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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 20, 2003 as doi:10.1096/fj.02-0689fje. |
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,2
* Faculty of Pharmacy, University of Toronto; and
Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada
2Correspondence: Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ontario, Canada M5S 2S2. E-mail: pg.wells{at}utoronto.ca
SPECIFIC AIMS
To determine the in vivo role that the particular oxidative DNA lesion 8-oxo-7,8-dihydrodeoxyguanosine (8-oxo-dG) plays in promoting reactive oxygen species (ROS)-related diseases, we engineered a binary line of transgenic mice conditionally expressing the highly active bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (fpg) (Fig. 1
, insert). The central objective was to determine whether expression of this repair enzyme in transgenic mice can reduce endogenous levels of the 8-oxo-dG DNA lesion in vivo and, if so, whether this heterologous repair can be regulated.
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PRINCIPAL FINDINGS
1. Fpg can be expressed and is functional in mammalian cells
HEK293 cells were transiently transfected with fpg under the control of the CMV promoter and assayed for DNA repair activity, using a double-stranded oligonucleotide containing a single internal 8-oxo-dG residue as the substrate. A sevenfold increase in repair activity was observed relative to controls, indicating that fpg can be expressed and is functional in mammalian cells. Cells were subsequently transfected with components of the "tet-off" tetracycline-dependent transactivator (tTA)/tetracycline operator (tetO) system described elsewhere, in which the addition of tetracycline blocks gene expression by binding to and inactivating the tTA element (Fig. 1
, insert). DNA repair activity was found to be 30-fold higher in cells cotransfected with both CMV-tTA and tetO-fpg than controls.
2. 8-Oxo-dG repair can be regulated by tetracycline in stable cell lines
CHO-AA8 cells were stably cotransfected with CMV-tTA and tetO-fpg and the fpg gene was confirmed by Southern blot. Individual clones were cultured in the presence of tetracycline or its solvent control in order to examine their tTA-mediated expression of fpg (Fig. 1
, insert). DNA repair activity was up to ninefold higher in stable cell lines relative to the parental line (Fig. 2
A). With the addition of tetracycline, repair activity was reduced by up to 80%. The bacterial fpg enzyme possesses an apyrimidinic/apurinic (AP) lyase activity that catalyzes successive ß and 
elimination reactions (in our experiment, visualized on the membrane as the two lower bands) that convert an AP site to a single-strand break. In contrast, the mammalian Ogg1 enzyme carries out only the ß elimination reaction. In our experiment, this latter biochemical activity is visualized as a single band, the higher of the two produced by fpg. Thus, the disappearance of the lower band of the pair by the addition of tetracycline to stable clones is an indication of the disappearance of fpg-mediated, rather than Ogg-mediated oligonucleotide excision. We observed that bacterial fpg-mediated DNA repair could be regulated with tetracycline in a mammalian system.
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3. tTA is expressed in the neonatal cortex in actin-tTA transgenic mice
Transgenic mice were engineered in which tTA was placed under the control of a human ß-actin promoter (Fig. 1
, insert). To test the temporal and spatial localization of tTA, transgenic actin-tTA mice were cross-bred to transgenic tetO-LacZ reporter mice (developed elsewhere) and resulting embryos/offspring were stained for ß-galactosidase activity (Fig. 3
). Prenatal expression was minimal (Fig. 3B
, 3D
) as was staining in adult (P50) brains (Fig. 3O
), where it was restricted to the dentate gyrus (Fig. 3O
). tTA expression was much more widespread in neonatal brain (P2) of binary transgenic actin-tTA:tetO-LacZ mice and was high throughout the neocortex (Fig. 3G
, 3J
). Low levels of cortical staining were also observed in tetO-LacZ animals (Fig. 3F
, 3I
). However, this was much lower than that observed in binary transgenic actin-tTA:tetO-LacZ animals (Fig. 3G
, 3J
). The staining observed within the cerebellum of these animals was largely due to leakiness of the tetO-LacZ transgene (Fig. 3F
). No tTA expression was observed outside the central nervous system (CNS) of the neonate or adult (data not shown).
