wild type wt jurkat  (ATCC)

Journal: Molecular Pharmacology

Article Title: Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

doi: 10.1124/mol.116.104000

Figure Lengend Snippet: TNFα-induction of necroptosis or extrinsic apoptosis induces release of adenine nucleotides from Jurkat cancer cells via mechanistically distinct pathways. (A) A schematic of cell death signaling and adenine nucleotide release pathways regulated by Fas and TNF receptors. (B, D, E) WT Jurkat cells were treated with either 250 ng/ml αFas for the indicated times or were pretreated with 3 μM of the Smac-mimetic BV6 for 2 hours followed by stimulation with 20 ng/ml TNF-α for the indicated times. (C, F) FADD-deficient Jurkat cells were pretreated with 3 μM BV6 for 2 hours followed by stimulation with 20 ng/ml TNF-α for the indicated times. Parallel samples were stimulated with death receptor ligands in the presence or absence of 20 μM zVAD or 1 μM NSA as indicated. (B, C) Samples of conditioned extracellular media were collected at 0, 2, 4, 8, and 12 hours after treatment and assayed for LDH activity. LDH released from TNFα + Smac mimetic (TS)-treated cells was assayed, analyzed, and normalized to the LDH released from detergent-lysed cells. Experiments with each agent were repeated 3–4 times with data indicating mean ± S.E. for n = 4 (WT) and n = 3 (FADD-deficient) independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: TS-stimulated +zVAD versus −zVAD; b: TS-stimulated +NSA versus −NSA. (D–F) Cells were treated with either αFas (D) or TS (E, F), and conditioned extracellular media samples were collected at 0, 2, 4, 8, and 12 hours after treatment and analyzed for summed ATP + ADP + AMP. Data indicate mean ± S.E. of n = 3 independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: αFas- or TS-treated +zVAD versus −zVAD; b: TS-stimulated +NSA versus −NSA. (G–I) Whole-cell lysates were prepared at the indicated times after treatment of Western blot analysis as described in Materials and Methods and probed for Panx1, PARP, and actin. Data are representative of three experiments for each condition. All panels: ns, not statistically significant; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The FADD-deficient human Jurkat T-cell acute leukemia cell line (I 2.1) and the wild-type (WT) human Jurkat T-cell acute leukemia cells were obtained from the American Type Culture Collection (Manassas VA).

Techniques: Activity Assay, Comparison, Western Blot

Journal: Molecular Pharmacology

Article Title: Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

doi: 10.1124/mol.116.104000

Figure Lengend Snippet: Intrinsic apoptotic signaling and Panx1 channel cleavage in FADD-deficient Jurkat cancer cells is uncoupled from accumulation of extracellular adenine nucleotides. WT and FADD-deficient cells were treated with 3 μM STS, 20 μM Etop, or 250 ng/ml αFas in the presence of 100 μM zVAD or 1 μM NSA as indicated. (A, B) Whole-cell lysates were prepared at the indicated times after treatment of Western blot analysis and probed for Panx1, PARP, and actin. Data are representative of three experiments for each cell type and condition. (C, D) Conditioned extracellular media samples were collected from STS-treated WT cells (C-i), Etop-treated WT cells (C-ii), STS-treated FADD-deficient (FADD-def) cells (D-i), or Etop-treated FADD-def cells (D-ii) at 0, 2, 4, 8, and 12 hours after treatment and assayed for release of LDH as described in Fig 1. Data indicate mean ± S.E. of three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: STS-stimulated or Etop-stimulated +zVAD versus −zVAD; b: STS- or Etop-stimulated +NSA versus −NSA. (E, F) WT and FADD-def cells were treated with 3 μM STS or 20 μM Etop in the presence or absence of 100 μM zVAD or 1 μM NSA where indicated. Conditioned extracellular media samples were collected from STS-treated WT cells (E-i), Etop-treated WT cells (E-ii), STS-treated (FADD-def cells (F-i), or Etop-treated FADD-def cells (F-ii) at 0, 2, 4, 8, and 12 hours after treatment and assayed for total ATP + ADP + AMP as described in Materials and Methods. Data indicate mean ± S.E. of three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: STS- or Etop-stimulated +zVAD versus −zVAD; b: STS- or Etop-stimulated +NSA versus −NSA. All panels: ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The FADD-deficient human Jurkat T-cell acute leukemia cell line (I 2.1) and the wild-type (WT) human Jurkat T-cell acute leukemia cells were obtained from the American Type Culture Collection (Manassas VA).

