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First published online 21 February 2006
doi: 10.1242/jcs.02821

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Disruption of G1-phase phospholipid turnover by inhibition of Ca²⁺-independent phospholipase A₂ induces a p53-dependent cell-cycle arrest in G1 phase

¹ Division of Experimental Diabetes and Aging, Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, NY 10029, USA
² Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA

View larger version (25K): [in a new window]   Fig. 1. Inactivation of iPLA2 inhibits INS-1 cell proliferation. (A) BEL inhibits INS-1-cell proliferation in a concentration-dependent manner. Cells (105) were treated with different BEL concentrations for up to 6 days and counted daily. Error bars represent the mean ± standard deviation (s.d.) of three experiments. (B) Proliferation of BEL-treated cells recovers upon BEL withdrawal. INS-1 cells were cultured in the presence of BEL (15 µM) for 2 days and then continuously cultured with (red) or without (green) BEL. Untreated cells were used as controls (blue). Error bars represent standard deviation from the mean of three experiments. (C) Transient expression of ARD-iPLA2-GFP reduces INS-1 cell proliferation. INS-1 cells (2x105) were transfected with ARD-iPLA2-GFP (5 µg/100 mm plate), then counted daily, starting 24 hours after transfection. Mock-transfected cells (no DNA) were used as controls. The cell lysates were prepared and analyzed for expression of ARD-iPLA2-GFP over 6 days, by western blotting with anti-GFP and anti-actin antibodies (top). Graph shows analysed data (bottom). Error bars represent the mean ± s.d. of three experiments. (D) Expression of Mut-iPLA2-GFP inhibits the proliferation of INS-1 cells. iPLA2-expressing INS-1 cells were transfected with Mut-iPLA2-GFP (labelled Mut or Mut-iPLA2 at 10 µg/100 mm plate) or mock transfected (no DNA) with FuGENE. Counting of cells began on the second day after transfection (bottom). After counting, cells lysates were prepared and analyzed by western blotting for GFP and actin (top). Error bars represent the mean ± s.d. of three experiments. *P<0.05. (E) Expressing siRNA against iPLA2 decreases cell proliferation. Mock transfected INS-1 cells, and those transfected with psiRNA-iPLA2 were counted on the fifth day after transfection (bottom), and the cell lysates were prepared for Western blot analysis for iPLA2 (top). *P<0.05. A scrambled siRNA construct was transfected to serve as a negative control. Error bars represent the mean ± s.d. of three experiments.

View larger version (47K): [in a new window]   Fig. 2. Inactivation of iPLA2 by BEL arrests INS-1 cells in G1 phase. (A) Inactivation of iPLA2 inhibits DNA synthesis. Cells were cultured for 24 hours in with or without BEL (12.5 µM) and labelled with BrdU or not. Cells were stained with fluorescein-conjugated anti-BrdU for BrdU and PI for DNA. R2 indicates cells in S phase. (B) BEL prevents cell progression to G2-M transition. INS-1 cells were treated with BEL (12.5 µM) followed by immunofluorescent staining of the mitosis marker phosphohistone H3. Treatment of cells with nocodazole served as a control for G2-M-transition arrest. Cells were fixed and stained for phosphohistone H3 and for iPLA2. Magnification, 40x. (C) Inhibition of iPLA2 in the presence of nocodazole increases the number of cells in G1 phase. INS-1 cells were treated either with nocodazole (0.5 µg/ml) only or with both nocodazole and different concentrations of BEL for 20 hours and 30 hours. Nontreated cells were used as controls. The DNA content of the cells was then analyzed by FACS after PI staining. The percentages of each indicated phase represents the mean of three FACS analyses.

View larger version (39K): [in a new window]   Fig. 3. Enforced expression of the iPLA2 ankyrin-repeat domain induces G1-phase arrest in INS-1 cells. (A) Cells with higher expression levels of ARD-iPLA2-GFP have a higher number of cells in G1 phase than cells with lower expression levels. INS-1 cells were transfected with ARD-iPLA2-GFP or iPLA2-GFP constructs; 42 hours after transfection, cells were stained with PI and subjected to FACS analysis for GFP-fluorescence (FL1-H) and DNA content (FL2-A). R1, R2 and R3 represent the gated cells with increasing GFP-fusion protein expression (left). Phasic distribution of the gated GFP cells is displayed (right). Figures represent one of four experiments and data represent the mean of four experiments. (B) Cells expressing ARD-iPLA2-GFP remain in G1 phase even in the presence of nocodazole. INS-1 cells were transfected with ARD-iPLA2-GFP or iPLA2-GFP constructs (green), stained for the nuclear pore protein mAB414 (red), counterstained for DNA with DAPI (blue) and analyzed by fluorescence microscopy (Zeiss Axioskop). Arrows in b and f indicate the condensed chromosomes; arrows in c and g indicate the nuclear-envelope-breakdown events; arrows in d and h indicate merged cells of a, b and c, and of e, f and g, respectively. Magnification, 100x. (C) The number of ARD-iPLA2-GFP-expressing cells in G1 phase far exceeds that of iPLA2-GFP-expressing cells, even in the presence of nocodazole. Cells of the same experiments as in B were collected and their DNA content was subjected to FACS analysis. When GFP-intensity levels were equalized (FL1-H) in the presence of nocodazole, 61.5% of ARD-iPLA2-GFP-expressing cells versus 37% of iPLA2-GFP-expressing cells were in G1 phase. By contrast, 27.7% of ARD-iPLA2-GFP-expressing cells versus 57.4% of iPLA2-GFP-expressing cells were in G2-M transition. A total of 200,000 total events were analysed. Error bars represent the mean ± s.d. of three experiments.

