Pathogenic Mechanisms of Cancer
Causing MLH1 Mutations
Functional Relationship between DNA Mismatch Repair and Cancer-Risk
Eddie O’Donnell
Image: Ribbon diagram of E. Coli MutL Protein (PDB)
Laboratory of Dr. Andrew B. Buermeyer
Department of Environmental and
Molecular Toxicology
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 Cancer
Approximate 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 Syndrome
1993 – MSH2 mutations linked to HNPCC
1994 – MLH1 mutations linked to HNPCC
*Account for majority of HNPCC occurrences
Mechanism & Functions of DNA MMR
• DNA mismatches arise from errors during DNA Replication
• MMR corrects replication errors
• MMR Stimulates apoptosis in response to DNA damage
• Basic Mechanism:
• Mismatch
recognition
G
•MutS family
MSH2/MSH6
MSH2/MSH3
T
G
• Strand choice
T
*
•MutL family
MLH1/PMS2
MLH1/PMS1
MLH1/MLH3
• PCNA
• RPA
• Excision
T
A
• Resynthesis
T
• Exonucleases
•Replicative
DNA
polymerase
ATP
Dependant
Mutations Prevented by MMR
DNA Synthesis Error
Mutation
 Base Substitution Mutations
Base Mismatches
Incorrect insertion
of base
A
T
G
T
No Repair,
Additional
Replication
A
T
Successful Repair
Insertion / Deletion Loops
Dinucleotide Loop Insertion
via slip-mispairing
AC
TG
Successful Repair
G
C
 Microsatellite Instability (MSI)
No Repair,
Additional
Replication
Insertion Mutation
Implications of MMR Deficiency
for Cancer Screening & Treatment
• Chemotherapy
• Microsatellite Instability - An Effective Screening Tool
• Clinical Relevance of MLH1: HNPCC cases without MSI?
Loss of
repeats
36, 694 - 699 (2004)
D132H
MLH1 amino acid site 132 changed
from D (Aspartic Acid) to H (Histidine)
Initial Data
Data from recent publications
• D132H apparently associated with 5-fold
increased cancer risk
• Modest decrease in ATPase function in D132H
Hypothesis:
Attenuated MLH1 function of
D132H increases cancer risk
•
Increased mutation rate not dramatic enough for MSI detection
•
Base substitutions more affected than microsatellites
•
Apoptosis signaling function more affected than error correction
Central Question
Is there an observable phenotype
associated with MLH1-D132H?
Research Goals
1. Use Cellular assays to evaluate the effect of the MLH1
mutation D132H in vivo
2. Determine in vitro repair capabilities for MLH1 mutant D132H
using biochemical assays
Project Outline
Research will involve in vitro MMR reactions to model presumed replication
errors and score repair efficiency of MMR proteins
І
Cellular
Assays
Mutant MLH1 &
Repair proteins
hMLH1expressing
cells
Mlh1-/MEFs
ІІ
In Vitro
Repair
Indirect Measurement of MLH1 activity
Cellular Assays
1. Forward Mutation Rate
2. Cytotoxic Response
Direct Measurement of MLH1 activity
G
ІІІ
In Vitro repair
T
Mismatch
Substrates
Measure Repair
Efficiency
+
Cell-free
extracts
Identification of Cell Lines Expressing MLH1 Mutants
Transfection
MLH1
hMLH1
Neo-R
Drug Resistance
Mlh1-/- MEFs
Drug Selection
• 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.
Western Blot Analysis of Extract Preparation
Cell-free
extracts
PMS2
MLH1
Fluctuation Analysis: Forward Mutation to OuabainR
Expansion,
Accumulation
of Mutants
12 Cultures
(1000 OuabainS cells)
Exposure to
Ouabain
Count number of Ouabain
Resistant Clones,
Calculate Rate of mutation
~5 x 106 cells, includes
some OuabainR cells
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
**
Conclusion: Expression of D132H decreased rate of base substitution
* *** Rates
- in MEF cell line determined by Dr. Andrew Buermeyer, 1999.
** Assay Repeated Twice
Response to Cytotoxic Agents: 6-Thioguanine Response
24 Hours
24 Hours
6-10 Days
Count Surviving
Colonies
Remove
6-Thioguanine
6-Thioguanine
0-6 uM Doses
300-3000 Cells
100.00%
% Survival
10.00%
1.00%
0.10%
MC2A
D132H-8
MLH1-2
0.01%
0
1
2
3
4
5
6
6-Thioguanine Dose (24 Hr Exposure, [uM] 6-TG in 15% BCS supplemented DMEM)
Conclusion: Expression of D132H increased cytotoxic response to 6-Thioguanine
In Vitro Mismatch Repair Assay
Xho1
CT C GAG
GA G CTC
T GAG
GA G CTC
CT
nick
Pvu1
Mismatch substrate
incubated with repair
factors from extracts
- Mismatch Blocks activity
of Restriction Endonuclease
- 3’ Nick initiates repair, facilitates
Strand choice
Mismatch dependant
nick directed excision
Resynthesis leads
to restoration of
Xho1 site Pvu1 Site
used to facilitate
analysis
Preparation of Mismatch Substrates
Starting
Plasmid
Xho1
T GAG
GA G CTC
CT
A
B
-CTLoop
G/T
Mismatch
A
B
A
B
Linear
(Pvu1 Cut)
nick
A
Pvu1
A – Closed Circular Substrate
Xho1 &
Pvu1 Cut
B – Double Digest
Conclusions
Substrate Preparation yields >95% Mismatch Substrate
Successful Preparation for G/T and CT Loop mismatches
Substrate preparation protocol developed in the Hay’s Laboratory, OSU
Gels 1% TAE 8 cm, 170V, 30’ w/Stain & w/Destain (10’,30’)
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 Hoffman
Huixian Wang
• Howard Hughes Medical Institute
• Dr. Kevin Ahern