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Absence of Wip1 partially rescues Atm deficiency phenotypes in mice.

Darlington Y, Nguyen TA, Moon SH, Herron A, Rao P, Zhu C, Lu X, Donehower LA - Oncogene (2011)

Bottom Line: WIP1 may have a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair.Additionally, double knockout splenocytes displayed reduced chromosomal instability compared with Atm mice.These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular.

View Article: PubMed Central - PubMed

Affiliation: Interdepartmental Graduate Program in Cell and Molecular Biology, Houston, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
Wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may have a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared with Atm mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared with their Atm counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared with Atm mice. Finally, doubly mice were partially rescued from gametogenesis defects observed in Atm mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular.

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Absence of Wip1 enhances p53 and DNA damage responses in Atm  mice(A) DNA damage-induced phosphorylation of p53 and H2AX is enhanced in thymic tissues of mice lacking Wip1. Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice were treated with 5 Gy of IR. Six hours after radiation, thymus tissue was harvested from each mouse. Protein lysates from individual mouse thymi were subjected to Western blot analysis with antibodies specific for the indicated proteins or their phosphorylated forms. (B) Quantitation of p53 phosphorylation at serine 18 in thymus lysates from untreated and IR-treated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice. (C) Quantitation of H2AX phosphorylation (γ-H2AX) at serine 140 in thymus lysates from untreated and IR-treated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice. (D) Quantitation of of p21 RNA expression in unirradiated and irradiated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mouse thymi by real-time PCR. For each of the genotype/radiation cohorts, results were calculated from four different animals and averaged for the graphs. Only two animals for each genotype radiation cohort are shown in Figure 2A, however. Asterisks (**P<0.01, *P<0.05) indicate significant differences in the magnitude of IR-induced increases compared to IR-induced increases in the wildtype mice.
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Figure 2: Absence of Wip1 enhances p53 and DNA damage responses in Atm mice(A) DNA damage-induced phosphorylation of p53 and H2AX is enhanced in thymic tissues of mice lacking Wip1. Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice were treated with 5 Gy of IR. Six hours after radiation, thymus tissue was harvested from each mouse. Protein lysates from individual mouse thymi were subjected to Western blot analysis with antibodies specific for the indicated proteins or their phosphorylated forms. (B) Quantitation of p53 phosphorylation at serine 18 in thymus lysates from untreated and IR-treated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice. (C) Quantitation of H2AX phosphorylation (γ-H2AX) at serine 140 in thymus lysates from untreated and IR-treated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice. (D) Quantitation of of p21 RNA expression in unirradiated and irradiated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mouse thymi by real-time PCR. For each of the genotype/radiation cohorts, results were calculated from four different animals and averaged for the graphs. Only two animals for each genotype radiation cohort are shown in Figure 2A, however. Asterisks (**P<0.01, *P<0.05) indicate significant differences in the magnitude of IR-induced increases compared to IR-induced increases in the wildtype mice.

Mentions: The reduced tumor incidence in the Atm−/−Wip1−/− mice compared to Atm mice is consistent with enhanced DNA damage and p53 responses. To examine this further, Atm+/+Wip1+/+, Atm+/+Wip1−/−, Atm−/−Wip1+/+, and Atm−/−Wip1−/− eight week old mice were irradiated with 5 Gy of ionizing radiation (IR). Thymi were harvested six hours after IR and analyzed for phosphorylation status of known Wip1 dephosphorylation targets. Lysates from normal thymi and spleens were assessed by Western blot analysis with antibodies to p53 and H2AX as well as phospho-specific antibodies for p53 (pS18) and γ H2AX (pS140). Both of these phosphorylation events are markers for an activated DNA damage response. Basal levels of γ-H2AX and phospho-p53 were low in unirradiated Atm+/+Wip1+/+ lymphoid tissues but were induced to moderate levels six hours after IR treatment (Fig. 2A; Fig. S1). Irradiated Atm+/+Wip1−/− thymi and spleens exhibited increased phosphorylation of H2AX and p53 compared to irradiated Atm+/+Wip1+/+ thymi and spleens. Surprisingly, deletion of Atm did not impair IR-induced phosphorylation of H2AX and p53 and was comparable to Atm+/+Wip1+/+ levels (Fig. 2A-C). This is likely a result of compensatory phosphorylation by other PIKKs. In the presence of IR damage, the Atm−/−Wip1−/− thymi exhibited high phosphorylation levels of H2AX and p53 comparable to Atm+/+Wip1−/− thymi (Fig. 2A-C). In addition, IR treatment resulted in increased p53 protein levels across all four genotypes, as expected. Absence of Wip1 in Atm+/+ and Atm−/− mice conferred modestly increased p53 protein stability after IR compared to wildtype and Atm mice (Fig. 2A). Finally, irradiation of the different Atm/Wip1 genotype mice resulted in similar patterns of enhanced phosphorylation of Brca1 Ser1423 in the absence of Wip1 (Fig. S2). This Brca1 phosphorylation site, targeted by Atm, is also dephosphorylated by Wip1 (Nguyen and Donehower, unpublished data).


Absence of Wip1 partially rescues Atm deficiency phenotypes in mice.

