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Nitric oxide induces apoptosis in megakaryocytic cell lines

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Presentation on theme: "Nitric oxide induces apoptosis in megakaryocytic cell lines"— Presentation transcript:

1 Nitric oxide induces apoptosis in megakaryocytic cell lines
by Elisabeth Battinelli, and Joseph Loscalzo Blood Volume 95(11): June 1, 2000 ©2000 by American Society of Hematology

2 GSNO and HEL cell endonucleosomal DNA cleavage
GSNO and HEL cell endonucleosomal DNA cleavage.(A) Autoradiographic analysis of total cellular DNA prepared from HEL cells after treatment with 100 μmol/L GSNO for different times and 3′-end labeled (1 μg DNA/lane). GSNO and HEL cell endonucleosomal DNA cleavage.(A) Autoradiographic analysis of total cellular DNA prepared from HEL cells after treatment with 100 μmol/L GSNO for different times and 3′-end labeled (1 μg DNA/lane). The characteristic endonucleosomal fragmentation of DNA into 185-bp multiples is visible as early as 30 minutes after treatment with 100 μmol/L GSNO. 1, Untreated HEL cells; 2, HEL cells treated with 100 μmol/L GSNO for 30 minutes; 3, for 1 hour; 4, for 2 hours; 5, for 3 hours; 6, for 4 hours; and 7, for 5 hours. (B) Autoradiographic analysis of total cellular DNA prepared from HEL cells after induction of endogenous iNOS, as described in “Materials and methods.” 1, Cytokine induction of iNOS; 2, cytokine induction of iNOS in the presence of L-NMMA. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95: ©2000 by American Society of Hematology

3 GSNO or iNOS induction and Meg-01 TUNEL assay
GSNO or iNOS induction and Meg-01 TUNEL assay.(A) Untreated Meg-01 cells (YOYO-1 DNA stain; 40× magnification). GSNO or iNOS induction and Meg-01 TUNEL assay.(A) Untreated Meg-01 cells (YOYO-1 DNA stain; 40× magnification). (B) Meg-01 cells treated with 100 μmol/L GSNO showing TUNEL-positive cells (YOYO-1 DNA stain; 40× magnification). (C) Percentage apoptotic cells visible by TUNEL assay. To calculate the percentages in each case, 5 to 10 random fields were examined, and each bar graph on the plot represents the average of these experiments (*P < .05). Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95: ©2000 by American Society of Hematology

4 GSNO induction and HEL TUNEL assay
GSNO induction and HEL TUNEL assay.(A) HEL cells treated with cytokines to stimulate the expression of iNOS and TUNEL-positive cells (YOYO-1 DNA stain; 40× magnification). GSNO induction and HEL TUNEL assay.(A) HEL cells treated with cytokines to stimulate the expression of iNOS and TUNEL-positive cells (YOYO-1 DNA stain; 40× magnification). (B) HEL cells treated with cytokines to stimulate expression of iNOS and incubated with 100 μmol/L L-NMMA showing decreased TUNEL-positive cell staining (YOYO-1 DNA stain; 40× magnification). (C) Percentage of apoptotic cells that were visible by TUNEL assay. 5 to 10 random fields were examined to calculate the percentages in each case, and each bar in the plot represents the average of 3 experiments (*P < .05). Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95: ©2000 by American Society of Hematology

5 Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459
©2000 by American Society of Hematology

6 Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459
©2000 by American Society of Hematology

7 HEL cell CPP32 activity.CPP32 enzymatic activity present in untreated HEL cells, HEL cells treated with a CPP32 inhibitor, and HEL cells treated with 100 μmol/L GSNO. HEL cell CPP32 activity.CPP32 enzymatic activity present in untreated HEL cells, HEL cells treated with a CPP32 inhibitor, and HEL cells treated with 100 μmol/L GSNO. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95: ©2000 by American Society of Hematology

8 Meg-01 cell Bcl-2 and Bax expression
Meg-01 cell Bcl-2 and Bax expression.(A) Western blot analysis of Bcl-2 expression in Meg-01 cell lysates after treatment with 100 μmol/L GSNO for varying times. Meg-01 cell Bcl-2 and Bax expression.(A) Western blot analysis of Bcl-2 expression in Meg-01 cell lysates after treatment with 100 μmol/L GSNO for varying times. Lane 1, untreated cells; lane 2, 1 hour of treatment; lane 3, 2 hours of treatment; lane 4, 4 hours of treatment. (B) Western blot analysis of Bax expression in Meg-01 cell lysates after treatment with 100 μmol/L GSNO for varying times. Lane 1, untreated; lane 2, 1 hour of treatment; lane 3, 2 hours of treatment. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95: ©2000 by American Society of Hematology

9 Peroxynitrite and Meg-01 COMET formation
Peroxynitrite and Meg-01 COMET formation.(A) Control Meg-01 cells or (B) Meg-01 cells exposed to 8 μmol/L peroxynitrite for 10 minutes. Peroxynitrite and Meg-01 COMET formation.(A) Control Meg-01 cells or (B) Meg-01 cells exposed to 8 μmol/L peroxynitrite for 10 minutes. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95: ©2000 by American Society of Hematology

10 HEL cells COMET formation and Tiron
HEL cells COMET formation and Tiron.Percentage of B and C COMET after treatment of HEL cells with 10 μmol/L GSNO for 120 minutes in the presence of various concentrations of Tiron (circles). HEL cells COMET formation and Tiron.Percentage of B and C COMET after treatment of HEL cells with 10 μmol/L GSNO for 120 minutes in the presence of various concentrations of Tiron (circles). Percentage of B and C COMET after cytokine induction of iNOS in HEL cells for 16 hours in the presence of various concentrations of Tiron (squares). Each point represents the average of 3 experiments. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95: ©2000 by American Society of Hematology

11 Effect of antioxidants on NO-induced HEL cell apoptosis
Effect of antioxidants on NO-induced HEL cell apoptosis.Percentage of B and C COMET achieved by exposure to NO produced in co-culture by fibroblasts in which iNOS had been induced in the presence of various superoxide inhibitors, including superoxide dismut... Effect of antioxidants on NO-induced HEL cell apoptosis.Percentage of B and C COMET achieved by exposure to NO produced in co-culture by fibroblasts in which iNOS had been induced in the presence of various superoxide inhibitors, including superoxide dismutase (SOD), catalase, oxypurinol, indomethacin, and Tiron (*P < .02). Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95: ©2000 by American Society of Hematology

12 Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459
©2000 by American Society of Hematology

13 Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459
©2000 by American Society of Hematology

14 Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459
©2000 by American Society of Hematology

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