Techniques: Western Blot, Comparison

Journal: Molecular Pharmacology

Article Title: Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

doi: 10.1124/mol.116.104000

Figure Lengend Snippet: Caspase-3–cleaved Panx1 channels are functionally active in FADD-deficient Jurkat cancer cells during intrinsic apoptosis. (A) Schematic illustrates caspase-3–mediated proteolytic activation of Panx1 channel function as a pathway for ATP release or efflux/influx of charged organic dyes. (B, D) WT or FADD-deficient Jurkat cells were treated with 3 μM STS for 4 hours or 20 μM Etop for 12 hours. The cells were then washed, resuspended in BSS supplemented with 1 μM YO-PRO2+, and incubated for 20 minutes before quantification of YO-PRO2+ fluorescence per well or phase-contrast/epifluorescence imaging. (B) Data indicate mean ± S.E. from three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +STS versus −STS; b: FADD-def versus WT. (D) Images are representative of two to three independent experiments with each stimulus. (C) WT or FADD-deficient Jurkat cells were loaded with 1 μM calcein-AM and then treated with STS for 4 hours in the presence or absence of 100 μM zVAD or 50 μM Trova. Cells were then washed and resuspended in BSS, and the calcein fluorescence was quantified. Data indicate mean ± S.E. from three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +STS versus −STS; b: FADD-def versus WT; c: +STS with zVAD or Trova versus +STS alone. (E) WT or FADD-deficient Jurkat cells were treated with STS for 4 hours and then stained with MitoTracker Red and DAPI. Cells were imaged by confocal microscopy at original magnification 60×. Data are representative images of n = 15–19 individual cells. All panels: ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The FADD-deficient human Jurkat T-cell acute leukemia cell line (I 2.1) and the wild-type (WT) human Jurkat T-cell acute leukemia cells were obtained from the American Type Culture Collection (Manassas VA).

Techniques: Activation Assay, Incubation, Fluorescence, Imaging, Comparison, Staining, Confocal Microscopy

Journal: Molecular Pharmacology

Article Title: Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

doi: 10.1124/mol.116.104000

Figure Lengend Snippet: CD73 ecto-5′-nucleotidase is up-regulated in FADD-deficient Jurkat cells to rapidly metabolize AMP released during apoptosis or necroptosis. (A–C) WT and FADD-deficient cells were treated with 3 μM STS (A-i and A-ii), 20 μM Etop (B-i and B-ii), or TNFα plus BV6 Smac-mimetic (TS) (C-i and C-ii). Conditioned medium samples were collected at 0, 2, 4, 8, and 12 hours and assayed for either summed ATP + ADP + AMP (A-i; B-i, C-i) or ATP only (A-ii, B-ii, C-ii). Data indicate mean ± S.E. of three experiments and statistical analyses (two-way analysis of variance with Tukey post-test comparison) of the values of FADD-def versus WT at each time point. (D) Schematic of interactions between Panx1 channel-mediated adenine nucleotide efflux and metabolism of the released nucleotides by CD39 and CD73 ectonucleotidases (APCP) during extrinsic or intrinsic apoptosis. (E) qRT-PCR analysis of CD73 mRNA expression in WT and FADD-deficient cells. Signals in FADD-def were normalized to signals in WT and represent values from three independent experiments; analysis by two-tailed t test. (F) We added 1 μM exogenous AMP to suspensions (2 × 106/ml) of untreated WT or FADD-deficient Jurkat cells in the presence or absence of 50 μM APCP. Cell-free supernatants were collected after 30 minutes and analyzed for the concentration of remaining [AMP]. Data indicate mean ±S.E. of four independent experiments with WT cells and three independent experiments with FADD-def cells. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +APCP versus −APCP; b: FADD-def versus WT. (G) WT or FADD-deficient Jurkat cells were treated with 3 μM STS for 4 hours in the absence or presence of 50 μM APCP. Conditioned medium samples were collected and assayed for summed ATP + ADP + AMP. Data indicate mean ± S.E. of six independent experiments with WT cells and five independent experiments with FADD-def cells. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +STS versus −STS; b: FADD-def versus WT; c: +STS with APCP versus STS alone. All panels: ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The FADD-deficient human Jurkat T-cell acute leukemia cell line (I 2.1) and the wild-type (WT) human Jurkat T-cell acute leukemia cells were obtained from the American Type Culture Collection (Manassas VA).

