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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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DFO induced cell cycle arrest in wild-type HCT116 cells and isogenic cell lines. Cells were treated with DFO (300 μM) or HU (3 mM) for 24 h. Cell cycle was quantified by single-parameter flow cytometry after PI staining for DNA content. Graphs represent percentages of cells in sub-G1, G1, S, and G1/M phases of cell cycle. Data shown here are representative of three experiments performed
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fig4: DFO induced cell cycle arrest in wild-type HCT116 cells and isogenic cell lines. Cells were treated with DFO (300 μM) or HU (3 mM) for 24 h. Cell cycle was quantified by single-parameter flow cytometry after PI staining for DNA content. Graphs represent percentages of cells in sub-G1, G1, S, and G1/M phases of cell cycle. Data shown here are representative of three experiments performed

Mentions: It is shown that anoxia or hypoxia induces a G1 and intra-S phase arrest.10 As DFO is widely used to induce hypoxia condition in cell culture,25 we studied the role of ATR in DFO-induced cell cycle checkpoint. Parental, HCT116-p53(−), HCT116-DNA-PKcs(−), or HCT116-ATR(−/flox) cells were treated with DFO or HU for 24 h and collected, then stained with propidium iodide (PI) for cell cycle analysis using flow cytometer (Figure 4).


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

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

DFO induced cell cycle arrest in wild-type HCT116 cells and isogenic cell lines. Cells were treated with DFO (300 μM) or HU (3 mM) for 24 h. Cell cycle was quantified by single-parameter flow cytometry after PI staining for DNA content. Graphs represent percentages of cells in sub-G1, G1, S, and G1/M phases of cell cycle. Data shown here are representative of three experiments performed
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101822&req=5

fig4: DFO induced cell cycle arrest in wild-type HCT116 cells and isogenic cell lines. Cells were treated with DFO (300 μM) or HU (3 mM) for 24 h. Cell cycle was quantified by single-parameter flow cytometry after PI staining for DNA content. Graphs represent percentages of cells in sub-G1, G1, S, and G1/M phases of cell cycle. Data shown here are representative of three experiments performed
Mentions: It is shown that anoxia or hypoxia induces a G1 and intra-S phase arrest.10 As DFO is widely used to induce hypoxia condition in cell culture,25 we studied the role of ATR in DFO-induced cell cycle checkpoint. Parental, HCT116-p53(−), HCT116-DNA-PKcs(−), or HCT116-ATR(−/flox) cells were treated with DFO or HU for 24 h and collected, then stained with propidium iodide (PI) for cell cycle analysis using flow cytometer (Figure 4).

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