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

Depletion of Artemis causes apoptosis in cancer cell lines(A) Immunoblot showing depletion of Artemis causes upregulation of the apoptotic marker Bax. (B,C) Taxol and nocodazole enhance apoptosis induced by Artemis depletion as measured by PARP degradation and cellular sub-G1 content in U2OS cells. (D) FACS analysis (left panel) and quantitation (right panel) of annexin V staining in HCT116 cells shows absence of p21 enhances apoptosis induced by Artemis depletion. The ratio of apoptotic cells was quantitated as described in Fig. 1A.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2692457&req=5

Figure 2: Depletion of Artemis causes apoptosis in cancer cell lines(A) Immunoblot showing depletion of Artemis causes upregulation of the apoptotic marker Bax. (B,C) Taxol and nocodazole enhance apoptosis induced by Artemis depletion as measured by PARP degradation and cellular sub-G1 content in U2OS cells. (D) FACS analysis (left panel) and quantitation (right panel) of annexin V staining in HCT116 cells shows absence of p21 enhances apoptosis induced by Artemis depletion. The ratio of apoptotic cells was quantitated as described in Fig. 1A.

Mentions: In addition to cell cycle arrest, activated p53 also induces programmed cell death (Vogelstein et al., 2000). We, therefore, examined markers of apoptosis after Artemis depletion. As shown, the pro-apoptotic gene Bax (Zhang et al., 2000) was upregulated (Fig. 2A), and both PARP cleavage and sub-G1 cells were increased (Fig. 2B,C). Furthermore, simultaneous treatment with either one of the spindle poisons Taxol or nocadazole significantly enhanced the level of apoptosis observed with Artemis depletion. To further confirm this phenotype, we examined p21 cells (Waldman et al., 1995) for apoptosis mediated by Artemis depletion. Others have shown that in the absence of p21 apoptosis due to cellular stress is enhanced (Sohn et al., 2006; Tian et al., 2000). In the absence of p21, Artemis-depleted HCT116 cells exhibited a significantly enhanced level of apoptosis (Fig. 2D) indicating that reduction of Artemis levels strongly induces programmed cell death.


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

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

Depletion of Artemis causes apoptosis in cancer cell lines(A) Immunoblot showing depletion of Artemis causes upregulation of the apoptotic marker Bax. (B,C) Taxol and nocodazole enhance apoptosis induced by Artemis depletion as measured by PARP degradation and cellular sub-G1 content in U2OS cells. (D) FACS analysis (left panel) and quantitation (right panel) of annexin V staining in HCT116 cells shows absence of p21 enhances apoptosis induced by Artemis depletion. The ratio of apoptotic cells was quantitated as described in Fig. 1A.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Depletion of Artemis causes apoptosis in cancer cell lines(A) Immunoblot showing depletion of Artemis causes upregulation of the apoptotic marker Bax. (B,C) Taxol and nocodazole enhance apoptosis induced by Artemis depletion as measured by PARP degradation and cellular sub-G1 content in U2OS cells. (D) FACS analysis (left panel) and quantitation (right panel) of annexin V staining in HCT116 cells shows absence of p21 enhances apoptosis induced by Artemis depletion. The ratio of apoptotic cells was quantitated as described in Fig. 1A.
Mentions: In addition to cell cycle arrest, activated p53 also induces programmed cell death (Vogelstein et al., 2000). We, therefore, examined markers of apoptosis after Artemis depletion. As shown, the pro-apoptotic gene Bax (Zhang et al., 2000) was upregulated (Fig. 2A), and both PARP cleavage and sub-G1 cells were increased (Fig. 2B,C). Furthermore, simultaneous treatment with either one of the spindle poisons Taxol or nocadazole significantly enhanced the level of apoptosis observed with Artemis depletion. To further confirm this phenotype, we examined p21 cells (Waldman et al., 1995) for apoptosis mediated by Artemis depletion. Others have shown that in the absence of p21 apoptosis due to cellular stress is enhanced (Sohn et al., 2006; Tian et al., 2000). In the absence of p21, Artemis-depleted HCT116 cells exhibited a significantly enhanced level of apoptosis (Fig. 2D) indicating that reduction of Artemis levels strongly induces programmed cell death.

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