ToxSci Advance Access originally published online on December 27, 2006
Toxicological Sciences 2007 96(2):279-284; doi:10.1093/toxsci/kfl197
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Comet Fluorescence in situ Hybridization Analysis for Oxidative Stress–Induced DNA Damage in Colon Cancer Relevant Genes
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* Institute for Nutrition, Dornburger Strasse 25, Friedrich-Schiller-University Jena, 07743 Jena, Germany
Institute for Human Genetics, Kollegiengasse 10, Friedrich-Schiller-University Jena, 07743 Jena, Germany
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
1 To whom correspondence should be addressed. Fax: +49(0)3641-949672. E-mail: b8pobe{at}uni-jena.de.
Received September 20, 2006; accepted December 20, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 TP53 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 toward 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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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is widely accepted that diet might be responsible for 30–60% of cancers in the developed countries (Doll, 1992
; World Cancer Research Fund and American Institute for Cancer Research, 1997). The most frequent nutrition-associated type of cancer is colon cancer which is strongly linked to carcinogens in our diet (Berlau et al., 2004). Also endogenous metabolites are expected to contribute to risk. Iron load, alcohol metabolism, and inflammatory bowel conditions can increase oxidative stress in the gut. Oxidative stress, in turn, is associated with an enhanced formation of reactive oxygen species (ROS). ROS oxidate lipids found in cellular membranes leading to the release of compounds, such as trans-2-hexenal and 4-hydroxy-2-nonenal (HNE) (Cerutti, 1985; Dreher and Junod, 1996; Erhardt et al., 1998; Lund et al., 1999). Trans-2-hexenal and HNE are well-investigated reactive products which cause DNA adducts and genomic damage (Gölzer et al., 1996; Schäferhenrich et al., 2003a,b).
The genetic process of human colon carcinogenesis involves mutations in oncogenes (KRAS [12p]) and in tumor suppressor genes (APC [5q], DCC [18q], SMAD2/4, TP53 [17p]) as well as multiple additional types of alterations (DNA hypermethylation) (Potter, 1999). It is thought that risk factors initiate colon cancer by first damaging the APC gene in the stem cells of the crypts (Fodde et al., 2001). Loss of heterozygosity leads to a loss of growth control, and the cells are pushed upward to the crypt surface to form dysplasia, aberrant crypts, polyps, and microadenoma. The tissue can protrude increasingly into the gut lumen and is then exceptionally well exposed to genotoxic and toxic ingredients (Bach et al., 2000), thus facilitating the additional alterations of KRAS and SMAD2/4 (Fodde et al., 2001). Mutations in TP53 are then responsible for converting adenoma to carcinoma. Additionally, approximately 10–15% of colorectal cancers arise via microsatellite instability and share morphological characteristics (Ionov et al., 1993; Jass, 2001; Jass et al., 1998).
APC, KRAS, and TP53, as genes that are most commonly mutated in colorectal tumors, could provide relevant targets to investigate carcinogens for their potentials to initiate and to enhance progression of human colon cells. Therefore, we have recently further refined the method of fluorescence in situ hybridization (Comet FISH) to detect TP53 damage in human colonocytes induced by products of endogenous oxidative stress (hydrogen peroxide [H2O2]) and lipid peroxidation (trans-2-hexenal and HNE) (McKelvey-Martin et al., 1998; Schäferhenrich et al., 2003a,b). To date, however, there are no data available on genotoxicity of diet-related risk factors in APC and KRAS genes. Therefore, using Comet FISH, we have now studied the sensitivities of APC, KRAS, and TP53 in the colon adenoma cell line LT97 and in primary human colon cells toward the genotoxic actions of H2O2, trans-2-hexenal and HNE. The general objective of this work was to develop a new technique for obtaining an improved basis for elucidating associations between genotoxic damage and initiation or progression of colorectal carcinogenesis in humans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell line and culture condition.
