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Phosphorylation of SMC1 by ATR is required for desferrioxamine (DFO)-induced apoptosis.

So EY, Ausman M, Saeki T, Ouchi T - Cell Death Dis (2011)

Bottom Line: Among these sites, phosphorylation of SMC1, NBS1, and Chk1 by DFO are mediated by ATR as it is greatly reduced in both ATR-deficient human fibroblasts and HCT116 human colon cancer cells in which ATR is heterozygously mutated, whereas these proteins are phosphorylated in cells deficient for ATM and DNA-PKcs.DFO-induced apoptosis is decreased in ATR-mutant HCT116 cells, although p53 is normally activated in those cells.These results suggest that DFO induces apoptosis through the ATR-SMC1 arm of the pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, NUHS, Systems Biology Program, Pritzker School of Medicine, The University of Chicago, Evanston, IL 60201, USA.

ABSTRACT
DNA damage signaling pathways are initiated in response to chemical reagents and radiation damage, as well as in response to hypoxia. It is implicated that structural maintenance of chromosomes 1 (SMC1) is not only a component of the cohesion complex but also facilitates the activation of DNA damage checkpoint proteins. Here, we studied the mechanism of DNA damage checkpoint activated by ATR-SMC1 pathway when cells are treated with desferrioxamine (DFO), a hypoxia-mimetic reagent. We show that DFO treatment induces phosphorylation of SMC1 at Ser966, NBS1 at Ser343, Chk1 at Ser317, Chk2 at Thr68, and p53 at Ser15. Among these sites, phosphorylation of SMC1, NBS1, and Chk1 by DFO are mediated by ATR as it is greatly reduced in both ATR-deficient human fibroblasts and HCT116 human colon cancer cells in which ATR is heterozygously mutated, whereas these proteins are phosphorylated in cells deficient for ATM and DNA-PKcs. DFO-induced apoptosis is decreased in ATR-mutant HCT116 cells, although p53 is normally activated in those cells. Expression of SMC1 S966A in which Ser966 is substituted to Ala attenuates apoptosis and phosphorylation of Chk1 at Ser317 after DFO treatment, although levels of HIF1α are not significantly changed. These results suggest that DFO induces apoptosis through the ATR-SMC1 arm of the pathway.

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Transfection of 293T cells and HCT116 cells with S966A-mutant SMC1 inhibited the DFO-induced apoptosis. (a) 293T cells and (b) HCT116 cells were transfected with myc-tagged mutant SMC1 plasmid (Myc-SMC1S966A) or a control vector. After 48 h, cells were treated with DFO (300 μM) or NCS (0.5 μg/ml) for additional 24 h. Total extracts were studied for western blotting with indicated antibodies to detect level of HIF1α and phosphorylated proteins of NBS1 S343P and Chk1 at S317P. Actin blot serves as a loading control. (c) 293T cells and (d) HCT116 cells were transfected with plasmid containing mutant SMC1 (Myc-SMC1S966A) or a control vector. After 48 h, cells were treated with DFO (300 μM) for additional 24 h and then apoptosis was analyzed using a flow cytometer (left panel). Dot plot represents two independent experiments, indicating Annexin V staining (right panel). DFO-induced apoptosis was expressed as fold-induction compared with untreated samples (right panel, *P<0.05, Student's t-test). (e) Levels of Myc-tagged SMC1S966A and phosphorylation of endogenous SMC1 at Ser966 were compared in HCT116 and 293T cell lines. The phosphorylation and expression of proteins were analyzed by densitometry as described in Figure 1
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fig6: Transfection of 293T cells and HCT116 cells with S966A-mutant SMC1 inhibited the DFO-induced apoptosis. (a) 293T cells and (b) HCT116 cells were transfected with myc-tagged mutant SMC1 plasmid (Myc-SMC1S966A) or a control vector. After 48 h, cells were treated with DFO (300 μM) or NCS (0.5 μg/ml) for additional 24 h. Total extracts were studied for western blotting with indicated antibodies to detect level of HIF1α and phosphorylated proteins of NBS1 S343P and Chk1 at S317P. Actin blot serves as a loading control. (c) 293T cells and (d) HCT116 cells were transfected with plasmid containing mutant SMC1 (Myc-SMC1S966A) or a control vector. After 48 h, cells were treated with DFO (300 μM) for additional 24 h and then apoptosis was analyzed using a flow cytometer (left panel). Dot plot represents two independent experiments, indicating Annexin V staining (right panel). DFO-induced apoptosis was expressed as fold-induction compared with untreated samples (right panel, *P<0.05, Student's t-test). (e) Levels of Myc-tagged SMC1S966A and phosphorylation of endogenous SMC1 at Ser966 were compared in HCT116 and 293T cell lines. The phosphorylation and expression of proteins were analyzed by densitometry as described in Figure 1

