DNA Mismatch Repair-Dependent Suppression in Genotoxicity of Complex Environmental

Carcinogenic Mixtures

Casey KernanMentor: Dr. Andrew BuermeyerDepartment: Environmental & Molecular ToxicologyOregon State University

Colorectal Cancer (CRC)• 2nd leading cause of cancer deaths worldwide• CRC claims nearly 50,000 lives/year in U.S.• American Cancer Society estimates 147,000

new cases by 2011

20%

80%

Hereditary Sporadic

Lynch Syndrome (HNPCC)• Autosomal dominant genetic condition• Mutation in one or more of the 4 MMR genes:

MLH1 & PMS2 (MutLα) and MSH2 & MSH6 (MutSα)

MSH2MSH6/3

PMS2MLH1

Mismatch Repair

Apoptosis

A number of cellular pathways, processes and environmental genotoxins interact to influence an individual’s susceptibility and risk for developing cancer.

Recognition of Mismatch

MSH2 MSH6/3

ATP ADP

•DNA replication error rate = 1 mispairing / 104-105 basepairs

•MutS heterodimer locates and binds mismatch

•MutL heterodimer recruited and endonuclease activated

MSH2MSH6/3

PMS2MLH1

Excision of Mismatch

MSH2MSH6/3

PMS2MLH1

RPA

hEXO1

hEXO1

•RPA (Replication Protein A)• Binds ssDNA, prevents degradation, promotes Polymerase δ,

•Exonuclease1 Activity: 5’ 3’ directed

Correction of Mismatch

c

c

RPA

Pol δ/ε

RFC

PCNA

DNALigase

NickPCNA = Proliferating Cell Nuclear AntigenRFC = Replication Factor CPol δ/ε = DNA Polymerase Delta/Epsilon

Mutator PhenotypeMutations are a driving force behind cancer development

Mutated MMR genes

MLH1

AberrantMMR proteins

Replication errors bypass defective MMR systems

Mutations inactivate tumor suppressor genes and enable onco-genes (APC gene)

Enhanced proliferation

Mutated cells divide

•unchecked growth•loss of apoptotic ability•acquisition of metastic ability•resistance to chemotherapeutic agents (6-TG, MNNG, 5-FU)

PAHs – The Environmental Influence• Mutagenic and carcinogenic - large nonpolar compounds • Exposure: diet, smoking, grilling food, fossil fuel processing• Metabolized forming highly reactive diol epoxides (DE) • Benzo[a]pyrene is metabolically activated to benzo[a]pyrene diol

epoxide (BPDE) which binds to DNA forming bulky DNA adducts

Big QuestionGlobal HypothesisSpecific combinations of environmental exposures and cellular deficiencies interact to influence cancer risk in individuals

Specific HypothesisMMR is a key pathway for reducing deleterious consequences (mutations) from PAH exposure

PredictionCells lacking MMR will show increased PAH-induced mutation

3 Questions:

1.) General phenomena of MMR-deficiency?2.) What are the extra mutations induced?3.) General phenomena of PAH’s, in complex mixtures?

Hypothesis

•We hypothesize that results seen with HCT 116 lines do reflect differences in MMR status rather than other potential known or unknown differences in the cell lines.

-Verify using DLD1 cell lines proficient and deficient in MSH6

•We hypothesize that MMR-dependent suppression of BPDE-induced mutations represent a phenomenon generalizable to other PAH’s, including environmentally relevant complex mixtures.

BPDE-Induced MutationForward mutations induced by exposure to PAH’s are measured using the reporter gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) 657bp

HAT media – 5 passages 1 hour BPDE exposure

Doses: 0, 25, 50, 100 nM

Bulky PAH-DNA adducts

Gene HPRT-Protein HPRT+/-

Grown 8 days to insure no HPRT+ protein present

HPRT+ HPRT-

Clear pre-existing mutants

HPRT- mutant cells survive in 6-Thioguanine selective media

HPRT+ HPRT-

Cell Lines

HCT 116 + Ch3

WTMLH1+

HCT 116 + Ch2

MLH1-

MMR Proficient MMR Deficient

WTMSH6+

MSH6-

DLD1+Ch2 DLD1

Mutant Frequency Calculation

MF=6-TG resistant colonies formed/(PE x # of plates)MF=mutant frequencyPE = plating efficiency

MMR Proficient

MLH1

MMR Deficient

MLH1

135,000 cells 300 cells

6-TG selective media Non-selective media

135,000 cells 300 cells

6-TG selective media Non-selective media

few colonies ~150 colonies more colonies ~150 colonies

Results: PAH-induced mutation in MSH6- deficient cells, similar to previous MMR+ proficient cells

•Technical issue with low plating efficiency in MSH6+

vMutation Identification

trypsinized cloning disc

1 2 3 45 6 7 89 10 11 12

1 2 3

4 5 6

Total RNA Purification

Centrifuge

agarose gel electrophoresis

RNA cDNA PCR sequence

PrimersP3: -36 to -17 5’ CCTGAGCAGTCAGCCCGCGC 3’P4: 701 to 682 5’ CAATAGGACTCCAGATGTTT 3’

Reverse Transcriptase - PCR

cDNA

PCR – amplify HPRT gene

Sequencing

Agarose Gel Electrophoresis

Mutant: 45 18 44 6 13 22 24 26 27 49 50 80 81

CONTROLS

Batch 3 PCR Products – HCT 116+2 HPRT Mutants

HPRT product657bp

Spectrum of HPRT Mutations

Deletion of one nucleotide base

AT → GC transition

Insertion of one nucleotide baseGC → CG transversionGC → TA transversion

GC → AT transition

Spectrum Key58.3%

12.5%25.0%

4.2%

ConclusionPreliminary data suggest:

•BPDE-induced spectra in MLH1 deficient cells different from spontaneous mutations

•Too soon to tell if induced spectra differs in MMR proficient vs. deficient cell lines

Future InvestigationsContinue mutant analysis on remaining clones:

•HCT 116+Ch2•HCT 116+Ch3•DLD1•DLD1+Ch2

Complex environmental mixtures

•Mutant frequency•Individual mutation analysis

GoalsResearch Goals & Significance •Understand MMR functions as well as genetic influences and their combined role in normal responses to carcinogens•Accurate evaluation of an individuals susceptibility and risk to developing CRC•Provide insight for more effective and practical CRC screening methods•Develop novel models for studying other genetic and environmentally linked diseases

Acknowledgements

• Howard Hughes Medical Institute• Environmental Health Sciences Center• Dr. Andrew Buermeyer– Jacki Coburn– Fatimah Almousawi– Kimberly Sarver

• Dr. Vidya Schalk• Dr. Kevin Ahern, program coordinator