The human colon adenoma cell line LT97 was established from colon micro adenomas of a patient with "familial adenoma polyposis". LT97 cells have lost both alleles of the APC tumor suppressor gene. In addition, they carry a mutated Ki-ras oncogene, while TP53 is normal. LT97 growth characteristics are thus representative of early adenomas (Richter et al., 2002
). LT97 was maintained in a culture medium (MCDB 302) containing 20% L15 Leibovitz medium, 2% fetal calf serum, 1% penicillin/streptomycin, 0.2nM triiodo-L-thyronine, 1 g/ml hydrocortisone (302 basic medium) supplemented with 10 µg/ml insulin, 2 µg/ml transferrin, 5nM sodium selenite, and 30 ng/ml epidermal growth factor. For these studies, we used passages 12–24.
Primary human colon cells from surgical tissue.
Colon tissue was obtained from patients admitted to the hospital for surgery of colorectal tumors, diverticulitis, and colon polyps (Schäferhenrich et al., 2003b). The Ethical Committee of the Friedrich-Schiller-University Jena approved the study, and the tissue was made available only from patients who had given their informed consent. The average age (± SD) of all donors was 62 ± 12 years (n = 36). Forty-one percentage of the donors were male and 59% were female. Samples for cell isolation were parts of the excised, nontumorous tissue removed together with the tumor for medical indications. Work up of the colon tissue included separation of the epithelium from underlying layers and isolation of primary cells by enzymatic digestion of the minced epithelium (Schäferhenrich et al., 2003b). Viability of the cells and cell yields were determined using the method of trypan blue exclusion, and the cell numbers were adjusted to 2 x 106 cells/ml.
Treatment of the cells with chemicals.
Stock solutions of H2O2 [CAS 7722-84-1] in phosphate-buffered saline were added to cell suspensions containing 2 x 106 cells/ml. Final concentrations were 0–150µM. H2O2 suspensions were incubated for 5 min at 4°C to avoid repair of the induced oxidative DNA damage (Collins et al., 1995). Trans-2-hexenal [CAS 6728-26-3] was dissolved in dimethyl sulfoxide and HNE [CAS 75899-68-2] in ethanol. These stock solutions were added to the cell suspensions containing 2 x 106 LT97 cells or primary colon cells per milliliter (Rousset, 1986). The cell suspensions (0–1600µM trans-2-hexenal and 0–250µM HNE) were incubated for 30 min in a shaking water bath at 37°C. The substances were removed by centrifugation, and the viability of the cells was tested by trypan blue exclusion. The cell pellets were taken up in agarose, distributed onto slides, and then processed according to the protocol for the microgel electrophoresis assay as described below.
Determination of DNA damage (Comet Assay and Comet FISH).
DNA damage was detected using the alkaline version of the Comet Assay (Singh et al., 1988) as described previously (Schäferhenrich et al., 2003a,b). For the Comet FISH experiments, Texas red-labeled probes were used. They were specific for genomic sequences of the APC (5q13-q31), KRAS (12pter-p11.2), and TP53 locus (17pter-p12). The dehydrated slides from the Comet Assay were processed by rehydration and staining as previously described (Schäferhenrich et al., 2003a,b). SYBR-Green was used to counterstain total DNA.
APC, KRAS, or TP53 signals were counted in each cell using a ZEISS Axiovert M100 (Carl Zeiss Jena GmbH, Jena, Germany). The number of signals in each cell and the localization of the signals in the comet head or comet tail were recorded. The percentage of signals in the tail was then calculated for each slide. The migration of APC, KRAS, or TP53 genes was compared with the migration of total DNA into the comet tail.
Statistics.
Statistics were carried out using the mean values of the independently reproduced experiments which are also presented (mean ± SD) in the "Results" section. Pairwise comparisons of all the treatment groups versus the control group were done using one-way ANOVA with Bonferroni posttest. The comparison between test compounds, cells, and genes was made by two-way ANOVA with Bonferroni posttest.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of H2O2, trans-2-Hexenal, and HNE on Total DNA Damage (Comet Assay)
The dose-dependent increase of the total DNA in the comet tail (%) was revealed for H2O2, trans-2-hexenal, and HNE in primary colon cells (Tables 1A–1C, first column) and LT 97 cells (Tables 2A–2C, first column) without concomitantly causing cytotoxicity (viability
80%; results not shown). The three compounds were assessed in different concentrations, but they were of equal genotoxic potency. Maximal tail intensities ranged from 21 to 25% in primary colon cells and from 25 to 30% in LT97 cells.
