Pathogenic Mechanisms of Cancer Causing hMLH1 Mutations

Functional Relationship between DNA Mismatch Repair and Tumor Formation

Eddie O’Donnell

Laboratory of Dr. Andrew B. BuermeyerDepartment 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, 2003.

Men285,900

Women270,600

•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 & bronchus 31%

Prostate 10%

Colon & rectum 10%

Pancreas 5%

Non-Hodgkin 4%lymphoma

Leukemia 4%

Esophagus 4%

Liver/intrahepatic 3%bile duct

Urinary bladder 3%

Kidney 3%

All other sites 22%

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 HNPCC1993 – MSH2 mutations linked to 35% of HNPCC1994 – 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

GT

T

TA

GT *

DNA Synthesis Error Mutation

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

GT

No Repair, Continued Replication

AT

AT

GC

Incorrect DNA sequence, mutation

in genome copy

Incorrect Placement

of Base

Successful Repair

Successful Repair

Dinucleotide Loop Insertion

ACTG

No Repair, Continued Replication

Incorrect DNA sequence, mutation

in genome copy

• hMLH1 significancehMLH1 is an essential protein for the prevention ofmutations. Exact function is unknown.

• TreatmentsMMR deficient cancers may respond differently to chemotherapeutic drugs.

• DetectionMMR deficient cancers are commonly detectedthrough 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

E578GK618AD132HV716M

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, containingMLH1, 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 transfectionscreate 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.

Original Plasmid DNA

Mismatch Substrate

Restored Plasmid

Restriction Endonuclease Site

Base-Base mismatch or

Dinucleotide Loop

Substrate Formation

Functional In Vitro Mismatch Repair Reaction

Non-Functional In Vitro Mismatch Repair Reaction

Digestion of DNA at endonuclease sites to

produce linear fragments

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 SelectionSelected Cell LinesPositive ControlNegative 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

TG

+ -

ContaminatingPlasmid

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 FormationDiagnostics 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 – E578GD – 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