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p53-dependent stimulation of redox-related genes in the lymphoid organs of gamma-irradiated--mice identification of Haeme-oxygenase 1 as a direct p53 target gene.

Meiller A, Alvarez S, Drané P, Lallemand C, Blanchard B, Tovey M, May E - Nucleic Acids Res. (2007)

Bottom Line: Results presented in this article illustrate an additional degree of complexity.Moreover, induction of HO-1 occurs later than that of Waf1/p21.Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

View Article: PubMed Central - PubMed

Affiliation: Commissariat à l'Energie Atomique (CEA), Centre National de la Recherche Scientifique (CNRS), UMR217, route du Panorama BP6, 92265 Fontenay-aux-Roses Cedex and CNRS FRE2937, Institut André Lwoff, 7, rue Guy Moquet, BP8, 94801 Villejuif Cedex, France.

ABSTRACT
Recent data showed that p53 stimulates the expression of genes encoding not only pro- but also antioxidant enzymes. It was suggested that antioxidant genes could be induced under physiologic levels of stress while the prooxidant ones respond to higher level of stress. Results presented in this article illustrate an additional degree of complexity. We show that the expression of Haeme-oxygenase 1 (HO-1), a stress-inducible gene that codes for an enzyme having antioxidant properties, is stimulated in a p53-dependent manner in the thymus and spleen of irradiated mice. We prove that HO-1 is a direct p53 target gene by showing that the p53RE identified within human and mouse genes is specifically bound by p53. The threshold of irradiation dose required to induce a significant response of HO-1 in the lymphoid organs of the irradiated mice is higher than that for Waf1/p21 that encodes an universal inhibitor of cell cycle. Moreover, induction of HO-1 occurs later than that of Waf1/p21. Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

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Gamma-irradiation induced the expression of HO-1 in p53+/+ but not in p53−/− mice. (A) Total RNA was extracted from thymus of p53+/+ and p53−/− mice either non-irradiated or whole body irradiated at a dose of 1 Gy. The thymus was taken 6 h post-irradiation. Hybridization to cDNA arrays of mouse stress/toxicology genes (AtlasTM filter arrays, Clontech, Palo Alto, CA, USA) was performed as described in Materials and Methods section. Signal intensity was normalized as described in Materials and Methods section. The normalized signal calculated for the thymus of irradiated mice was expressed relative to the thymus of non-irradiated mice. Results from microarray analysis (array) were confirmed by real-time quantitative RT-PCR as described in Materials and Methods section. The relative level of HO-1 mRNA measured by RT-PCR was normalized relative to GAPDH mRNA and the level of stimulation was calculated relative to non-irradiated animals. (B) HO-1 mRNA induction in thymus and spleen of mice at 3 h following whole body irradiation at a dose of 5 Gy. The level of HO-1 mRNA estimated by real-time quantitative RT-PCR was normalized as described below. The graphs represent the mean of four independent experiments with their respective standard deviation. NI, non-irradiated mice; IR, irradiated mice.
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Figure 1: Gamma-irradiation induced the expression of HO-1 in p53+/+ but not in p53−/− mice. (A) Total RNA was extracted from thymus of p53+/+ and p53−/− mice either non-irradiated or whole body irradiated at a dose of 1 Gy. The thymus was taken 6 h post-irradiation. Hybridization to cDNA arrays of mouse stress/toxicology genes (AtlasTM filter arrays, Clontech, Palo Alto, CA, USA) was performed as described in Materials and Methods section. Signal intensity was normalized as described in Materials and Methods section. The normalized signal calculated for the thymus of irradiated mice was expressed relative to the thymus of non-irradiated mice. Results from microarray analysis (array) were confirmed by real-time quantitative RT-PCR as described in Materials and Methods section. The relative level of HO-1 mRNA measured by RT-PCR was normalized relative to GAPDH mRNA and the level of stimulation was calculated relative to non-irradiated animals. (B) HO-1 mRNA induction in thymus and spleen of mice at 3 h following whole body irradiation at a dose of 5 Gy. The level of HO-1 mRNA estimated by real-time quantitative RT-PCR was normalized as described below. The graphs represent the mean of four independent experiments with their respective standard deviation. NI, non-irradiated mice; IR, irradiated mice.