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Effects of H2O2, trans-2-Hexenal, and HNE on APC, KRAS, and TP53 (Comet FISH)
Comet FISH experiments were evaluated by first determining the total number of APC, KRAS, and TP53 signals per cell. The expected number of spots in a normal metaphase or interphase nucleus should be two. Additionally, cells without signals or with only one signal were counted, and the hybridization efficiency was determined to be 83.8 ± 2.7% for APC, 81.0 ± 2.7% for KRAS, and 80.9 ± 4.6% for TP53. Only about 2.2% of all comets (APC: 1.9%, KRAS: 2.4%, TP53: 2.2%) had three signals, which indicated breakage and isolated migration of APC, KRAS, or TP53 fragments. This effect was neither gene related nor compound related (results not shown). Only cells with two fluorescent spots were used for the further evaluation.
Comet FISH results are shown in Tables 1 and 2A–C (second columns) for primary colon and LT97 cells, respectively. Statistically significant dose-related migration of the FISH signals into the comet tails was observed for all tested compounds in both cell types and for all three genes. Gene responses were as follows (see Tables 1 and 2):
The APC.
There were clear-cut compound-induced effects on overall APC migration, which were significant (in comparison to the "0" controls) after treatment with 75–150µM of H2O2, 800–1600µM of trans-2-hexenal (primary colon cells and LT97 cells), and 200–250µM of HNE (LT97 cells) or 150–250µM of HNE (primary colon cells). When comparing the total DNA migration (TI [%]) to the migration of APC, significant differences were apparent between the compounds and between the cells. APC was more susceptible than total DNA to both lipid peroxidation products, but only in primary colon cells.
The KRAS.
This gene was also susceptible, indicated by an enhanced migration of KRAS in both primary colon cells and LT97 cells, for all three investigated compounds (37.5–150µM of H2O2, 800–1600µM of trans-2-hexenal, and 150–250µM of HNE). Significant differences between migration of total DNA and KRAS were again only apparent in primary colon cells for lipid peroxidation products, but not for H2O2.
The TP53.
Clear-cut H2O2-, trans-2-hexenal-, and HNE-induced effects on TP53 migration were found in primary colon cells (37.5–150µM of H2O2, 800–1600µM of trans-2-hexenal, and 150–250µM of HNE). In LT97 cells, the TP53 migration into the comet tails was also significantly different from the negative controls for all three compounds at similar concentrations. TP53 damage in comparison with damage of total DNA was significant in primary human colon cells treated with all three compounds and in LT97 cells treated with trans-2-hexenal and HNE.
Comparison of the genes.
Figures 1A–1C compare the relative sensitivities of APC, KRAS, and TP53. Significant differences between TP53 and APC/KRAS were found in H2O2-treated primary colon cells (Fig. 1A), in which TP53 was more sensitive to the action of 150µM H2O2 than APC and KRAS. In all experiments with the lipid peroxidation products, TP53 (Figs. 1B and 1C) was significantly more sensitive than APC or KRAS. This particular sensitivity is especially apparent in LT97 cells treated with HNE.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endogenously formed risk factors, such as ROS, products of lipid peroxidation, nitroso compounds, and bile acids (DeKok and van Maanen, 2000
) may enhance an individual's risk of developing colorectal cancer (World Cancer Research Fund and American Institute for Cancer Research, 1997). Basic studies are needed to understand how putative carcinogens interact with the relevant target genes of human colon cells. These types of studies are largely unavailable, mainly due to the lack of appropriate experimental techniques. The quantification of gene migration using Comet FISH, as reported here, however, offers a unique possibility to study the sensitivity of genes toward genotoxic compounds (Bock et al., 1999; McKelvey-Martin et al., 1998). Since initiation and progression of colorectal carcinogenesis involves damage in the tumor suppressor genes APC and TP53 as well as in the protooncogene KRAS (Vogelstein et al., 1988), it was a logical step to add these genes to the panel of Comet FISH targets and to use the method to study cancer risk factors in appropriate human colon cells.
