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I count 1, 2, 3, 4 , 5 p53 mutants and…oh…~300,000 wild-type DNA molecules. DEVELOPING TUMOR-ASSOCIATED SOMATIC MUTATIONS AS BIOMARKERS OF CANCER RISK. Barbara Parsons Division of Genetic and Reproductive Toxicology National Center for Toxicological Research. ACB-PCR.
I count 1, 2, 3, 4 , 5 p53 mutants and…oh…~300,000 wild-type DNA molecules.
DEVELOPING TUMOR-ASSOCIATED SOMATIC MUTATIONS AS BIOMARKERS OF CANCER RISK
Division of Genetic and Reproductive Toxicology
National Center for Toxicological Research
“Reliance on animal tumor incidence data with its inherent need to extrapolate to humans introduces considerable uncertainty into the risk assessment process.”
guidelines indicate risk assessment
should be based on an understanding
of chemical mode-of-action and
key events in carcinogenesis.
effect (tumor-associated mutations considered early biomarkers of disease state).
Priming strategy for K-RAS codon 12 GGT GTT mutation
Synthesize first-round PCR product
Aliquot, quantify all unknowns and standards
Isolate genomic DNA from tissue
ACB-PCR of duplicate MF standards, no mutant, no DNA controls, and ~24 unknowns in parallel, per experiment
Polyacrylamide gel electrophoresis, measure fluorescence, use MF standards to a construct a standard curve, calculate the MF (ratio of mutant to wild-type sequence of unknowns, repeat assay 3 times
no mutant control
Albertini et al., 2003
ACB-PCR was used to investigate relationship between DNA adducts, H-Ras codon 61 CAA to AAA mutation, and tumor induction in newborn B6C3F1 and C57BL/6 mice treated with 0.3 mol 4-aminobiphenyl
“The more we understand about the applied dose metabolism mutation effect continuum, the more confidence we can have in a cancer risk assessment”
Molecular Carcinogenesis 42:193-201 (2005)
Spontaneous H-Ras codon 12 CAA to AAA MF in mouse liver is closer to 10-5 than 10-7 or 10-8 as was expected based on reporter gene MFs.
Albertini et al., 2003
Compared the levels of two different K-Ras codon 12 mutations in livers of Big Blue rats treated with N-hydroxy-2-acetylaminofluorene in an attempt to distinguish between chemical-induced induction of de novo mutation vs. chemically-induced amplification of pre-existing mutation.G T mutation, the predominant mutagenic specificity of N-OH-AAFG A mutation, the most common spontaneous mutation
induction of K-Ras codon 12
GGT GTT and GGT GAT
mutation in liver DNA using
McKinzie et al. (2006) Mutagenesis 21: 391-397
Albertini et al., 2003
Conducted a dose-response assessment, measuring the induction of p53 codon 270 CGT to TGT mutation by simulated solar light (SSL).
Filtered 6.5 kW xenon arc light mimics terrestrial exposure to sunlight
Measured the p53 codon 270 CGT to TGT mutational response induced by 4 different doses of SSL – after three different durations of exposure
P = 0.008
The increase in p53 MF with cumulative dose is highly-significant
P = <0.0001
MF = 10α100.0016*d, where α is the spontaneous MF at a
particulartimepoint and d is the dose of SSL in mJ CIE/cm2
Tumors induced by 32 weeks of exposure at the high dose had significantly more p53 mutation than skin exposed for 28 weeks at the high dose (t-test, P = 0.0006).
Albertini et al., 2003
VEGF which induces angiogenesis.
mutation in sporadic colonic adeno-
carcinoma has been associated with
Literature review of APC and K-RAS mutation prevalence in sporadic colon cancer
GAT mutation in ~20% of colonic adenocarcinoma
GTT mutation in ~7% of colonic adenocarcinoma
Fodde et al. (2001) Nature Reviews Cancer 1:55-67
Luebeck and Moolgavkar (2002) PNAS
Assumes clonal amplification after acquiring multiple mutations
Normal (individuals w/o colon ca.) 2
Tumor distal mucosa (5 or more cm from tumor) 17
Tumor proximal mucosa (2 to 5 cm from tumor) 14
Tumor margin (normal tumor-adjacent) 10
Tumor distal mucosa
K-RAS mutations as
MF during tumor progression
suggests that tumors are
composed of more than one
clone of tumor cells (K-RAS
mutant and non-K-RAS
mutant) and that during
progression to adenocarcinoma,
K-RAS mutant cells are either
dying or other clone(s) are
NCI Teacher’s Guide, “Understanding Cancer”…cancer cells are indigenous cells—abnormal cells that arise from the body's normal tissues. Furthermore, virtually all malignant tumors are monoclonal in origin, that is, derived from a single ancestral cell that somehow underwent conversion from a normal to a cancerous state.
One Renegade Cell (Science Masters) (Paperback) by Robert A. Weinberg (Author)
Thliveris et al., PNAS 102:6960 (2005).
