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First published online November 21, 2007
doi: 10.1242/10.1242/jcs.015834

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The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR

Division of Experimental Diabetes and Aging, Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, NY 10029, USA

View larger version (56K): [in this window] [in a new window]   Fig. 1. Inhibition of iPLA2 induces phosphorylation of p53 at Ser15. (A) Dose-dependent induction of p53 phosphorylation at Ser15 (p53-P) by BEL. HCT116 cells were prepared and treated with BEL for 6 hours. The cell lysates were prepared and the levels of p53-P and total p53 were determined by western blotting. Actin was used as an internal protein control. (B) siRNA silencing of iPLA2 expression induced phosphorylation of p53. HCT116 cells were transfected with mock, scramble siRNA and siRNA specifically targeting iPLA2. The samples were analyzed by western blotting for iPLA2, p53-P and actin. (C) Time course of BEL-induced p53-P in HCT116 cells. HCT116 cells were treated with 15 µM BEL for the times indicated. p53-P levels were assessed at each time point by western blotting. (D) BEL-induced p53 activation and MDM2 expression. HCT116 cells were incubated with BEL (12.5 µM) or vehicle for up to 20 hours and the levels of p53, p53-P and MDM2 were analyzed by western blotting. (E) BEL-induced p53 phosphorylation in primary human foreskin fibroblast BJ PD27 cells. BJ PD27 cells were prepared and treated with BEL for 10 hours. The cell lysates were prepared and the levels of iPLA2, p53-P and actin were determined by western blotting.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Detection of DNA damage by phosphorylation of H2AX at ser 139, TUNEL, and comet assay. (A) Western blot analyses of H2AX-P. HCT116 cells were treated with increasing concentration of BEL for 8 or 28 hours. H2AX-P and p53-P, p21, total p53 and PCNA levels were analyzed by western blotting. Cells treated with doxorubicin (Dox) (1 µg/ml) for 28 hours were used as a positive control for dsDNA breaks. (B) Western blot analysis of H2AX-P in HCT116-p21–/– cells. HCT116-p21–/– cells were treated with increasing concentrations of BEL for 8 hours and H2AX-P levels were analyzed by western blotting. HCT116-p21–/– cells were next incubated with and without caspase inhibitor (Z-VAD-FMK, 20 µM) for 30 minutes as indicated before being continuously cultured in the presence or absence of 12.5 µM BEL for 6 hours. H2AX-P levels in these cells were analyzed by western blotting. (C) Immunofluorescent staining of H2AX-P in multiple HCT116 cells. Cells were treated with vehicle (control), Dox (0.2 µg/ml) for 8 hours, BEL (12.5 µM) for 8 hours. Samples were stained for DAPI (blue) and H2AX-P (red) and analyzed by a confocal microscope at x20 magnification. Merged cells are shown in pink. (D) Immunofluorescent staining of H2AX-P in a single nucleus. BEL (12.5 µM, 8 hours) and Dox (0.2 µg/ml, 8 hours) treated cells were stained with anti-H2AX-P antibody (red) and DAPI (blue), and individual cells were analyzed using a confocal microscope at x100 magnification. Merging of H2AX-P and DAPI staining in the nucleus appears pink. (E) TUNEL analysis in HCT116 cells. HCT116 cells were treated with 50 J/m2 UV light and BEL (10 and 15 µM) for 8 hours, and stained using TUNEL (green) and DAPI (blue) followed by the confocal fluorescence microscopy analysis at x63 magnification. Merging of TUNEL and DAPI staining in the nucleus appear light blue. (F) Comet assay to detect DNA damage in individual cells. HCT116 cells were treated with vehicle (control), 50 J/m2 of UV light, and 10 µM BEL for 8 hours, respectively. The cells were subjected to an analysis by comet assay. The upper panel showed the results visualized by a fluorescent microscope. The cells with UV light treatment showed the typical `comet pattern' of damaged DNA. The lower panel showed the plot of tail moments (TM) analyzed by the CometScore. More than 50 cells were scored in each condition. There was no significant difference between the BEL treatment and the control. *, significant difference of UV-light treatment from the control or BEL treatment.

View larger version (31K): [in this window] [in a new window]   Fig. 3. BEL-induced phosphorylation of p53 is sensitive to caffeine. (A) BEL-induced phosphorylation in HCT116 cells. HCT116 cells were cultured in the presence and absence of 2.5 mM caffeine for 12 hours and continuously cultured in the presence or absence of 12.5 µM BEL for 12 hours. Cell lysates were analyzed for p53-P, p53, p27 and p21 by western blotting. (B) BEL induced p53-P and accumulation of p53 in rat pancreatic -cell line INS-1 cells. INS-1 cells were treated as described in A and cell lysates were analyzed for p53-P, p53, PCNA and actin by western blotting.

