Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy

Soultanakis, Rebecca P; Melamede, Robert J.; Bespalov, Ivan A.; Wallace, Susan S.; Beckman, Kenneth B.; Ames, Bruce N.; Taatjes, Douglas J.; Janssen–Heininger, Yvonne M. W.

doi:10.1016/s0891-5849(00)00185-4

Cited by 79 publications

(84 citation statements)

References 18 publications

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“…For comparison, the fluorescence of scr-siRNA cells exposed to 500 M H 2 O 2 for 30 min, a situation that induces high levels of 8-oxoG, is presented. Other laboratories using the same methodology have also demonstrated a punctuate pattern, with the signal more intense on the periphery of the nucleus, probably at heterochromatic DNA (7,32,40; see also the Trevigen web site). For the control, the results for the DAPI signal and the secondary antibody-only control are also presented.…”

mentioning

confidence: 86%

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Souza-Pinto

Maynard

Hashiguchi

et al. 2009

Molecular and Cellular Biology

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Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.Oxidative DNA damage is generated at high levels in mammalian cells, even in cells not exposed to exogenous sources of reactive oxygen species. Several kinds of DNA modifications are formed upon oxidative stress (8). The most prevalent modifications, quantitatively, are single-strand breaks and oxidized bases. Clustered DNA damage, when two or more modifications are closely positioned in opposite strands, is detectable after gamma irradiation and has recently been shown to be generated by normal oxidative metabolism (3,35). One unique aspect of such clustered lesions is that they can be converted into double-strand breaks (DSB) if a DNA glycosylase removes the two opposite bases and an apurinic/apyrimidinic (AP)-endonuclease cleaves the resulting abasic sites. Thus, although quantitatively minor, DSB are possible outcomes of oxidative DNA damage.

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confidence: 86%

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Souza-Pinto

Maynard

Hashiguchi

et al. 2009

Molecular and Cellular Biology

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“…Immunohistochemical staining of 8-oxoguanine in DNA Sections stained with Neutral Red and then with antilaminin b2/c1 chain monoclonal antibody were next immunostained for 8-oxoG in DNA using a specific mouse monoclonal recombinant antibody Fab 166, generated by repertoire cloning and combinatorial phage display as previously reported (Bespalov et al 1996(Bespalov et al , 1999Soultanakis et al 2000).…”

Section: Immunohistochemical Staining Of Lamininmentioning

confidence: 99%

“…However, the data obtained with these techniques can be compromised because of artefacts derived in isolating DNA and processing samples (Yin et al 1995;Loft and Poulsen 1999;Dizdaroglu et al 2002). On the other hand, immunohistochemical staining of 8-oxodG and 8-oxoG in nuclear and mitochondrial DNA in situ in tissues or cells in culture using, respectively, antibodies specific for 8-oxodG and 8-oxoG (Yarborough et al 1996;Nehls et al 1997;Tanaka et al 1997;Toyokuni et al 1997Toyokuni et al , 1999Soultanakis et al 2000) have provided data of their localisation in tissue sections and cells. The merits of the histochemical technique are that it requires only small pieces of tissue and a small number of culture cells and is not subject to the artefacts caused by in vitro techniques.…”

Section: Introductionmentioning

confidence: 99%

A new technique for the quantitative assessment of 8-oxoguanine in nuclear DNA as a marker of oxidative stress. Application to dystrophin-deficient DMD skeletal muscles

Nakae

Stoward

Bespalov

et al. 2005

Histochem Cell Biol

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This is the first report on the development of an immunohistochemical technique, combined with quantitative image analysis, for the assessment of oxidative stress quantitatively in nuclear DNA in situ, and its application to measure DNA damage in Duchenne muscular dystrophic (DMD) muscles. Three sequential staining procedures for cell nuclei, a cell marker, and a product of oxidative DNA damage, 8-oxoguanine (8-oxoG), were performed. First, the nuclei in muscle sections were stained with Neutral Red followed by the capture of their images with an image analysis system used for absorbance measurements. Second, the same sections were then immunostained for laminin in basement membranes as the cell marker. Next, the sections were treated with 2 N HCl to remove the bound Neutral Red and to denature tissue DNA. Third, the sections were immunostained for 8-oxoG in DNA, using diaminobenzidine (DAB) to reveal the antibody complex. This was followed by capture of the images of the immunostained sections as previously. The absorbances at 451.2 nm of bound Neutral Red and DAB polymer oxides, the final product of 8-oxoG immunostaining, were measured in the same myonuclei in the sections. Analysis of these absorbances permitted indices of the 8-oxoG content, independent of the nuclear densities, to be determined in nuclear DNA in single myofibres and myosatellite cells surrounded by basement membranes. We found that the mean index for the myonuclei in biceps brachii muscles of 2- to 7-year-old patients was 14% higher than that in age-matched normal controls. This finding of the increased oxidative stress in the myonuclei in young DMD muscles agrees with the previous reports of increased oxidative stress in the cytoplasm in the DMD myofibres and myosatellite cells. The present technique for the quantitative assessment of oxidative stress in nuclear DNA in situ is applicable not only in biomedical research but also in the development of effective drugs for degenerative diseases related to oxidative stress.

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“…It was shown that DNA damaged by H202 and ionizing radiation contained antigenic sites, and such sites co-localized with nuclear and mitochondrial DNA. In addition, the antigenic sites were removed when the cells were treated with the bacterial repair enzyme Fpg, which strongly indicates that the antigen is, indeed, 8-oxo-dG (53).…”

Section: Methods Used For the Measurement Of Oxidative Dna Damagementioning

confidence: 95%

Mitochondria, oxidative DNA damage, and aging

Anson

Bohr

2000

AGE

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Protection from reactive oxygen species (ROS) and from mitochondrial oxidative damage is well known to be necessary to longevity. The relevance of mitochondrial DNA (mtDNA) to aging is suggested by the fact that the two most commonly measured forms of mtDNA damage, deletions and the oxidatively induced lesion 8-oxo-dG, increase with age. The rate of increase is species-specific and correlates with maximum lifespan.It is less clear that failure or inadequacies in the protection from reactive oxygen species (ROS) and from mitochondrial oxidative damage are sufficient to explain senescence. DNA containing 8-oxo-dG is repaired by mitochondria, and the high ratio of mitochondrial to nuclear levels of 8-oxo-dG previously reported are now suspected to be due to methodological difficulties. Furthermore, MnSOD -/+ mice incur higher than wild type levels of oxidative damage, but do not display an aging phenotype. Together, these findings suggest that oxidative damage to mitochondria is lower than previously thought, and that higher levels can be tolerated without physiological consequence.A great deal of work remains before it will be known whether mitochondrial oxidative damage is a "clock" which controls the rate of aging. The increased level of 8-oxo-dG seen with age in isolated mitochondria needs explanation. It could be that a subset of cells lose the ability to protect or repair mitochondria, resulting in their incurring disproportionate levels of damage. Such an uneven distribution could exceed the reserve capacity of these cells and have serious physiological consequences.

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Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy

Cited by 79 publications

References 18 publications

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

A new technique for the quantitative assessment of 8-oxoguanine in nuclear DNA as a marker of oxidative stress. Application to dystrophin-deficient DMD skeletal muscles

Mitochondria, oxidative DNA damage, and aging

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