Download Two distinct pathways of cell death triggered by oxidative damage to

Two distinct pathways of cell death triggered by oxidative damage to
nuclear and mitochondrial DNAs
Sugako Oka, Mizuki Ohno, Daisuke Tsuchimoto, Kunihiko Sakumi, Masato Furuichi, and
Yusaku Nakabeppu
Supplemental materials
Mouse embryonic fibroblasts
By means of gene targeting, we previously established Ogg1 gene knockout mice and
maintained heterozygous mice (Ogg1+/-) backcrossed to C57BL/6J for 5 generations
(Sakumi et al., 2003). MEFs were isolated from embryos (13.5 days postcoital) obtained by
mating Ogg1+/- mice, and cultured in DMEM (Invitrogen) supplemented with 10% heatinactivated FBS, 100 µg/ml streptomycin, and 100 units/ml penicillin, at 37˚C in 5% CO2.
MEFs were maintained for 40 passages and spontaneously immortalized. Their genotypes
were determined by genomic PCR as described previously (Sakumi et al., 2003), and
Ogg1+/+ (5L, 9L) and Ogg1-/- (7L, 12L) MEF lines were independently established.
Detection of reactive oxygen species
In order to confirm that menadione produces reactive oxygen species (ROS) within cells,
intracellular ROS such as hydroxyradical or peroxynitrite were detected using
hydroxyphenyl fluorescein (HPF, Daiichi Pure Chemicals, Japan). Thirty minutes after
exposure to 50 µM menadione, HPF (10 µM) was applied to each MEF culture in fresh
medium and the cells were further incubated for 30 min. Grayscale digital images were
obtained with an Axio-Skop2-equipped Axio Cam with Axio Vision 3.1 (Carl Zeiss), and are
shown in pseudocolor.
Construction of plasmids and transfection
pcDNA3.1Hyg(-):hOGG1-1a and pcDNA3.1Hyg(-):hOGG1-2a were constructed by
inserting the XbaI/BamHI fragment of pET8c:hOGG1-1a and pET8c:hOGG1-2a (Nishioka
et al., 1999) into the XbaI/BamHI region of pcDNA3.1 Hyg(-) (Invitrogen). To construct
pcDNA3.1PURO:hOGG-1a, a NaeI fragment carrying the coding sequence for hygromycin
B phosphotransferase was replaced with a SalI fragment of pPGK-PURU carrying the
coding sequence for puromycin-N-acetyltransferase (Tucker et al., 1996). Ogg1-/- MEFs
(7L) were transfected with plasmid DNA with the aid of EFFECTENETM Transfection
Reagent (Qiagen), according to the manufacturer’s instructions. Stable transfectants were
selected in the presence of 250 µg/ml hygromycin B or 25 µg/ml puromycin, and were
maintained in the presence of 100 µg/ml hygromycin B or 12.5 µg/ml puromycin,
respectively.
RT-PCR
RT-PCR for Mutyh and Gapdh mRNA was performed as described previously (Ichinoe et al.,
2004).
Single-stranded DNA index
To quantify immunoreactivity of single-stranded DNA, images of immunoreactivity were
converted to 256-level gray-scale images, whereas nuclear DAPI staining images were
converted to binary monochromes using Adobe PhotoShop 5.0J (Adobe Systems). From the
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gray-scale images of single-strand DNA immunoreactivity, the signals were detected in
areas corresponding to nuclei represented by the monochrome DAPI images, and then
measured using NIH Image 1.61. At least 30 cells were analyzed, and an average value of
the integrated pixel density per cell was calculated as the index for each sample.
Immunostaining
Anti-mitochondrial transcription factor A (TFAM) antibody was obtained from Molecular
Probes. To verify the specificity of 8-oxoG immunoreactivity in mitochondrial DNA, slides
were pretreated with 10 µg/ml MutM protein (Sigma) in nicking buffer (10 mM Tris-HCl, 5
µM ZnCl2, 0.5 mM DTT, 0.5 mM EDTA, 1.5% glycerol, 100 µg/ml bovine serum albumin)
or 1000 U/ml of DNase I (Sigma) in reaction buffer (50 mM Tris-HCl [pH 7.5], 0.1 mM
MgCl2) at 37˚C for 1 h after RNase treatment. They were then subjected to
immunofluorescence microscopy with anti-8-oxoG antibody.
Supplemental references
Ichinoe A, Behmanesh M, Tominaga Y, Ushijima Y, Hirano S, Sakai Y, Tsuchimoto D,
Sakumi K, Wake N and Nakabeppu Y (2004) Identification and characterization of
two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts.
Nucleic Acids Res 32: 477-487
Nishioka K, Ohtsubo T, Oda H, Fujiwara T, Kang D, Sugimachi K and Nakabeppu Y (1999)
Expression and differential intracellular localization of two major forms of human 8oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs. Mol
Biol Cell 10: 1637-1652
Sakumi K, Tominaga Y, Furuichi M, Xu P, Tsuzuki T, Sekiguchi M and Nakabeppu (2003)
Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene
disruption. Cancer Res 63: 902-905
Tucker KL, Beard C, Dausmann J, Jackson-Grusby L, Laird PW, Lei H, Li E and Jaenisch R
(1996) Germ-line passage is required for establishment of methylation and
expression patterns of imprinted but not of nonimprinted genes. Genes Dev 10:
1008-1020
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