Department of Pharmacology and Toxicology and School of Environmental Studies, Queen's University, Kingston, Ontario, Canada 27L3N6
Received February 13, 2004; accepted June 10, 2004
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ABSTRACT |
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Key Words: TCDD; homologous recombination; AhR; DNA repair.
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INTRODUCTION |
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Damage to DNA is well known to contribute to the development of cancer. Although the cell has developed an array of mechanisms to repair damaged DNA, inherited defects in DNA repair mechanism such as those seen in patients with xeroderma pigmentosum, ataxia telangiectasia, and Nijmegen breakage syndrome, enhance the susceptibility of these patients to certain types of cancers (Rotman and Shiloh, 1998). One particular form of DNA damage, DNA double strand breaks (DSBs), which can occur "spontaneously" in the genome, or as a consequence of ionizing radiation or chemical exposure, are thought to be particularly detrimental to the cell (Jackson, 2002
).
DSBs can be repaired by two distinct repair pathways: non-homologous end-joining and homologous recombination (HR). Non-homologous end-joining does not require extensive homology and involves the direct rejoining of the two ends of the broken DNA, which is accomplished with the cooperation of a number of different proteins involved in the recognition and targeting of the damaged DNA followed by the removal or addition of a few base pairs and finally ligation (van Gent et al., 2001). On the other hand, repair of DSBs via HR requires homologous donor duplex DNA, which serves as a template for repair. The donor DNA can be an undamaged allele, sister chromatid, or an ectopic DNA region that shares significant homology with the damaged site. Consequently, if the donor DNA is completely homologous, this type of repair will result in a normal functional gene, indistinguishable from the gene prior to the DSB. However, DSBs can induce HR between DNA that are not completely homologous and thus result in genetic changes. HR can also involve gene deletion events that can lead to the loss of critical genetic information (Bishop and Schiestl, 2000
, 2001
; Nickoloff and Brenneman, 2001
). Thus, DSB repair via HR can lead to various deleterious events, including the loss of heterozygosity, translocations, and gene deletions or amplifications (Jackson and Loeb, 2001
; Pierce et al., 2001
; Van den et al., 2002
).
Since TCDD does not directly damage DNA and is not considered a potent genotoxin, unlike many known carcinogens, the molecular events leading to the development of cancer as a result of TCDD exposure still need to be elucidated. Several studies have demonstrated that while TCDD lacks the ability to initiate carcinogenesis, it can act as a potent tumor promoter (reviewed in Dragan and Schrenk, 2000). It is well known that cells are continuously exposed to DNA damaging agents including ultraviolet light, xenobiotics and endogenously produced reactive oxygen species. While the cell does have mechanisms to repair this damage, these repair mechanisms are not error free. Similarly, as discussed above DSB repair via HR may result in detrimental genetic changes. Therefore, we hypothesize that TCDD exerts its carcinogenicity, in part, by affecting the repair of DNA DSBs caused by either endogenous and exogenous DNA damaging agents. Furthermore, given that the specific binding of TCDD to the AhR and the subsequent downstream signaling pathway, appear to mediate a variety of the toxic effects seen upon exposure to TCDD (Fernandez-Salguero et al., 1996
; Gonzalez and Fernandez-Salguero, 1998
), we hypothesize that the AhR plays a role in TCDD's effects on HR repair of DSBs.
In order to investigate our hypothesis, we used a previously characterized model (Taghian and Nickoloff, 1997) that utilizes the Saccharomyces cerevisiae mitochondrial endonuclease, I-SceI, to initiate a site specific DSB in a reporter tandem repeat neomycin (neo) recombination substrate which is stably integrated in the Chinese hamster ovary (CHO) cell line, strain 33. Previous studies have already shown that this yeast endonuclease efficiently induces a DSB in this model and induces a high rate of recombination (Choulika et al., 1995
; Taghian and Nickoloff, 1997
). Therefore, we were able to use this model to investigate the effect of TCDD exposure on the repair of an artificially created DSB in mammalian cells and evaluate the role of the AhR in this process.