Techniques: Comparison, Quantitative RT-PCR, Expressing, Two Tailed Test, Concentration Assay

Journal: Molecular Pharmacology

Article Title: Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

doi: 10.1124/mol.116.104000

Figure Lengend Snippet: Apoptotic signaling and Panx1 channel activation in RIP1-deficient Jurkat cancer cells is also uncoupled from accumulation of extracellular adenine nucleotides. (A–C) WT and RIP1-deficient Jurkat cells were treated with 3 μM STS for 4 hours (A), 20 μM Etop for 12 hours (B), or 250 ng/ml αFas for 4 hours (C). Conditioned extracellular media samples were collected and assayed for total ATP + ADP + AMP accumulation. Data indicate mean ± S.E. for three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: post-incubation ATP + ADP + AMP versus background ATP + ADP + AMP at time 0; b: RIP1-def versus WT. (D) WT or RIP1-deficient cells were treated with STS or Etop as described before preparation of whole-cell lysates for SDS-PAGE and Western blot analysis of Panx1, PARP, and actin. Data are representative of three experiments. (E) WT and RIP1-deficient Jurkat cells were treated with STS for 4 hours and then stained with MitoTracker Red and DAPI. Cells were imaged by confocal microscopy at a 60× magnification. Data are representative images of n = 15–19 individual cells. (F) RIP1-deficient cells were treated with STS or Etop for 4 or 12 hours, respectively, and cell suspensions were analyzed for YO-PRO2+ accumulation. Representative phase-contrast and epifluorescence microscopy images from three experiments. (G) WT and RIP1-deficient Jurkat cells were treated with 3 μM STS for 4 hours and then washed and resuspended in BSS supplemented with 1 μM YO-PRO2+; the cell suspensions were incubated for 20 minutes before quantification of YO-PRO fluorescence per well. Data indicate mean ± S.E. from three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +STS versus −STS; b: RIP1-def versus WT. (H) WT and RIP1-deficient Jurkat cells were loaded with 1 μM calcein-AM and then treated with STS for 4 hours in the presence or absence of 100 μM zVAD or 50 μM Trova. Cells were then washed and resuspended in BSS, and calcein4- fluorescence was measured. Data indicate mean ± S.E. from three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +STS versus −STS; b: RIP1-def versus WT; c: +STS with zVAD or Trova versus +STS alone. All panels: ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The FADD-deficient human Jurkat T-cell acute leukemia cell line (I 2.1) and the wild-type (WT) human Jurkat T-cell acute leukemia cells were obtained from the American Type Culture Collection (Manassas VA).

Techniques: Activation Assay, Comparison, Incubation, SDS Page, Western Blot, Staining, Confocal Microscopy, Epifluorescence Microscopy, Fluorescence

Journal: Molecular Pharmacology

Article Title: Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

doi: 10.1124/mol.116.104000

Figure Lengend Snippet: Ecto-AMPase activity, but not CD73 expression, is increased in RIP1-deficient Jurkat cells. (A) WT or RIP1-deficient Jurkat cells were treated without or with STS for 4 hours in the presence or absence of 50 μM APCP. Extracellular media samples were collected and analyzed for total ATP + ADP + AMP. Data indicate mean ± S.E. of six independent experiments with WT cells and five independent experiments with RIP1-def cells. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +STS versus −STS; b: RIP1-def versus WT; c: +STS with APCP versus STS alone. (B) WT or RIP1-deficient cells were suspended at 2 × 106/ml. We added 1 μM exogenous AMP in the presence or absence of 50 μM APCP, and cell-free supernatants were collected after 30 minutes and analyzed for remaining [AMP]. Data indicate mean ±S.E. of four independent experiments with WT cells and three independent experiments with RIP1-def cells. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +APCP versus −APCP; b: RIP1-def versus WT. (C) qRT-PCR analysis of CD73 mRNA expression in WT and RIP1-deficient cells. Signals in RIP1-def were normalized to signals in WT and represent values from three independent experiments; analysis by two-tailed t test. All panels: ns, not statistically significant; ***P < 0.001; ****P < 0.0001.