Mentions: Microarray analysis was used to identify genes involved in oxidative metabolism whose stimulation depends on p53 activation, in vivo. Recently, we showed that 1 Gy is the lowest dose of irradiation to give a significant p53-dependent stimulation of expression of the principal proapoptotic genes, in the thymus and spleen of whole body irradiated mice (39). We decided therefore to use this relative low dose of irradiation to search for oxidant genes whose expression is regulated in response to p53 activation. Wt-p53 (p53+/+) and (p53−/−) mice were irradiated at a dose of 1 Gy and sacrificed 6 h after irradiation. Radioactively labelled cDNA was synthesized from total RNA extracted from the thymus and hybridized to Altas™ mouse stress/toxicology array membranes as described in the Materials and Methods section. Among the 140 genes present in the membrane, HO-1 (still referred as Hmox1) is the only one that was found to be significantly up-regulated in response to irradiation in p53+/+ but not p53−/− mice. According to the microarray analysis, expression of HO-1 is stimulated by a factor of 7.5 in the thymus of irradiated p53+/+ mice compared to non-irradiated mice. Such stimulation is not observed in the thymus of p53−/− mice (Figure 1A). Microarray analysis was validated by quantitative real-time RT-PCR analysis using the same RNA samples (Figure 1A). The γ-ray induction of HO-1 in p53+/+ but not in p53−/− mice was confirmed in independent experiments, both in thymus and spleen of mice subjected to 5 Gy of whole body irradiation (Figure 1B). These results strongly suggest that the up-regulation of HO-1 in response to γ-irradiation requires a functional p53, at least in the lymphoid organs.Figure 1.


p53-dependent stimulation of redox-related genes in the lymphoid organs of gamma-irradiated--mice identification of Haeme-oxygenase 1 as a direct p53 target gene.

Meiller A, Alvarez S, Drané P, Lallemand C, Blanchard B, Tovey M, May E - Nucleic Acids Res. (2007)

