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ToxSci Advance Access published online on December 27, 2006
Toxicological Sciences, doi:10.1093/toxsci/kfl197
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© The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Comet FISH Analysis for Oxidative Stress Induced DNA Damage in Colon Cancer Relevant Genes

TOTAL DNA AND APC, KRAS, TP53 GENE DAMAGE

M Glei*, A Schaeferhenrich*, U Claussen{dagger}, A Kuechler{dagger},{ddagger}, T Liehr{dagger}, A Weise{dagger}, B Marian§, W Sendt and BL Pool-Zobel*

* Institute for Nutrition, Dornburger Strasse 25, Friedrich-Schiller-University Jena, 07743 Jena, Germany {dagger} Institute for Human Genetics, Kollegiengasse 10, Friedrich-Schiller-University Jena, 07743 Jena, Germany {ddagger} Department of Radiotherapy, School of Medicine, Bachstrasse 18, Friedrich-Schiller-University Jena, 07743 Jena, Germany § Institute for Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria St. Joseph-Stift, Schwachhauser Heerstraße 54, 28209 Bremen, Germany

Correspondence: Prof B L Pool-Zobel, Institute for Nutrition, Dornburger Strasse 25, Friedrich-Schiller-University Jena, 07743 Jena, Germany. E-mail: b8pobe{at}uni-jena.de, Phone: +49 (0) 3641 949670

Received August 17, 2006; accepted December 20, 2006


  Abstract

Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H2O2), trans-2-hexenal and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single cell gel electrophoresis assay, known as "Comet Assay". APC, KRAS and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS or TP53 signals into the comet tails was quantified and compared to total DNA damage.

All three substances were clearly genotoxic for APC, KRAS and TP53 genes and total DNA in both types of cells. In primary colon cells TP53 gene was more sensitive toward H2O2, trans-2-hexenal and HNE than total DNA was. In LT97 cells the TP 53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products, but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS towards HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.

Key Words: Colon cancer; DNA damage; Comet FISH; APC; KRAS; TP53.


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