Article Snippet: The FADD-deficient human Jurkat T-cell acute leukemia cell line (I 2.1) and the wild-type (WT) human Jurkat T-cell acute leukemia cells were obtained from the American Type Culture Collection (Manassas VA).

Techniques: Activity Assay, Expressing, Comparison, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular Pharmacology

Article Title: Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

doi: 10.1124/mol.116.104000

Figure Lengend Snippet: Increased ecto-AMPase activity of RIP-deficient Jurkat cells is not correlated with up-regulation of ecto-AMP deaminase. (A) Schematic of extracellular pathways, ectoenzymes (green), and inhibitors (red) that can mediate the metabolism/clearance of released AMP. (B) We added 100 μM AMP minus (B-i, upper traces) or plus (B-ii, lower traces) 50 μM dipyridamole to WT or RIP1-deficient cells (2 × 106 cells/ml in BSS + 5 mM glucose + 0.1% BSA). Cell-free supernatants were collected after 30 minutes and processed for separation and quantification of AMP, IMP, adenosine, and inosine by reverse phase HPLC and absorbance detection. Arrows indicate elution times of AMP, IMP, adenosine, and inosine standards; ? indicates an unidentified peak. Data are representative of four to six experiments. (C) WT or RIP1-deficient Jurkat cells were treated without or with STS for 4 hours in the presence or absence of 50 μM APCP, 200 μM pentostatin, or combined APCP plus pentostatin. Data indicate mean ±S.E. of three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +STS versus −STS; b:RIP1-def versus WT; c: +STS with pentostatin or pentostatin plus APCP versus STS alone. All panels: ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The FADD-deficient human Jurkat T-cell acute leukemia cell line (I 2.1) and the wild-type (WT) human Jurkat T-cell acute leukemia cells were obtained from the American Type Culture Collection (Manassas VA).

Techniques: Activity Assay, Comparison

Journal: Molecular Pharmacology

Article Title: Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

doi: 10.1124/mol.116.104000

Figure Lengend Snippet: Increased ecto-AMPase activity in RIP1-deficient Jurkat cells correlates with up-regulation of PAP. (A) 10 μM ε-AMP was added to WT or RIP1-deficient cells (2 × 106 cells/ml). Cell-free supernatants were collected after 30 minutes and processed for separation and quantification of ε-ADO and ε-AMP by anion exchange HPLC and fluorescence detection. Data are representative of three experiments. (B) We added 10 μM ε-AMP to WT or RIP1-deficient cells (2 × 106 cells/ml) in the absence or presence of 10 mM l-tartrate. Cell-free supernatants were collected after 30 minutes and processed for separation and quantification of ε-ADO and ε-AMP by anion exchange HPLC and fluorescence detection. Data are representative of four experiments. (C) WT, RIP1-deficient, or FADD-deficient Jurkat cells (2 × 106/ml) were plated in 96-well plates and suspended in BSS that was buffered to pH 7.5 or pH 6.5. The cell suspensions were supplemented with 500 μM p-nitrophenyl phosphate in the absence or presence of 10 mM l-tartrate and incubated for 18 hours at 25°C. Hydrolysis of nitrophenyl phosphate to nitrophenyl was assayed by measuring absorbance at 405 mm. Data indicate mean ±S.E. of three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +tartrate versus −tartrate; b:RIP1-def or FADD-def versus WT. (C, inset) Western blot analysis of PAP expression in whole-cell lysates from WT or RIP1-deficient Jurkat cells that were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes; mobility of molecular mass markers is indicated on the right. (D) WT, FADD-deficient, and RIP1-deficient Jurkat cells were treated with 3 μM STS for 4 hours. Extracellular media samples were collected and assayed for extracellular ATP. Data indicate mean ± S.E. of three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison. All panels: ns, not statistically significant; *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: The FADD-deficient human Jurkat T-cell acute leukemia cell line (I 2.1) and the wild-type (WT) human Jurkat T-cell acute leukemia cells were obtained from the American Type Culture Collection (Manassas VA).

Techniques: Activity Assay, Fluorescence, Incubation, Comparison, Western Blot, Expressing, SDS Page