Mentions: On the basis of the results demonstrated in Figure 1, we hypothesized that this ATR-mediated phosphorylation of SMC1is involved in DFO-induced apoptosis that is shown in Figure 5. We explored transient expression of a phospho-deficient mutant form of SMC1 in cell culture. 293T cells and HCT116 cells were transfected with an empty vector or a plasmid expressing a Myc-tagged mutant SMC1 in which Ser966 is substituted by Ala (SMC1S966A) (Figures 6a and b, see Kim et al14), and we analyzed apoptotic phenotype after DFO treatment. The expression of mutant SMC1 was confirmed by immunoblotting of anti-Myc antibody. As described in Figure 3, phosphorylation of both Chk1 at Ser317 and NBS1 at Ser343 were induced with DFO treatment in both cell types, but it was weakly decreased in when SMC1S966A was expressed (Figure 6a, lanes 2 and 6; Figure 6b, lanes 2 and 5). It is well illustrated that hypoxia condition induces HIF1α, a transcription factor that has an essential role in cellular and systemic responses to hypoxia.25 As shown in Figure 6a, DFO induced HIF1α in 293T cells, and levels of HIF1a were not significantly changed when SMC1 S966A was expressed (Figure 6a, lanes 2 and 6).


Phosphorylation of SMC1 by ATR is required for desferrioxamine (DFO)-induced apoptosis.

So EY, Ausman M, Saeki T, Ouchi T - Cell Death Dis (2011)

