578G D – CMV2 (negative control) E – No extract (negative control) Test Repair Assays

Further Research Questions - Do any of the MLH1 mutants show ability to differentiate Between different types of mismatches - At what efficiency relative to wildtype MLH1 do the mutants repair mismatches

Thank You HHMI URISC Dr. Andrew Buermeyer The Buermeyer Lab Group. “Good People” Dr. Kevin Ahern