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MATERIALS AND METHODS |
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Plating efficiency experiments (i.e., cell death experiments) were conducted in a similar manner as the recombination assay except that CHO 33 cells were plated at a density of 300 cells per 10 cm dish, cells were grown only in fresh cell culture media without G418, and the colonies were scored after one week.
Southern blot analysis. To determine the types of HR events (gene conversion or gene deletion) induced by TCDD, G418-resistant colonies were identified and isolated using a light microscope and colonies were expanded in one well of a six-well plate in culture media containing G418. Once confluent, genomic DNA was then isolated using a commercially available Qiagen DNeasy Tissue Kit (Qiagen Incorporated, CA). The isolated genomic DNA was digested with EcoRI and I-SceI (New England BioLabs Inc., Mississauga, ON). Digested DNA was run on a 1.5% agarose gel and transferred to a nylon membrane and a neo cDNA was used as the probe (Taghian and Nickoloff, 1997). A single 10.3 KB band represents the product of a gene conversion event while a deletion event results in a single 5.2 KB band. The I-SceI site is lost in both types of HR event and thus digestion with I-SceI should not alter the size of the band.
Aryl hydrocarbon receptormediated DSB repair studies. In order to evaluate the effects of the AhR on TCDD-mediated alterations in DSB repair, transfection assays were performed as described above and cells were exposed to TCDD (500 pM) alone or in the presence of the AhR antagonist, -naphthoflavone (
-NF), at a final concentration of 0.1 µM.
Statistical analysis. Results were analyzed using a standard, computerized statistical program (GraphPad Prism 3.0). Groups were compared using a one factor analysis of variance (ANOVA). The minimum level of significance used throughout was p < 0.05.
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RESULTS |
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DISCUSSION |
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DNA DSBs are generated when both strands of the DNA double helix are broken in close proximity. Sources of DNA DSBs include ionizing radiation and genotoxic xenobiotics (van Gent et al., 2001). HR is one mechanism by which DNA DSBs can be repaired using an undamaged sister chromatid or a homologous chromosome as a template for repair (van den et al., 2002
). While HR is a necessary repair mechanism, erroneous repair via HR can produce detrimental genetic changes, such as loss of heterozygosity and gene deletions or duplications, which can lead to genome instability and carcinogenesis. In this study we used the Saccharomyces cerevisiae mitochondrial endonuclease I-SceI to induce HR by initiating a site specific DSB in the neo tandem repeat recombination substrate located in the genome of the CHO 33 cell line (Taghian and Nickoloff, 1997
). The yeast endonuclease I-SceI recognition sequence is rare in the mammalian genome, making this model a very useful tool to study DSB repair by HR (Choulika et al., 1995
; Taghian and Nickoloff, 1997
; Theirry et al., 1991
). Our results support the fact that DSBs are potent inducers of HR since I-SceI transfected cells exhibited a significantly higher HR frequency than cells that were transfected with the control plasmid (Fig. 3). More importantly, here we demonstrate that exposure to TCDD can lead to an increase in the frequency of DNA DSB repair, since TCDD exposure alone did not lead to an increase in HR in CHO 33 cells when a DSB was not initiated while exposure of I-SceI transfected cells to TCDD resulted in a significant increase in HR compared to I-SceI transfected cells that were exposed to the vehicle (Fig. 3). As previously mentioned, TCDD can act as a tumor promoter and we hypothesize that TCDD can act by increasing the repair of DSBs created either endogenously or via exposure to DNA damaging agents. This increased frequency of HR can then lead to a greater possibility of repair mistakes and ultimately genomic instability.