Darlington Y, Nguyen TA, Moon SH, Herron A, Rao P, Zhu C, Lu X, Donehower LA - Oncogene (2011)

Absence of Wip1 enhances p53 and DNA damage responses in Atm  mice(A) DNA damage-induced phosphorylation of p53 and H2AX is enhanced in thymic tissues of mice lacking Wip1. Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice were treated with 5 Gy of IR. Six hours after radiation, thymus tissue was harvested from each mouse. Protein lysates from individual mouse thymi were subjected to Western blot analysis with antibodies specific for the indicated proteins or their phosphorylated forms. (B) Quantitation of p53 phosphorylation at serine 18 in thymus lysates from untreated and IR-treated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice. (C) Quantitation of H2AX phosphorylation (γ-H2AX) at serine 140 in thymus lysates from untreated and IR-treated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice. (D) Quantitation of of p21 RNA expression in unirradiated and irradiated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mouse thymi by real-time PCR. For each of the genotype/radiation cohorts, results were calculated from four different animals and averaged for the graphs. Only two animals for each genotype radiation cohort are shown in Figure 2A, however. Asterisks (**P<0.01, *P<0.05) indicate significant differences in the magnitude of IR-induced increases compared to IR-induced increases in the wildtype mice.
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Figure 2: Absence of Wip1 enhances p53 and DNA damage responses in Atm mice(A) DNA damage-induced phosphorylation of p53 and H2AX is enhanced in thymic tissues of mice lacking Wip1. Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice were treated with 5 Gy of IR. Six hours after radiation, thymus tissue was harvested from each mouse. Protein lysates from individual mouse thymi were subjected to Western blot analysis with antibodies specific for the indicated proteins or their phosphorylated forms. (B) Quantitation of p53 phosphorylation at serine 18 in thymus lysates from untreated and IR-treated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice. (C) Quantitation of H2AX phosphorylation (γ-H2AX) at serine 140 in thymus lysates from untreated and IR-treated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mice. (D) Quantitation of of p21 RNA expression in unirradiated and irradiated Atm+/+Wip1+/+, Atm−/−Wip1+/+, Atm+/+Wip1−/−, and Atm−/−Wip1−/− mouse thymi by real-time PCR. For each of the genotype/radiation cohorts, results were calculated from four different animals and averaged for the graphs. Only two animals for each genotype radiation cohort are shown in Figure 2A, however. Asterisks (**P<0.01, *P<0.05) indicate significant differences in the magnitude of IR-induced increases compared to IR-induced increases in the wildtype mice.
Mentions: The reduced tumor incidence in the Atm−/−Wip1−/− mice compared to Atm mice is consistent with enhanced DNA damage and p53 responses. To examine this further, Atm+/+Wip1+/+, Atm+/+Wip1−/−, Atm−/−Wip1+/+, and Atm−/−Wip1−/− eight week old mice were irradiated with 5 Gy of ionizing radiation (IR). Thymi were harvested six hours after IR and analyzed for phosphorylation status of known Wip1 dephosphorylation targets. Lysates from normal thymi and spleens were assessed by Western blot analysis with antibodies to p53 and H2AX as well as phospho-specific antibodies for p53 (pS18) and γ H2AX (pS140). Both of these phosphorylation events are markers for an activated DNA damage response. Basal levels of γ-H2AX and phospho-p53 were low in unirradiated Atm+/+Wip1+/+ lymphoid tissues but were induced to moderate levels six hours after IR treatment (Fig. 2A; Fig. S1). Irradiated Atm+/+Wip1−/− thymi and spleens exhibited increased phosphorylation of H2AX and p53 compared to irradiated Atm+/+Wip1+/+ thymi and spleens. Surprisingly, deletion of Atm did not impair IR-induced phosphorylation of H2AX and p53 and was comparable to Atm+/+Wip1+/+ levels (Fig. 2A-C). This is likely a result of compensatory phosphorylation by other PIKKs. In the presence of IR damage, the Atm−/−Wip1−/− thymi exhibited high phosphorylation levels of H2AX and p53 comparable to Atm+/+Wip1−/− thymi (Fig. 2A-C). In addition, IR treatment resulted in increased p53 protein levels across all four genotypes, as expected. Absence of Wip1 in Atm+/+ and Atm−/− mice conferred modestly increased p53 protein stability after IR compared to wildtype and Atm mice (Fig. 2A). Finally, irradiation of the different Atm/Wip1 genotype mice resulted in similar patterns of enhanced phosphorylation of Brca1 Ser1423 in the absence of Wip1 (Fig. S2). This Brca1 phosphorylation site, targeted by Atm, is also dephosphorylated by Wip1 (Nguyen and Donehower, unpublished data).

Bottom Line: WIP1 may have a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair.Additionally, double knockout splenocytes displayed reduced chromosomal instability compared with Atm mice.These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular.

View Article: PubMed Central - PubMed

Affiliation: Interdepartmental Graduate Program in Cell and Molecular Biology, Houston, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
Wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may have a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared with Atm mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared with their Atm counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared with Atm mice. Finally, doubly mice were partially rescued from gametogenesis defects observed in Atm mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular.

Show MeSH
Related in: MedlinePlus