View larger version (20K): [in a new window]   Fig. 4. LPA or Lyso-PC treatment cannot overcome the inhibition of cellular proliferation by BEL. INS-1 cells were treated with or without BEL, and 20 µM of LPA and lyso-PC were added to the BEL-treated group. BrdU incorporation was then measured. All data represent the mean of duplicates from three individual experiments. Error bars represent the mean ± s.d. of six experiments; *P<0.05.

View larger version (38K): [in a new window]   Fig. 5. Inhibition of iPLA2 induces accumulation of p53 and expression of p21cip1. (A) Expression of ARD-iPLA2-GFP in INS-1 cells induces the accumulation of p53 and expression of p21. Cells were mock-transfected or transfected with increasing amounts of DNA (2, 5 and 10 µg/100 mm plate). Cell lysates were prepared and analyzed by western blotting with antibodies against GFP, p53, p21 and actin. Recombinant TNT p53 and ARD-iPLA2-GFP proteins were loaded as controls. (B) Treatment with BEL for 30 hours increases p53, p21 and p27 levels and decreases cyclin A levels in INS-1 cells. Cells were treated with or without BEL (15 µM) for 30 hours. Cell lysates were prepared and analyzed by western blotting for p53, p21, p27, cyclin A and actin. (C) p53 accumulates in the nuclei of BEL-treated INS-1 cells. Cells treated with 15 µM BEL were fixed and stained with anti-p53 (FL-393, Santa Cruz Biotechnology) and counterstained with DAPI followed by analysis with a confocal scanning microscope (Zeiss LSM 510 META). Magnification, 100x.

View larger version (39K): [in a new window]   Fig. 6. G1-phase arrest induced by inhibition of iPLA2 requires p53. (A) BEL treatment for 10 hours increases the number of HCTp53+/+ cells but not of HCTp53-/- cells in G1 phase. Both HCTp53+/+ and HCTp53-/- cells were treated with increasing concentrations of BEL for 10 hours. The DNA contents of each sample was analyzed by FACS. Data represent the mean of three experiments. (B) Accumulation of p53 and expression of p21 increases with increasing concentrations of BEL. HCTp53+/+ and HCTp53-/- cells were treated with increasing concentrations of BEL for 10 hours. Cell lysates were prepared and analyzed by western blot for p53, p21, p27 and actin. (C) Inhibition of iPLA2 with increasing concentrations of BEL dramatically inhibits the proliferation of HCTp53+/+ but only mildly inhibits the proliferation of HCTp53-/- cells. HCTp53+/+ cells (blue bars) and HCTp53-/- cells (red bars) were cultured with increasing concentrations of BEL in 24-well microplates for 28 hours, followed by 6 hours of BrdU labelling. Cell proliferation was then determined on a µQuant microplate reader. Error bars represent the mean ± s.d. of three experiments; *P<0.05, **P<0.005. (D) Accumulation of p53 and expression of p21 increases with increasing concentrations of BEL in HCTp53+/+ (left). In HCTp53-/- cells, by contrast, p21 expression increased only slightly (right). Both HCTp53+/+ and HCTp53-/- cells were treated with increasing concentrations of BEL for 28 hours in 24-well microplates. Cell lysates were prepared and analyzed by western blot for p53, p21, p27 and actin.

View larger version (18K): [in a new window]   Fig. 7. BEL treatment induces p53-dependent apoptosis in p21-deficient HCT cells. (A) HCTp21-/- cells have a high death rate after inactivation of iPLA2 by BEL. HCTp21-/- cells (5x105) were treated with varying concentrations of BEL for 28 hours. Viable and nonviable cells were determined by Trypan Blue staining and counted. Error bars represent the mean ± s.d. of six experiments. (B) Inactivation of iPLA2 induces apoptosis of p21-deficient cells. Both wild-type HCT and HCTp21-/- cells were incubated with or without BEL for 10 hours and then analyzed for apoptosis by Annexin-V-Fluos staining and FACS. R1 represents living cells. R2 represents cells undergoing early apoptosis, and R3 representes secondary necrotic cells. Each panel shows a typical flow-cytometric histogram of 10,000 cells/sample from one of three experiments. (C) BEL-associated death of HCTp21-/- cells can be prevented by p53 depletion. HCTp21-/- cells were cultured overnight in the presence of BEL with 1 nmol of SMARTpool-p53. dsRNA was used as a negative control. The cells were harvested for both western blot analysis for p53 (top) and cell death analysis (bottom). Recombinant TNT p53 was loaded as indicated. *P<0.05.