The protocol used in our Comet FISH studies resulted in a good hybridization efficiency (sensitivity and stringency) comparable to the initial studies reported by Rapp et al. (2001). Despite the potentials of this technique, only very few studies have employed Comet FISH to investigate damage induced by chemical carcinogens. One reason probably is that the Comet FISH method requires the use of cells with a stable diploid karyotype, which is not a common property of most human cell lines. Instead, they usually have very unstable karyotypes (Eisenbrand et al., 2002), which results in unaccounted for FISH signals that cannot be adequately interpreted. Our approach has therefore been based on the use of primary human colon cells isolated from healthy, nontumor tissues of surgical samples (Schäferhenrich et al., 2003b). Moreover, we have established a human adenoma cell line which is characterized by a largely stable karyotype (Schäferhenrich et al., 2003a).
In the current study and in our previous work (Schäferhenrich et al., 2003a,b), we have shown that the main lipid peroxidation products trans-2-hexenal and HNE cause damage of APC, KRAS, and TP53 genes in human colon cells, while no increase of gene damage was seen after H2O2 treatment of the cells. The concentrations of the individual compounds were different, but all investigated dosages were of similar genotoxic potency. TP53 was damaged more by the investigated risk factors than APC and KRAS. The particular sensitivity of TP53 was apparent in primary colon cells as well as in LT97 adenoma cells. This may be due to the fact that LT97 cells normally carry damaged APC and KRAS, but undamaged TP53 (Richter et al., 2002). In normal colonocytes, APC and KRAS were also sensitive to damage. These findings are highly interesting when considering the sequence of mutational events that occur during human colon carcinogenesis (Vogelstein et al., 1988). APC and KRAS mutations transform normal epithelial (stem) cells into initiated, more rapidly proliferating cells to yield dysplasia and small adenoma. TP53 mutations in adenoma are then crucial alterations leading to further progression and to carcinoma. Based on our studies, we may conclude that HNE could potently contribute to both cancer initiation and progression in the colon, if it is formed there in sufficient amounts.
We may also conclude that the technique of combining the Comet Assay and FISH offers the unique possibility to study simultaneously the damage of total DNA and certain genes in vitro. APC, KRAS, and TP53 genes were significantly damaged by oxidative risk factors in nontransformed primary human colon cells. TP53 was particularly sensitive to HNE in adenoma cells. Alterations in APC and KRAS are probably the first steps of initiation in normal colon cells, whereas mutations in TP53 are pivotal for the transition from adenoma to carcinoma. Therefore, all risk factors may potentially contribute to cancer initiation, if exposure occurs, whereas HNE seems to be important for cancer progression. It still remains to be studied to which extent the observed genotoxicity is related to mutagenicity. Altogether, however, our findings on the enhanced migration of APC, KRAS, and TP53 genes indicate that there is an interaction of the chemicals with these particular genes in human colon cells. Thus, we have obtained an improved basis for understanding the links between the investigated genotoxic agents and events related to initiation and progression of colorectal cancer in humans.
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ACKNOWLEDGMENTS |
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This work was supported by Bundesministerium für Ernährung, Landwirtschaft und Forsten 99 HS 039—"Toxizität von 2-Alkylcyclobutanon;" Deutsche Forschungsgemeinschaft PO 284/8-1—"Molecular mechanisms of colon cancer chemoprevention: studies on the potential of intestinal fermentation products to induce glutathione S-transferases in colonic epithelium cells." We are obliged to Dr W. M. Pool and Dr G. Hovhannisyan for editorial assistance.
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