Heme oxygenase-1 and carbon monoxide modulate DNA repair through ataxia-telangiectasia mutated (ATM) protein - PubMed (original) (raw)
Heme oxygenase-1 and carbon monoxide modulate DNA repair through ataxia-telangiectasia mutated (ATM) protein
Leo E Otterbein et al. Proc Natl Acad Sci U S A. 2011.
Abstract
Stability and repair of DNA is of principal importance in cell survival. Heme oxygenase-1 (HO-1; Hmox1) is critical in maintaining cellular homeostasis, in large part through its ability to generate CO, but neither molecule has been studied in the setting of DNA damage. Naïve Hmox1(-/-) mice exhibit excessive tissue levels of γ-histone H2A, whereas administration of genotoxic stressors or irradiation in HO-1-deficient cells resulted in loss of ataxia-telangiectasia mutated/ataxia telangiectasia and Rad3-related protein and breast cancer 1, early onset induction with dysfunctional γ-H2AX foci and marked elevations in DNA damage. HO-1 induction or exposure to CO induced homologous recombination-mediated DNA repair through ataxia-telangiectasia mutated/ataxia telangiectasia and Rad3-related protein. In vivo, exposure of mice to CO followed by genotoxin (Adriamycin) or radiation-induced injury led to diminished tissue DNA damage and improved survival. We characterize a joint role for HO-1 and the gasotransmitter CO for appropriate DNA repair and provide a mechanism for their potent cytoprotective effects in various pathologies.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Levels of H2AXγ in tissues from Hmox1−_/_− mice. (A and B) Immunohistochemical analysis of H2AXγ in kidney, lung, liver, and spleen of wild-type (Hmox1+/+; black bars) and Hmox1_−/_− mice (white bars). (A) Quantification of the number of H2AXγ foci per field of view. (n = 5–10 fields of view from three or four mice; *P = 0.03; **P < 0.001.) (B) Representative images. (Scale bar: 100 μm.) (C) Immunoblotting with antibodies against H2AXγ and HO-1 in the spleen lysates of wild-type (+/+), Hmox1+/− (+/−), and Hmox1_−/− (−/_−) mice. Western blot shown is representative of two experiments.
Fig. 2.
Lack of HO-1 results in altered H2AXγ activation in response to DNA damage. (A) Fibroblasts from Hmox1+/+ and Hmox1_−/− mice were isolated and treated with camptothecin (1 μg/mL) or doxorubicin (10 μg/mL) for 1 h and stained with antibodies against H2AXγ. Images are shown representative of at least two independent experiments conducted in duplicate. (Scale bar: 25 μm.) (B and C) Immunoblotting analysis of H2AXγ in the lysates of Hmox1+/+, Hmox1_−/+, and Hmox1_−/_− fibroblasts treated with doxorubicin (10 μg/mL) for 2–4 h. (B) Representative immunoblot. (C) Quantitation. Data are representative of at least two independent experiments. *P < 0.05, Hmox1+/+ 4-h treatment versus control; #P < 0.05, Hmox1_−/_− versus Hmox1+/+.
Fig. 3.
DNA repair signaling in the absence of HO-1. (A and B) HEK293 cells with stable knockdown of HO-1 (mirHO-1) and control cells were treated with camptothecin (1 μg/mL) for 2 min to 2 h. The activation levels of HO-1 and DNA repair signaling proteins were measured by Western blot. (C) Immunoblot analysis in HEK mirHO-1 and control cells treated with doxorubicin (10 μg/mL) for 5 min to 2 h. Data are representative of two experiments. (D) Immunoblot analysis with antibodies against P-ATM, P-Brca1, P-H2AX, and P-p53 in the lysates of HEK293 mirHO-1 and control cells treated with 10 Gy γ-irradiation and harvested 5 min to 1 h after irradiation. Akt is a loading control for the immunoblot for P-p53. Data are representative of two independent experiments conducted in triplicate.
Fig. 4.
