Limits...
Artemis is a negative regulator of p53 in response to oxidative stress.

Zhang X, Zhu Y, Geng L, Wang H, Legerski RJ - Oncogene (2009)

Bottom Line: Treatment with ionizing radiation or hydrogen peroxide did not cause activation of this signaling pathway, whereas inhibitors of mitochondrial electron transport were effective in reducing its activation.In addition, we show that p53-inducible genes involved in reducing reactive oxygen species are upregulated by Artemis depletion.These findings indicate that Artemis and DNA-PKcs participate in a new, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

ABSTRACT
Artemis is a multifunctional phospho-protein with roles in V(D)J recombination, repair of double-strand breaks by nonhomologous end-joining and regulation of cell-cycle checkpoints after DNA damage. Here, we describe a new function of Artemis as a negative regulator of p53 in response to oxidative stress in both primary cells and cancer cell lines. We show that depletion of Artemis under typical culture conditions (21% oxygen) leads to a spontaneous phosphorylation and stabilization of p53, and resulting cellular G1 arrest and apoptosis. These effects are suppressed by co-depletion of DNA-PKcs, but not ATM, indicating that Artemis is an inhibitor of DNA-PKcs-mediated stabilization of p53. Culturing of cellsat 3% oxygen or treatment with an antioxidant abrogated p53 stabilization, indicating that oxidative stress is the responsible cellular stimulus. Treatment with ionizing radiation or hydrogen peroxide did not cause activation of this signaling pathway, whereas inhibitors of mitochondrial electron transport were effective in reducing its activation. In addition, we show that p53-inducible genes involved in reducing reactive oxygen species are upregulated by Artemis depletion. These findings indicate that Artemis and DNA-PKcs participate in a new, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.

Show MeSH

Related in: MedlinePlus

Artemis acts at the posttranslational level and interacts with p53(A) Immunoblot showing that accumulation of p53 induced by Artemis depletion is not inhibited by the translation inhibitor cycloheximide (CHX) in U2OS cells. P53 accumulation occurs in the presence of the 26S proteosome inhibitor MG132 with or without Artemis depletion. (B) Reciprocal co-IP assays performed in HCT-116 cells between Artemis and p53. “IgG” indicates a nonspecific immunoglobulin. (C) Co-IP assay showing that Artemis interacts with p53 before and after IR treatment in HCT-116 cells. IR dose was 6 Gy, and the “0” lane indicates cell that were not irradiated. “Beads” indicates assay performed without Artemis antibody. (D) Co-IP between p53 and Artemis is not observed in p53-deficient HCT-116 cells. (E) Co-IP between p53 and Artemis occurs after depletion of DNA-PKcs. “C+EB” indicates control siRNA and that the co-IP was performed in the presence of ethidium bromide.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2692457&req=5

Figure 3: Artemis acts at the posttranslational level and interacts with p53(A) Immunoblot showing that accumulation of p53 induced by Artemis depletion is not inhibited by the translation inhibitor cycloheximide (CHX) in U2OS cells. P53 accumulation occurs in the presence of the 26S proteosome inhibitor MG132 with or without Artemis depletion. (B) Reciprocal co-IP assays performed in HCT-116 cells between Artemis and p53. “IgG” indicates a nonspecific immunoglobulin. (C) Co-IP assay showing that Artemis interacts with p53 before and after IR treatment in HCT-116 cells. IR dose was 6 Gy, and the “0” lane indicates cell that were not irradiated. “Beads” indicates assay performed without Artemis antibody. (D) Co-IP between p53 and Artemis is not observed in p53-deficient HCT-116 cells. (E) Co-IP between p53 and Artemis occurs after depletion of DNA-PKcs. “C+EB” indicates control siRNA and that the co-IP was performed in the presence of ethidium bromide.

Mentions: We next examined whether Artemis depletion upregulated p53 by a transcriptional or posttranslational mechanism. Knockdown of Artemis in HeLa cells did not alter the levels of p53 transcripts compared to control cells as determined by quantitative RT-PCR (Fig. S1). However, in the presence of the translational inhibitor cycloheximide stabilization of p53 was observed with Artemis depletion by siRNA (Fig. 3A). In the presence of the 26S proteosome inhibitor MG132, stabilization of p53 was observed after treatment with either control or Artemis siRNA (Fig. 3A). In addition, the half-life of p53 was increased in U2OS cells treated with Artemis siRNA compared to control siRNA (Fig. S2). Taken together, these results indicate that Artemis depletion causes stabilization of p53 by a posttranslational mechanism. This latter finding suggested that Artemis and p53 might physically interact. We, therefore, performed reciprocal co-immunoprecipitation experiments, and as shown (Fig. 3B-D), the results indicated that Artemis and p53 reside in a common complex. Furthermore, depletion of DNA-PKcs did not affect the interaction between Artemis and p53, nor did the inclusion of ethidium bromide indicating that the interaction is not mediated by DNA (Fig. 3E). Taken together these results suggest that Artemis may directly regulate p53.


