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First published online 22 March 2005
doi: 10.1242/jcs.01728

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STAT-1 facilitates the ATM activated checkpoint pathway following DNA damage

Paul A. Townsend^1,*, Mark S. Cragg², Sean M. Davidson¹, James McCormick¹, Sean Barry¹, Kevin M. Lawrence¹, Richard A. Knight¹, Michael Hubank³, Phang-Lang Chen⁴, David S. Latchman¹ and Anastasis Stephanou^1,

¹ Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, UK
³ Molecular Haematology, Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, UK
² Cancer Sciences Division, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, UK
⁴ Department of Molecular Medicine and Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78245, USA

View larger version (20K): [in a new window]   Fig. 1. STAT-1 functions in the S-phase and G2/M checkpoint. (A) U3A cells lacking STAT-1 display an RDS phenotype. The method involves pre-pulsing 2fTGH, U3A or U3A-ST1 cells with 14C thymidine, irradiation, and then assessing 3H uptake after 2-5 Gy -irradiation (IR); DNA synthesis was assessed 2 hours later. The upper panel shows expression levels of STAT-1 (ST1) and also induction of phospho-STAT-1Y701 (pST1) in response to -interferon for 30 minutes in 2fTGH, U3A or U3A-ST1 cells. (B) Dose effect of ionising -irradiation (1-10 Gy) and DNA synthesis in 2fTGH, U3A or U3A-ST1 cells. (C) Analysis of the G2/M checkpoint in 2fTGH, U3A or U3A-ST1 cells exposed to 10 Gy -irradiation (IR). The mitotic index of cells was assessed by histone H3 phosphorylation 4 hours after irradiation. (D) Analysis of the G2/M checkpoint in 2fTGH, U3A or U3A-ST1 cells exposed to 10 Gy -irradiation (IR), where the mitotic index of cells was assessed by DAPI staining of chromosomal metaphase spreads of treated versus untreated cells. (E) Cell survival was assessed following exposure to 10 Gy -irradiation in 2fTGH, U3A or U3A-ST1 cells for 72 hours. After Crystal Violet staining, the percentage of cell survival was determined. Data are representative of three separate experiments.

View larger version (47K): [in a new window]   Fig. 2. STAT-1 is required for activation of the ATM-Chk2-Cdc25A and ATM-NBS1 pathways. (A) 2fTGH, U3A and U3A-ST1 cells were exposed to 0, 2 or 10 Gy -IR. Cells were harvested after 2 hours and extracts were incubated with relevant antibodies against the proteins indicated on the western blot. (B) 2fTGH, U3A and U3A-ST1 cells were exposed to -IR (2 Gy), fixed after 2 hours and immunofluorescence analysis with antibody against phosphorylated Chk2-T68 was carried out. (C) U3A-ST1 and U3A cells were exposed to 2 or 10 Gy -IR; cells were harvested 2 hours later and extracts immunoblotted with antibodies against the proteins indicated. (D) U3A-ST1 and U3A cells were exposed to -IR (2 Gy), fixed after 2 hours and immunofluorescence analysis was carried out with antibodies against the proteins indicated. The dimensions of the field of view are 40 mM x40 mM.

View larger version (30K): [in a new window]   Fig. 3. Modulation of 53BP1 and MDC1 levels by STAT-1. (A) Cell lysates from 2fTGH, U3A and U3A-ST1 cells were immunoblotted for MDC1 and 53BP1. 53BP1 and MDC1 expression is reduced in STAT-1-deficient U3A cells. (B) RT-PCR showing reduced 53BP1 and MDC1 mRNA levels in U3A cells. MDC1 and 53BP1 mRNA levels were assessed from 2fTGH, U3A or U3A-ST1 cells. (C) 2fTGH, U3A or U3A-ST1 cells were exposed to -IR (2 Gy), fixed after 2 hours and stained with anti-53BP1 or anti-MDC1 antibody. The dimensions of the field of view are 40 mM x40 mM. (D,E) The MDC1 promoter is modulated by STAT-1. The MDC1-reporter construct was transfected into STAT-1+/+ and STAT-1–/– MEF cells, and U3A and U3A-ST1 cells together with either full length STAT-1 (ST1), STAT-1ß (ST1B) or a control vector. Upper panels in D and E show immunoblots of transfected cells for STAT-1 (ST1) or STAT-1ß (ST1ß).

View larger version (20K): [in a new window]   Fig. 4. Association of STAT-1 with Chk2, MDC1 and 53BP1 following DNA damage. Immunoprecipitations were carried out with an anti-STAT-1 antibody (IP-ST1) in untreated 2fTGH cells (A) or 2fTGH cells exposed to -IR (B) and immunoblotted with antibodies against the target proteins indicated.

View larger version (44K): [in a new window]   Fig. 5. In cells lacking STAT-1, overexpression of MDC1 restores ATM-dependent phosphorylation, which requires the FHA domain. (A) U3A cells were transfected with GFP control, wild-type GFP-MDC1 (WT) or a mutant GFP-MDC1-FHA (FHA), lacking the FHA domain. Cells were immunoblotted with antibodies against the proteins indicated. (B,C) Effect of increasing amounts of (B) wild type (MDC1-WT) or (C) mutant (MDC1-FHA) on ATM-dependent phosphorylation as assessed by immunoblotting with antibodies against the proteins indicated. (D) Quantification of the Chk2-T68 phosphorylation shown in B and C by densitometry. Data represent three independent experiments. (E) MEF STAT-1–/– cells were transfected with GFP control, wild-type GFP-MDC1 (WT) or a mutant GFP-MDC1-FHA (FHA). Cells were immunoblotted with an antibody against phosphorylated p53-S15, p53 and actin (control). (F) MDC1 RNAi reduces expression of endogenous MDC1 in 2fTGH cells. (G) Transfection of MDC1 RNAi in 2fTGH cells reduced ATM-dependent phosphorylation following -IR (5 Gy) as assessed by immunoblotting with antibodies against the proteins indicated.

View larger version (41K): [in a new window]   Fig. 6. Western blot analysis demonstrates that elevated expression of STAT-1 is associated with enhanced expression of MDC1 and 53BP1, and constitutive phosphorylation of ATM, Chk2 and NSB1 in cells that lack p53 or carry a mutation for p53. (A) Lysates of Soas2 (p53-deficient), HCT15 (p53 mutant), IMR90 (p53 wild type) or SKNSH (p53 wild type) cells were immunoblotted with antibodies against the proteins indicated. (B) STAT-1 levels are enhanced in MEF p53–/– cells compared with MEF p53+/+ cells as assessed in a western blot. (C,D,E) Western blots show that, transfection of HCT15 and Soas2 cells (C and D, respectively) with STAT-1 RNAi reduces levels of STAT-1 protein, levels of MDC1 and 53BP1, and also reduces the level of phosphorylated ATM (pATM-S1981). Overexpression of STAT-1, by contrast, increases levels of phosphorylated ATM and the phosphorylated forms of downstream phosphorylation-substrates of ATM. IMR90 cells were transfected with a STAT-1 expression vector; cells were harvested 48 hours later and lysates immunoblotted with antibodies against the proteins indicated.