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To determine whether expression of the LacZ reporter gene could be regulated in this system in vivo by tetracylines, pregnant females were given doxycycline in the drinking water throughout gestation; newborn pups were injected with doxycycline and analyzed for their pattern of ß-galactosidase activity reflecting tTA function on postnatal day 2. Actin-tTA:tetO-LacZ pups treated with doxycycline (Fig. 3M
) exhibited a reduction in cortical ß-galactosidase activity similar to that observed in doxycycline-treated littermates containing tetO-LacZ alone (Fig. 3L
), implying that the tTA-mediated expression of ß-galactosidase is inhibited by doxycycline. Since the highest level of ß-galactosidase expression was observed within neonatal cortex, this tissue was chosen for the subsequent analysis of DNA repair activity in binary transgenic actin-tTA:tetO-fpg mice.
4. Doxycycline regulates the endogenous level of 8-oxo-dG in vivo in binary transgenic actin-tTA:tetO-fpg mice
Transgenic mice were engineered in which fpg was placed under the control of a minimal promoter containing heptad repeats of the tet operator sequence (tetO). To assess the efficacy of transgenic fpg to enhance DNA repair in vivo, we determined the endogenous level of 8-oxo-dG in binary transgenic actin-tTA:tetO-fpg mice as well as in control littermates containing the tetO-fpg or actin-tTA transgene alone. The level of 8-oxo-dG analyzed by HPLC-EC in postnatal day 2 cortex and hippocampus was reduced by 55% in the binary actin-tTA:tetO-fpg mice compared with control littermates (Fig. 3P
) (P<0.001). Consistent with this, 8-oxo-dG levels in control littermates (actin-tTA-alone and tetO-fpg-alone mice) were not significantly different from one another. These data indicate that tTA-driven expression of fpg results in a reduction of 8-oxo-dG levels in vivo. In vivo administration of doxycycline resulted in a down-regulation of DNA repair activity to control levels in binary transgenic actin-tTA:tetO-fpg mice. Taken together, these results demonstrate that expression of bacterial fpg reduces endogenous levels of 8-oxo-dG within the murine cerebral cortex during a specific developmental window to roughly half that of control levels and that this extra repair activity can be regulated by the administration of doxycycline.
CONCLUSIONS AND SIGNIFICANCE
8-Oxo-dG is a pervasive oxidative DNA lesion formed by endogenous oxidative stress and enhanced by drugs and environmental chemicals (Fig. 1)
. This lesion results in transcriptional errors and mutations and is linked to neurodegeneration, teratogenesis, cancer, and other pathologies. However, a major question remains regarding the mechanistic relevance of oxidative DNA damage, particularly specific oxidative lesions, as opposed to other effects such as alternative kinds of DNA damage (e.g., adducts, methylation), damage to other cellular macromolecules (e.g., proteins, lipids), or reversible alterations in ROS-dependent signaling pathways, all of which could contribute to disease. The original aim of the study was to directly address this fundamental question by engineering a mouse that transgenically expresses a highly active bacterial pathway for the repair of a specific type of oxidative DNA damage potentially relevant to ROS-dependent diseases. This approach complements the development of knockouts for a particular DNA repair pathway yet avoids their potential for embryolethality. Its regulable design (Fig. 1
, insert) also allows for partial fpg suppression should excessive repair prove harmful. From a clinical perspective, the results suggest an important role for interindividual variability in DNA repair as a risk factor for disease.
The most significant and novel finding reported in our paper is the demonstration of a substantial 50% reduction in the endogenous level of 8-oxo-dG in the CNS of newborn mice transgenically expressing higher repair activity. This is the first demonstration that endogenous oxidative DNA damage can be reduced via genetic engineering. In other studies of in utero embryonic death and birth defects, this degree of modulation in 8-oxo-dG levels has been shown to have substantial pathological implications. Our results are interesting in that these mice and their controls have normal intrinsic DNA repair activity and have not been exposed to ROS-initiating xenobiotics, so the ability of the mice transgenically expressing higher repair activity to substantially reduce their normal endogenous levels of 8-oxo-dG is remarkable. We further show that DNA repair can be regulated in a temporally and spatially specific manner, allowing its role as a modulatory factor in developmental brain dysfunction and neurodegenerative disease to be determined in vivo. The findings reported here provide an important advance in the field of reactive oxygen species and disease, namely, the first experimental model for determining in vivo the pathophysiological relevance of reducing a specific oxidative DNA lesion.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0689fje; doi: 10.1096/fj.02-0689fje 
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