Artemis is a negative regulator of p53 in response to oxidative stress.

Zhang X, Zhu Y, Geng L, Wang H, Legerski RJ - Oncogene (2009)

Artemis acts at the posttranslational level and interacts with p53(A) Immunoblot showing that accumulation of p53 induced by Artemis depletion is not inhibited by the translation inhibitor cycloheximide (CHX) in U2OS cells. P53 accumulation occurs in the presence of the 26S proteosome inhibitor MG132 with or without Artemis depletion. (B) Reciprocal co-IP assays performed in HCT-116 cells between Artemis and p53. “IgG” indicates a nonspecific immunoglobulin. (C) Co-IP assay showing that Artemis interacts with p53 before and after IR treatment in HCT-116 cells. IR dose was 6 Gy, and the “0” lane indicates cell that were not irradiated. “Beads” indicates assay performed without Artemis antibody. (D) Co-IP between p53 and Artemis is not observed in p53-deficient HCT-116 cells. (E) Co-IP between p53 and Artemis occurs after depletion of DNA-PKcs. “C+EB” indicates control siRNA and that the co-IP was performed in the presence of ethidium bromide.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2692457&req=5

Figure 3: Artemis acts at the posttranslational level and interacts with p53(A) Immunoblot showing that accumulation of p53 induced by Artemis depletion is not inhibited by the translation inhibitor cycloheximide (CHX) in U2OS cells. P53 accumulation occurs in the presence of the 26S proteosome inhibitor MG132 with or without Artemis depletion. (B) Reciprocal co-IP assays performed in HCT-116 cells between Artemis and p53. “IgG” indicates a nonspecific immunoglobulin. (C) Co-IP assay showing that Artemis interacts with p53 before and after IR treatment in HCT-116 cells. IR dose was 6 Gy, and the “0” lane indicates cell that were not irradiated. “Beads” indicates assay performed without Artemis antibody. (D) Co-IP between p53 and Artemis is not observed in p53-deficient HCT-116 cells. (E) Co-IP between p53 and Artemis occurs after depletion of DNA-PKcs. “C+EB” indicates control siRNA and that the co-IP was performed in the presence of ethidium bromide.
Mentions: We next examined whether Artemis depletion upregulated p53 by a transcriptional or posttranslational mechanism. Knockdown of Artemis in HeLa cells did not alter the levels of p53 transcripts compared to control cells as determined by quantitative RT-PCR (Fig. S1). However, in the presence of the translational inhibitor cycloheximide stabilization of p53 was observed with Artemis depletion by siRNA (Fig. 3A). In the presence of the 26S proteosome inhibitor MG132, stabilization of p53 was observed after treatment with either control or Artemis siRNA (Fig. 3A). In addition, the half-life of p53 was increased in U2OS cells treated with Artemis siRNA compared to control siRNA (Fig. S2). Taken together, these results indicate that Artemis depletion causes stabilization of p53 by a posttranslational mechanism. This latter finding suggested that Artemis and p53 might physically interact. We, therefore, performed reciprocal co-immunoprecipitation experiments, and as shown (Fig. 3B-D), the results indicated that Artemis and p53 reside in a common complex. Furthermore, depletion of DNA-PKcs did not affect the interaction between Artemis and p53, nor did the inclusion of ethidium bromide indicating that the interaction is not mediated by DNA (Fig. 3E). Taken together these results suggest that Artemis may directly regulate p53.

Bottom Line: Treatment with ionizing radiation or hydrogen peroxide did not cause activation of this signaling pathway, whereas inhibitors of mitochondrial electron transport were effective in reducing its activation.In addition, we show that p53-inducible genes involved in reducing reactive oxygen species are upregulated by Artemis depletion.These findings indicate that Artemis and DNA-PKcs participate in a new, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

ABSTRACT
Artemis is a multifunctional phospho-protein with roles in V(D)J recombination, repair of double-strand breaks by nonhomologous end-joining and regulation of cell-cycle checkpoints after DNA damage. Here, we describe a new function of Artemis as a negative regulator of p53 in response to oxidative stress in both primary cells and cancer cell lines. We show that depletion of Artemis under typical culture conditions (21% oxygen) leads to a spontaneous phosphorylation and stabilization of p53, and resulting cellular G1 arrest and apoptosis. These effects are suppressed by co-depletion of DNA-PKcs, but not ATM, indicating that Artemis is an inhibitor of DNA-PKcs-mediated stabilization of p53. Culturing of cellsat 3% oxygen or treatment with an antioxidant abrogated p53 stabilization, indicating that oxidative stress is the responsible cellular stimulus. Treatment with ionizing radiation or hydrogen peroxide did not cause activation of this signaling pathway, whereas inhibitors of mitochondrial electron transport were effective in reducing its activation. In addition, we show that p53-inducible genes involved in reducing reactive oxygen species are upregulated by Artemis depletion. These findings indicate that Artemis and DNA-PKcs participate in a new, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.

Show MeSH
Related in: MedlinePlus