View larger version (49K): [in this window] [in a new window]   Fig. 4. BEL induced the phosphorylation of p53 in ATM+/+, ATM–/– cells and U2OS cells through doxycycline-stimulated expression of ATR-wt or ATR-kd. (A) Comparison of BEL-induced phosphorylation of p53 in GM01805 (ATM+/+) and GM01526 (ATM–/–) cells. ATM+/+ cells and ATM–/– cells were treated with or without BEL (15 µM) for 4 or 8 hours. Cell lysates were analyzed for p53 and p53-P by western blotting. (B) Response to BEL in ATM–/– (GM01526) cells over time. ATM–/– cells were cultured in the absence or presence of caffeine (2.5 mM) for 12 hours and continuously cultured in the presence or absence of BEL (12.5 µM) for 30 minutes and 3 hours. Levels of p53-P and p53 were examined by western blotting. (C) BEL-induced phosphorylation of p53-P in ATR-wt-inducible U2OS cells. ATR-wt-inducible U2OS cells (GW33) were cultured with or without doxycycline (1 µg/ml) for 1 day. The expression of FLAG-ATR-wt was determined using an anti-FLAG monoclonal antibody. The cells were first cultured in the absence or presence of caffeine (2.5 mM) for 12 hours and then in the absence or presence of BEL (12.5 µM) for 8 hours. Cells were collected and the levels of FLAG-ATR-wt, p53-P, p53, PCNA and actin were examined by western blotting. (D) BEL-induced p53-P in ATR-kd-inducible U2OS cells. ATR-kd-inducible U2OS cells (GK41) were cultured with or without doxycycline (1 µg/ml) for 1 day. The expression of FLAG-ATR-kd was examined using an anti-FLAG monoclonal antibody. The cells were treated with increasing concentrations of BEL for 8 hours and collected for analysis of FLAG-ATR-kd and p53-P by western blotting.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Lipid profile of BEL-treated HCT116 cells. (A) HCT116 cells were treated with 10 µM BEL or vehicle (Control) for 0.5, 8 or 24 hours and samples were prepared following the manufacturer's instructions. PC-fatty-acid composition was analyzed by TrueMass. Data represent the results from five repeats.*P<0.05. (B) The ratio of polyunsaturated to saturated fatty acids in PCs of BEL-treated HCT116 cells. Data were calculated using those shown in A. *P<0.05. (C) Cells were prepared as described in A and the composition of those PCs containing 18:2n6-fatty acids was analyzed by TrueMass. *P<0.05.

View larger version (50K): [in this window] [in a new window]   Fig. 6. PC induces phosphorylation of p53 at Ser15. (A) Time course of PC-induced p53-P in HCT116 cells. HCT116 cells were treated with 200 µM of PCs containing polyunsaturated fatty acids (18:2n6), and p53-P levels were analyzed at varying time points using western blotting. (B) Dose-dependent induction of p53-P by L--PC. U2OS (GW33) cells were treated with varying concentrations of L--PC for 3 hours and p53-P levels were analyzed by western blotting. (C) Effect of PCs containing saturated and monounsaturated fatty acids (8 hours) on p53-P in HCT116 cells. (D) PC induction of p53 phosphorylation requires ATR. ATR-kd-inducible U2OS (GK41) cells were induced for expression of ATR-kd with or without doxycycline and then treated with PC alone or a combination of both PC and BEL (7.5 µM) for 8 hours. TUNEL analysis in PC (18:2n6)-treated HCT116 cells. HCT116 cells were treated with 50 J/m2 UV light, and PCs (100 or 200 µM) for 4 and 8 hours and stained with TUNEL (green) and DAPI (blue) followed by the confocal fluorescence microscopy analysis at x63 magnification. In merged images of TUNEL and DAPI staining, the nucleus appear light blue. Only the merged images of TUNEL and DAPI staining were shown in the PC-treated cells.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Proposed model for activation of the ATR-p53-p21 pathway induced by increasing the amount of PCs that contain polyunsaturated fatty acids in membrane phospholipids. Under physiological conditions, the levels of polyunsaturated fatty acids in cell-membranous PCs in are precisely regulated by a iPLA2-mediated deacylation and reacylation cycle. Inhibition of iPLA2 results in an increase of phospholipids that contain polyunsaturated fatty acids, which may alter the fluidity of cell membranes especially nuclear envelopes. This leads to activation of the ATR-p53-p21 signalling pathway to prevent cells from entering the S phase of the cell cycle. In the absence of p21, the alteration in membrane phospholipids leads to apoptosis (Zhang et al., 2006). PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; LysoPC, lysophosphatidylcholine.