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
Lung & bronchus
31%
Prostate
10%
Men
285,900
Women
270,600
•25% Lung & bronchus
•15% Breast
Colon & rectum 10%
•11% Colon & rectum
Pancreas
5%
•6%
Pancreas
Non-Hodgkin
lymphoma
4%
•5%
Ovary
•4%
Leukemia
4%
Non-Hodgkin
lymphoma
Esophagus
4%
•4%
Leukemia
Liver/intrahepatic
bile duct
3%
•3%
Uterine corpus
•2%
Brain/ONS
Urinary bladder
3%
•2%
Multiple myeloma
Kidney
3%
•23% All other sites
All other sites
22%
ONS=Other nervous system.
*Excludes basal and squamous cell skin cancers and in situ carcinomas except urinary bladder.
Source: American Cancer Society, 2003.
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
HNPCC, FAP
Non Hereditary
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
G
T
G
T
• Excision
T
• Resynthesis
A
T
*
Mutation Types
DNA Synthesis Error
Mutation
Base Mismatches
 Base Substitution Mutations
A
T
Incorrect
Placement
of Base
No Repair,
Continued
Replication
G
T
Successful Repair
Insertion / Deletion Loops
Incorrect DNA
sequence, mutation
in genome copy
A
T
G
C
 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
Dinucleotide
Loop Insertion
AC
TG
Successful Repair
No Repair,
Continued
Replication
Incorrect DNA
sequence, mutation
in genome copy
Clinical relevance of the hMLH1 gene
• 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.
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
• Are mutations of hMLH1 responsible for the observed
molecular phenotypes?
The goal of the research is to determine the
mutation prevention capabilities of the MLH1
mutants E578G, K618A, V716M, and D132H using
biochemical assays
Project Overview
Stable transfections
Generation of substrates
create extracts containing repair
factors including mutant hMLH1
protein
to model DNA mismatches
Biochemical Assays
will elucidate the repair
efficiency of the mutant
hMLH1 protein
Biochemical Assay Procedure
Original Plasmid
DNA
Substrate
Formation
Mismatch
Substrate
Restored
Plasmid
Functional
In Vitro Mismatch
Repair Reaction
Non-Functional
In Vitro Mismatch
Repair Reaction
Restriction
Endonuclease Site
Base-Base mismatch or
Dinucleotide Loop
Digestion of DNA at
endonuclease sites to
produce linear fragments
Length of repair products will be measured with Analytical gel electrophoresis.
Cytoplasmic Extracts
Stable Transfection
•
•
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
Extracts
•
•
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
Selected Cell Lines
Positive Control
Negative Control
E578G Cell Line Selection
250 kD
150 kD
MSH6 (140.1 kD)
100 kD
75 kD
50 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
T
G
+
Contaminating
Plasmid
-
Purified
Substrate
Substrate Formation
A circular piece of DNA, known as a plasmid, serves
as the starting material for the mismatch repair
substrate.
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 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.
Substrate Formation
Diagnostics used at each step to ensure quality of 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 ?
Gap Molecule
Mismatch Substrate
Nicked Plasmid
Starting Plasmid
Linearized DNA
Supercoiled DNA
Linear DNA
from double
digest
Uncut Plasmid
Restriction Endonuclease Cut
Nicked DNA
Preliminary Repair Assays
•
Cellular extracts without MLH1 protein and extract with functional (wildtype) MLH1
protein will serve as experimental controls
Test Repair Assays
Extracts:
A – HeLa cells
(positive control)
B – WT22 cells
(positive control)
C – E578G
D – CMV2
(negative control)
E – No extract
(negative control)
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