In this study we also investigated the type of HR events that occurred as a result of the repair of a DSB followed by exposure to TCDD by Southern blot analysis. Similar to our previous studies using CHO 36 cells, our results using CHO 33 demonstrate that TCDD changes the proportion of gene conversion (80%) versus gene deletion (20%) events. This is based on previous studies (Kim et al., 2001; Taghian and Nickoloff, 1997
) which showed that CHO 33 cells underwent mainly gene conversion events (97%). While gene conversion events are considered a conservative process, which involves the direct copying of the genetic information from the donor template, this repair pathway can be detrimental if the donor DNA is not completely homologous. Gene conversion events involving heterologous DNA can lead to the loss of heterozygosity of key genes (e.g., tumor suppressor gene), which can promote tumorigenesis (Johnson and Jasin, 2000
; Pfeiffer et al., 2000
). Nevertheless, TCDD's ability to increase the frequency of gene deletion events may be particularly detrimental since there is a direct loss of genetic material and therefore a greater likelihood of important genes being deleted.
Since many of the toxicities induced by TCDD are believed to be mediated via the AhR, we investigated the role of the AhR in TCDD's ability to increase DSB repair. Our results suggest that the AhR does play a role in this increase since the AhR antagonist -NF significantly reduced TCDD-induced HR frequency (Fig. 4). The binding of TCDD to the AhR results in the induction or repression of many AhR-controlled genes, some of which are involved in DNA repair (Puga et al., 2000
). HR involves a number of proteins including members of the RAD50 group and the breast cancer susceptibility genes, BRCA1 and BRCA2, and it is likely that TCDD affects the transcription of these genes. While the function of BRCA1 remains unknown, many studies implicate this gene in cell-cycle control and DNA repair (Gowen et al., 1998
; MacLachlan et al., 2002
). Recently, Rattenborg et al. (2002)
demonstrated in vitro that TCDD down regulated the basal as well as the estradiol-inducible BRCA1 promoter activity. These studies are in agreement with earlier studies showing inhibition of BRCA1 expression by the polycyclic aromatic hydrocarbon benzo[a]pyrene (Jeffy et al., 1999
). Given that TCDD appears to increase the frequency of DSB repair in our studies, we suspect that while TCDD can repress BRCA1 activity, it can induce other proteins involved in HR.
While our results suggest an involvement of the AhR in TCDD's modulation of DSB repair, these results are somewhat in contrast to those demonstrated by Schiestl et al. (1997) where TCDD-induced deletion events did not correlate with induction of AH hydroxylase activity in embryos and in a lymphoblastoma cell line. A number of potential reasons may explain our conflicting results: (1) Our study investigated the role of TCDD in DSB repair induced by a specific insult (i.e., I-SceI-induced DSB) whereas the assumption in the Schiestl paper is that TCDD is either directly initiating the deletion events or that TCDD modulates the deletion event process initiated by an unknown mechanism. Our study uses DSBs as the initiating factor, which we and others have shown are strong inducers of HR in CHO 33 cells and as shown in Figure 3 TCDD does not increase HR in these cells but rather modulates the frequency of DSB-induced HR. (2) While TCDD alone did not effect HR in CHO 33 cells (Fig. 3) and our
-NF studies suggest a role for the AhR in TCDD's effects on DSB-induced HR, we cannot dismiss the effects of TCDD and
-NF on the glucocorticoid receptor which has been shown to bind with these two compounds and also bind to the MMTV promoter which drives the 5'-neo gene in the reporter construct in the CHO 33 cells (Fig. 1). (3) And furthermore, while our results using
-NF support a role for the AhR, it is important to remember that the use of all chemical probes have associated problems. While the concentration of
-NF used in our study was carefully chosen after a considerable literature review, studies have demonstrated that even at this low concentration
-NF also has partial agonist properties. However, we did not find that
-NF had a similar effect to that of TCDD but rather antagonzied the effects of TCDD. It is clear that additional studies are necessary to further implicate the AhR in TCDD's effects on DNA DSB repair.
In summary, we demonstrate that exposure to TCDD can affect the frequency and types of HR repair of DNA DSBs potentially through the AhR and propose that this may be a potential molecular mechanism mediating the carcinogenicity of this highly toxic environmental contaminant.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (613) 533-6412. E-mail: winnl{at}biology.queensu.ca.
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