Transfection of 293T cells and HCT116 cells with S966A-mutant SMC1 inhibited the DFO-induced apoptosis. (a) 293T cells and (b) HCT116 cells were transfected with myc-tagged mutant SMC1 plasmid (Myc-SMC1S966A) or a control vector. After 48 h, cells were treated with DFO (300 μM) or NCS (0.5 μg/ml) for additional 24 h. Total extracts were studied for western blotting with indicated antibodies to detect level of HIF1α and phosphorylated proteins of NBS1 S343P and Chk1 at S317P. Actin blot serves as a loading control. (c) 293T cells and (d) HCT116 cells were transfected with plasmid containing mutant SMC1 (Myc-SMC1S966A) or a control vector. After 48 h, cells were treated with DFO (300 μM) for additional 24 h and then apoptosis was analyzed using a flow cytometer (left panel). Dot plot represents two independent experiments, indicating Annexin V staining (right panel). DFO-induced apoptosis was expressed as fold-induction compared with untreated samples (right panel, *P<0.05, Student's t-test). (e) Levels of Myc-tagged SMC1S966A and phosphorylation of endogenous SMC1 at Ser966 were compared in HCT116 and 293T cell lines. The phosphorylation and expression of proteins were analyzed by densitometry as described in Figure 1
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fig6: Transfection of 293T cells and HCT116 cells with S966A-mutant SMC1 inhibited the DFO-induced apoptosis. (a) 293T cells and (b) HCT116 cells were transfected with myc-tagged mutant SMC1 plasmid (Myc-SMC1S966A) or a control vector. After 48 h, cells were treated with DFO (300 μM) or NCS (0.5 μg/ml) for additional 24 h. Total extracts were studied for western blotting with indicated antibodies to detect level of HIF1α and phosphorylated proteins of NBS1 S343P and Chk1 at S317P. Actin blot serves as a loading control. (c) 293T cells and (d) HCT116 cells were transfected with plasmid containing mutant SMC1 (Myc-SMC1S966A) or a control vector. After 48 h, cells were treated with DFO (300 μM) for additional 24 h and then apoptosis was analyzed using a flow cytometer (left panel). Dot plot represents two independent experiments, indicating Annexin V staining (right panel). DFO-induced apoptosis was expressed as fold-induction compared with untreated samples (right panel, *P<0.05, Student's t-test). (e) Levels of Myc-tagged SMC1S966A and phosphorylation of endogenous SMC1 at Ser966 were compared in HCT116 and 293T cell lines. The phosphorylation and expression of proteins were analyzed by densitometry as described in Figure 1
Mentions: On the basis of the results demonstrated in Figure 1, we hypothesized that this ATR-mediated phosphorylation of SMC1is involved in DFO-induced apoptosis that is shown in Figure 5. We explored transient expression of a phospho-deficient mutant form of SMC1 in cell culture. 293T cells and HCT116 cells were transfected with an empty vector or a plasmid expressing a Myc-tagged mutant SMC1 in which Ser966 is substituted by Ala (SMC1S966A) (Figures 6a and b, see Kim et al14), and we analyzed apoptotic phenotype after DFO treatment. The expression of mutant SMC1 was confirmed by immunoblotting of anti-Myc antibody. As described in Figure 3, phosphorylation of both Chk1 at Ser317 and NBS1 at Ser343 were induced with DFO treatment in both cell types, but it was weakly decreased in when SMC1S966A was expressed (Figure 6a, lanes 2 and 6; Figure 6b, lanes 2 and 5). It is well illustrated that hypoxia condition induces HIF1α, a transcription factor that has an essential role in cellular and systemic responses to hypoxia.25 As shown in Figure 6a, DFO induced HIF1α in 293T cells, and levels of HIF1a were not significantly changed when SMC1 S966A was expressed (Figure 6a, lanes 2 and 6).

Bottom Line: Among these sites, phosphorylation of SMC1, NBS1, and Chk1 by DFO are mediated by ATR as it is greatly reduced in both ATR-deficient human fibroblasts and HCT116 human colon cancer cells in which ATR is heterozygously mutated, whereas these proteins are phosphorylated in cells deficient for ATM and DNA-PKcs.DFO-induced apoptosis is decreased in ATR-mutant HCT116 cells, although p53 is normally activated in those cells.These results suggest that DFO induces apoptosis through the ATR-SMC1 arm of the pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, NUHS, Systems Biology Program, Pritzker School of Medicine, The University of Chicago, Evanston, IL 60201, USA.

ABSTRACT
DNA damage signaling pathways are initiated in response to chemical reagents and radiation damage, as well as in response to hypoxia. It is implicated that structural maintenance of chromosomes 1 (SMC1) is not only a component of the cohesion complex but also facilitates the activation of DNA damage checkpoint proteins. Here, we studied the mechanism of DNA damage checkpoint activated by ATR-SMC1 pathway when cells are treated with desferrioxamine (DFO), a hypoxia-mimetic reagent. We show that DFO treatment induces phosphorylation of SMC1 at Ser966, NBS1 at Ser343, Chk1 at Ser317, Chk2 at Thr68, and p53 at Ser15. Among these sites, phosphorylation of SMC1, NBS1, and Chk1 by DFO are mediated by ATR as it is greatly reduced in both ATR-deficient human fibroblasts and HCT116 human colon cancer cells in which ATR is heterozygously mutated, whereas these proteins are phosphorylated in cells deficient for ATM and DNA-PKcs. DFO-induced apoptosis is decreased in ATR-mutant HCT116 cells, although p53 is normally activated in those cells. Expression of SMC1 S966A in which Ser966 is substituted to Ala attenuates apoptosis and phosphorylation of Chk1 at Ser317 after DFO treatment, although levels of HIF1α are not significantly changed. These results suggest that DFO induces apoptosis through the ATR-SMC1 arm of the pathway.

Show MeSH
Related in: MedlinePlus