Albertini et al., 2003Comparing spontaneous levels of K-Ras mutation in rat and human colonic mucosa
*Mean age 67 years
#38 week-old rats (Page McKinzie)
Colon of a 38 week-old rat has a K-RAS MF
7 to 15 times the of a 67 year-old man
Can we model carcinogenesis based on the frequency and spatial distribution of different tumor-associated mutations?
Frequency and distribution of mutation relates to frequency of adenoma formation
Michael A. Newton, University of Wisconsin Seminar, On Polyclonality of Intestinal Tumors describes the “Random Collision Hypothesis” originally proposed by Armitage http://sprott.physics.wisc.edu/Chaos-Complexity/newton06.pdf
NTP Center for Phototoxicology
Rat K-Ras Studies
Human K-RAS Study
p53 SSL Study
Director, Division of Genetic and Reproductive Toxicology
Ablertini, R., H. Clewel, M.W. Himmelstein, E. Morinello, S. Olin, J. Preston, L. Scarano, M.T. Smith, J. Swenberg, R. Tice, and C. Travis (2003) The use of non-tumor data in cancer risk assessment: reflections on butadiene, vinyl chloride, and benzene. Regulatory Toxicology and Pharmacology 37: 105-132.
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Fearon, E.R. and B. Vogelstein (1990) A genetic model for colorectal tumorigenesis. Cell 61: 759-767.
Fodde, R., R. Smits, and H. Clevers (2001) APC, signal transduction and genetic instability in colorectal cancer. Nature Reviews Cancer 1: 55-67.
Hanahan, D., and R.A. Weinberg (2000) The hallmarks of cancer. Cell 100: 57-70.
Leedham, S.J., S. Schier, T. Thliveris, R.B. Halberg, M.A. Newton, and N.A. Wright (2005) From gene mutations to tumours – stem cells in gastrointestinal carcinogenesis. Cell Proliferation 38: 387-405.
Luebeck, E.G., and S.H. Moolgavkar (2002) Multistage carcinogenesis and the incidence of colorectal cancer. PNAS 99: 15095-15100.
McKinzie, P.B., R.R. Delongchamp, T. Chen, and B.L. Parsons (2006) ACB-PCR Measurement of K-ras codon 12 mutant fractions in livers of Big Blue rats treated with N-hydroxy-2-acetylaminofluorene. Mutagenesis 21: 391-397.
McKinzie, P.B., R. Delongchamp, R.H. Heflich, and B.L. Parsons (2001) Prospects for applying genotypic selection of somatic oncomutation to chemical risk assessment. Mutation Research 489: 47-78.
McKinzie, P.B., and B.L. Parsons (2002) Detection of rare K-ras codon 12 mutations using allele-specific competitive blocker PCR. Mutation Research 517: 209-220.
Newton, M.A (2006) On polyclonality of intestinal tumors. University of Wisconsin. Chaos and Complex Systems April 2006. http://sprott.physics.wisc.edu/Chaos-Complexity/newton06.pdf
Novelli, M.R., J.A. Williamson, I.P.M. Tomlinson, G. Elia, S.V. Hodgson, I.C. Talbot, W.F. Bodmer, N.A. Wright (1996) Polyclonal origin of colonic adenomas in an XO/XY patient with FAP. Science 272: 1187-1190.
Novelli, M., A. Cossu, D. Oukrif, A. Auaglia, S. Lakhani, R. Poulsom, P. Sasieni, P. Carta, M. Contini, A. Pasca, G. Palmieri, W. Bodmer, F. Tanda, and N. Wright (2003) X-inactivation patch size in human female tissue confounds the assessment of tumor clonality. PNAS 100: 3311-3314.
Parsons, B.L., F.A. Beland, L.S. Von Tungeln, R.R. Delongchamp, P. Fu, and R.H. Heflich (2005) Levels of 4-aminobiphenyl-induced somatic H-ras mutation in mouse liver DNA correlate with potential for liver tumor development. Molecular Carcinogenesis 42:193-201.
Parsons, B.L., R.R. Delongchamp, F.A. Beland, R.H. Heflich (2006) Levels of H-ras codon 61 CAAAAA mutation: response to 4-ABP-treatment and Pms2-deficiency. Mutagenesis 21: 29-34.
Parsons, B.L. and R.H. Heflich (1997) Genotypic selection methods for the direct analysis of point mutations. Mutation Research, 387: 97-121.
Parsons, B.L. and R.H. Heflich (1998) Detection of a mouse H-ras codon 61 mutation using a modified allele-specific competitive blocker PCR genotypic selection method. Mutagenesis 13: 581-588.
Parsons, B.L. P.B. McKinzie, and R.H. Heflich (2005) ACB-PCR detection of rare point mutation. In: Molecular Toxicology Protocols (Methods in Molecular Biology Series), P. Keohavong and S. Grant (eds.), Humana Press, Totawa, NJ.
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Verkler, T.L., L.H. Couch, P.C. Howard, and B.L. Parsons (2005) Quantifying levels of p53 mutation in mouse skin tumors. Environmental and Molecular Mutagenesis 45: 427-43.