Gamma-irradiation induced the expression of HO-1 in p53+/+ but not in p53−/− mice. (A) Total RNA was extracted from thymus of p53+/+ and p53−/− mice either non-irradiated or whole body irradiated at a dose of 1 Gy. The thymus was taken 6 h post-irradiation. Hybridization to cDNA arrays of mouse stress/toxicology genes (AtlasTM filter arrays, Clontech, Palo Alto, CA, USA) was performed as described in Materials and Methods section. Signal intensity was normalized as described in Materials and Methods section. The normalized signal calculated for the thymus of irradiated mice was expressed relative to the thymus of non-irradiated mice. Results from microarray analysis (array) were confirmed by real-time quantitative RT-PCR as described in Materials and Methods section. The relative level of HO-1 mRNA measured by RT-PCR was normalized relative to GAPDH mRNA and the level of stimulation was calculated relative to non-irradiated animals. (B) HO-1 mRNA induction in thymus and spleen of mice at 3 h following whole body irradiation at a dose of 5 Gy. The level of HO-1 mRNA estimated by real-time quantitative RT-PCR was normalized as described below. The graphs represent the mean of four independent experiments with their respective standard deviation. NI, non-irradiated mice; IR, irradiated mice.
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Figure 1: Gamma-irradiation induced the expression of HO-1 in p53+/+ but not in p53−/− mice. (A) Total RNA was extracted from thymus of p53+/+ and p53−/− mice either non-irradiated or whole body irradiated at a dose of 1 Gy. The thymus was taken 6 h post-irradiation. Hybridization to cDNA arrays of mouse stress/toxicology genes (AtlasTM filter arrays, Clontech, Palo Alto, CA, USA) was performed as described in Materials and Methods section. Signal intensity was normalized as described in Materials and Methods section. The normalized signal calculated for the thymus of irradiated mice was expressed relative to the thymus of non-irradiated mice. Results from microarray analysis (array) were confirmed by real-time quantitative RT-PCR as described in Materials and Methods section. The relative level of HO-1 mRNA measured by RT-PCR was normalized relative to GAPDH mRNA and the level of stimulation was calculated relative to non-irradiated animals. (B) HO-1 mRNA induction in thymus and spleen of mice at 3 h following whole body irradiation at a dose of 5 Gy. The level of HO-1 mRNA estimated by real-time quantitative RT-PCR was normalized as described below. The graphs represent the mean of four independent experiments with their respective standard deviation. NI, non-irradiated mice; IR, irradiated mice.
Mentions: Microarray analysis was used to identify genes involved in oxidative metabolism whose stimulation depends on p53 activation, in vivo. Recently, we showed that 1 Gy is the lowest dose of irradiation to give a significant p53-dependent stimulation of expression of the principal proapoptotic genes, in the thymus and spleen of whole body irradiated mice (39). We decided therefore to use this relative low dose of irradiation to search for oxidant genes whose expression is regulated in response to p53 activation. Wt-p53 (p53+/+) and (p53−/−) mice were irradiated at a dose of 1 Gy and sacrificed 6 h after irradiation. Radioactively labelled cDNA was synthesized from total RNA extracted from the thymus and hybridized to Altas™ mouse stress/toxicology array membranes as described in the Materials and Methods section. Among the 140 genes present in the membrane, HO-1 (still referred as Hmox1) is the only one that was found to be significantly up-regulated in response to irradiation in p53+/+ but not p53−/− mice. According to the microarray analysis, expression of HO-1 is stimulated by a factor of 7.5 in the thymus of irradiated p53+/+ mice compared to non-irradiated mice. Such stimulation is not observed in the thymus of p53−/− mice (Figure 1A). Microarray analysis was validated by quantitative real-time RT-PCR analysis using the same RNA samples (Figure 1A). The γ-ray induction of HO-1 in p53+/+ but not in p53−/− mice was confirmed in independent experiments, both in thymus and spleen of mice subjected to 5 Gy of whole body irradiation (Figure 1B). These results strongly suggest that the up-regulation of HO-1 in response to γ-irradiation requires a functional p53, at least in the lymphoid organs.Figure 1.

Bottom Line: Results presented in this article illustrate an additional degree of complexity.Moreover, induction of HO-1 occurs later than that of Waf1/p21.Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

View Article: PubMed Central - PubMed

Affiliation: Commissariat à l'Energie Atomique (CEA), Centre National de la Recherche Scientifique (CNRS), UMR217, route du Panorama BP6, 92265 Fontenay-aux-Roses Cedex and CNRS FRE2937, Institut André Lwoff, 7, rue Guy Moquet, BP8, 94801 Villejuif Cedex, France.

ABSTRACT
Recent data showed that p53 stimulates the expression of genes encoding not only pro- but also antioxidant enzymes. It was suggested that antioxidant genes could be induced under physiologic levels of stress while the prooxidant ones respond to higher level of stress. Results presented in this article illustrate an additional degree of complexity. We show that the expression of Haeme-oxygenase 1 (HO-1), a stress-inducible gene that codes for an enzyme having antioxidant properties, is stimulated in a p53-dependent manner in the thymus and spleen of irradiated mice. We prove that HO-1 is a direct p53 target gene by showing that the p53RE identified within human and mouse genes is specifically bound by p53. The threshold of irradiation dose required to induce a significant response of HO-1 in the lymphoid organs of the irradiated mice is higher than that for Waf1/p21 that encodes an universal inhibitor of cell cycle. Moreover, induction of HO-1 occurs later than that of Waf1/p21. Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

Show MeSH