Pathogenic Mechanisms of Cancer Causing MLH1 MutationsFunctional Relationship between DNA Mismatch Repair and Cancer-Risk

Eddie O’DonnellLaboratory of Dr. Andrew B. Buermeyer

Department of Environmental and Molecular ToxicologyImage: Ribbon diagram of E. Coli MutL Protein (PDB)

10%

DNA Mismatch Repair Deficiencies in Colorectal Cancer

• Causes of Cancer• Mutations within cells cause uncontrolled cell growth• Risk of cancer development can be inherited • Most cancers are sporadic (no family history)

• Colorectal CancerApproximate percentages of occurrence

15 % - Mismatch Repair (MMR) deficiency observed 90 % of sporadic cases linked to MMR deficiency

are MLH1 deficient (loss of expression) 2-5 % - Lynch Syndrome (HNPCC)

•Discoveries involving Lynch Syndrome1993 – MSH2 mutations linked to HNPCC1994 – MLH1 mutations linked to HNPCC*Account for majority of HNPCC occurrences

• DNA mismatches arise from errors during DNA Replication

• MMR corrects replication errors

• MMR Stimulates apoptosis in response to DNA damage

• Basic Mechanism:

• Mismatch recognition

•MutS familyMSH2/MSH6MSH2/MSH3

G

T

* MLH1/MLH3

• Strand choice•MutL family

MLH1/PMS2MLH1/PMS1

• Excision• Exonucleases

• PCNA• RPA

T

• Resynthesis•Replicative

DNA polymerase

A

T

Mechanism & Functions of DNA MMR

G

T

ATP Dependant

DNA Synthesis Error Mutation

Mutations Prevented by MMR

Successful Repair

Dinucleotide Loop Insertion via slip-mispairing

ACTG

No Repair, Additional Replication

Insertion Mutation

Insertion / Deletion Loops Microsatellite Instability (MSI)

GT

No Repair, Additional Replication

AT

AT

GC

Incorrect insertion of base

Successful Repair

Base Mismatches Base Substitution Mutations

Implications of MMR Deficiency for Cancer Screening & Treatment• Chemotherapy

• Microsatellite Instability - An Effective Screening Tool

• Clinical Relevance of MLH1: HNPCC cases without MSI?

36, 694 - 699 (2004)

D132HMLH1 amino acid site 132 changed from D (Aspartic Acid) to H (Histidine)

Loss of repeats

Normal

Tumor

Hypothesis:

Initial DataData from recent publications

• D132H apparently associated with 5-fold increased cancer risk

• Modest decrease in ATPase function in D132H

• Increased mutation rate not dramatic enough for MSI detection

• Base substitutions more affected than microsatellites

• Apoptosis signaling function more affected than error correction

Attenuated MLH1 function of D132H increases cancer risk

Is there an observable phenotype associated with MLH1-D132H?

Central Question

Research Goals

2. Determine in vitro repair capabilities for MLH1 mutant D132H using biochemical assays

1. Use Cellular assays to evaluate the effect of the MLH1 mutation D132H in vivo

Project Outline

Mutant MLH1 & Repair proteinsІ

ІІ

ІІІ

Cellular Assays

In Vitro repair

Research will involve in vitro MMR reactions to model presumed replication errors and score repair efficiency of MMR proteins

hMLH1-expressing

cells

Mlh1-/- MEFs

Cellular Assays

Measure Repair

Efficiency

G

T

+Mismatch

Substrates

In Vitro Repair

1. Forward Mutation Rate2. Cytotoxic Response

Indirect Measurement of MLH1 activity

Direct Measurement of MLH1 activity

Cell-free extracts

Identification of Cell Lines Expressing MLH1 Mutants

Western Blot Analysis of Extract Preparation

Cell-free extracts

Transfection

hMLH1

Neo-R

MLH1

Drug Resistance Mlh1-/- MEFs Drug Selection

D132H

-8

D132H

-9

MLH1

MLH1-

13 (+

)

MLH1-

2 (+

)

MC2A

(-)

PMS2

• Screen for MLH1 Expression with Western Blotting• Isolate and Expand Expressing Cell Lines for extract• 2 D132H Lines identified.

•Expression is less than MLH1 wildtype lines.

Fluctuation Analysis: Forward Mutation to OuabainR

Cell Line Events/Cell/Generation (Rate)

MLH1 (-/-) 60 x 10-7

+ WT hMLH1* ~ 1 x 10-7

+ Hmlh1- D132H 0.7 ± 0.2 x 10-7 **

* - ** -

12 Cultures(1000 OuabainS cells)

Expansion, Accumulation of Mutants

Exposure to Ouabain Count number of Ouabain

Resistant Clones, Calculate Rate of mutation

* Rates in MEF cell line determined by Dr. Andrew Buermeyer, 1999. ** Assay Repeated Twice

~5 x 106 cells, includes some OuabainR cells

Conclusion: Expression of D132H decreased rate of base substitution

Response to Cytotoxic Agents: 6-Thioguanine Response

300-3000 Cells

24 Hours

6-Thioguanine 0-6 uM Doses

24 Hours

Remove 6-Thioguanine

Count Surviving Colonies

6-10 Days

Conclusion: Expression of D132H increased cytotoxic response to 6-Thioguanine

In Vitro Mismatch Repair Assay

CT T GAG

GA G CTC

Mismatch substrateincubated with repair factors from extracts

- Mismatch Blocks activityof Restriction Endonuclease

- 3’ Nick initiates repair, facilitatesStrand choice

nick

Mismatch dependantnick directed excision

Xho1CT C GAGGA G CTC

Pvu1

Resynthesis leadsto restoration of Xho1 site Pvu1 Site used to facilitateanalysis

Substrate preparation protocol developed in the Hay’s Laboratory, OSUGels 1% TAE 8 cm, 170V, 30’ w/Stain & w/Destain (10’,30’)

Preparation of Mismatch Substrates

Xho1CT T GAG

GA G CTC

nick

A – Closed Circular Substrate

B – Double Digest

ConclusionsSubstrate Preparation yields >95% Mismatch SubstrateSuccessful Preparation for G/T and CT Loop mismatches

Starting Plasmid

A B A B A B

G/T Mismatch

-CT- Loop

Linear (Pvu1 Cut)

Xho1 & Pvu1 CutPvu1

A

Results & Discussion

I. Expression of D132H in MLH1 deficient cells:

1) Reduced mutation rate similar to wildtype expressing cells, suggesting good repair activity in vivo

2) Increased cytotoxic response to 6-Thioguanine with a modest decrease in response relative to wildtype expressing

cells -Protein Expression?

II. In Vitro Repair

1) Substrates Prepared, Assays in Progress

Future Work

Repair Assays Additional D132H expressing lines for cellular assays

Acknowledgments

• Dr. Andrew Buermeyer• Buermeyer Lab Group

Xin Huo• Hays Lab Group

Pete HoffmanHuixian Wang

• Howard Hughes Medical Institute• Dr. Kevin Ahern