HO-1/CO induces HR-mediated DNA repair. (A) U20S-SCR reporter cells were cotransfected transiently with SceI and HO-1 or control plasmid. HO-1 levels were measured by Western blot. (B_–_D) The number of GFP+ U20S-SCR reporter cells was measured by fluorescence microscopy (B) and flow cytometry (C and D). Cells were transfected with HO-1 construct, and the number of GFP+ cells was measured 48 h after transfection. Data shown are ± SD and are representative of at least five experiments conducted in duplicate. *P < 0.05, CO versus air. (Scale bar in B: 25 μm.) (E_–_G) U2OS-SCR reporter cells after transfection with SceI for 24 h were treated with CO (250 ppm) for 24 h. The level of GFP+ cells was measured by fluorescence microscopy (E) or flow cytometry (F and G). Data shown are averages ± SD of three independent experiments. (Scale bar: 25 μm.)
Fig. 5.
CO induces activation of DNA repair signaling. (A and B) Immunoblotting with antibody against P-Brca1 and P-ATM (A) and P-ATR (B) in the lysates of PC3 and HEK cells treated with CO (250 ppm) for 2 min to 1 h. Data are representative of three independent experiments. (C and D) U20S-SCR reporter cells were transfected with SceI for 24 h and treated with CGK733 (10 μM) or DMSO for 1 h before treatment with CO (250 ppm) for 24 h. The level of GFP+ cells was measured by flow cytometry. Quantitation and representative dot plot figures are shown in C and D, respectively. *P < 0.05, CO versus air; #P < 0.05, CO + CGK733 versus CO. (E) U20S-SCR reporter cells were cotransfected with SceI for 24 h and KU55933 (20 μM) or DMSO for 1 h before treatment with CO (250 ppm) for 24 h. The level of GFP+ cells was measured by flow cytometry. *P < 0.05, CO versus air; **P < 0.01, CO + KU55933 versus CO; (−), untransfected cells. (F) U20S-SCR reporter cells were cotransfected with SceI and HO-1 for 24 h; then CGK733 was applied for 24 h. The level of GFP+ cells was measured by flow cytometry. Data are representative of three independent experiments conducted in duplicate. *P < 0.05, HO-1 versus control (C), #P < 0.05, HO-1 + CGK733 versus HO-1.
Fig. 6.
CO blocks DNA damage in tissues of mice treated with doxorubicin (Dox). (A_–_E) Immunohistochemistry with antibody against H2AXγ was performed in colon (A), kidney (B), lung (C), liver (D), and spleen (E) tissue. Tissues from nude mice with established PC3 tumors (2 wk) were harvested 14 d after treatment with CO (control) or doxorubicin (8 mg/kg i.v., twice per week) ± CO (250 ppm, 1 h, daily). (Magnification: 40×.) (F) Quantitation of H2AXγ staining in the tissues. Air or CO, n = 3; doxorubicin ± CO, n = 5. Data shown are the number (average ± SD) of H2AXγ+ cells per field of view. (Magnification: 20× for colon and kidney; 40× for spleen, liver, and lung.) #P < 0.05, doxorubicin versus control; *P < 0.05, CO + doxorubicin versus air + doxorubicin; **P < 0.001, CO + doxorubicin versus air + doxorubicin.
Fig. 7.
CO decreased tissue damage induced by lethal-dose irradiation. (A and B) Immunoblot (A) and immunohistochemistry (B) with antibody against HO-1 in tissues from mice irradiated with 10 Gy. (A) Kidneys and spleen harvested 2 h after irradiation. (B) Liver and spleen harvested 1 and 3 d postirradiation. (C and D) Immunohistochemical analysis of P-H2AX in the spleens and intestines of mice pretreated with air or CO (250 ppm) 1 h before a lethal dose of irradiation (10 Gy) and treated daily with CO for 11 d. Representative images are shown in C, and quantitation of the number of γ-H2AX+ cells is shown in D. n = 3 or 4 views per section from three or four mice per group. *P < 0.05. (E and F) Immunofluorescence staining of P-ATM and P-p53 in mononuclear blood cells of mice treated with CO or air for 1 h before lethal irradiation. Tissues were harvested 2 h after irradiation. Representative images (Lower) and quantitation of the number of positive cells per field of view (Upper) are shown. n = 3 or 4 mice group. (G). Survival of mice after marginal BM transplantation from H2ax_−/_− and H2ax+/+ mice to wild-type recipients. Mice were treated with CO before a lethal dose of irradiation (10 Gy) and after receiving BM. n = 5–10 mice per group.
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