N(alpha)-tosyl-L-phenylalanine chloromethyl ketone induces caspase-dependent apoptosis in transformed human B cell lines with transcriptional down-regulation of anti-apoptotic HS1-associated protein X-1

Abstract

N(alpha)-tosyl-L-phenylalanine chloromethylketone (TPCK) has been widely used to investigate signal transduction pathways that are involved in gene expression and cell survival/cell death. However, contradictory effects of TPCK on apoptosis have been reported, and the underlying signaling events leading to TPCK-induced promotion or prevention of apoptosis are not fully understood. Here, we show that TPCK induces caspase-dependent apoptosis in Epstein-Barr virus (EBV)-transformed human B cell lines with release of pro-apoptotic proteins from mitochondria. TPCK treatment also results in down-regulation of the anti-apoptotic proteins, cIAP1, cIAP2, and HAX-1, and caspase-dependent cleavage of the anti-apoptotic proteins, Bcl-2 and XIAP. Quantitative PCR analysis confirmed that the TPCK-induced down-regulation of HAX-1 occurred at the transcriptional level, and experiments using the specific pharmacological inhibitor, Bay 11-7082, suggested that HAX-1 expression is subject to regulation by the transcription factor, NF-kappaB. B cell lines derived from patients with homozygous HAX1 mutations were more sensitive to TPCK-induced apoptosis when compared with normal donor cell lines. Furthermore, N-acetylcysteine effectively blocked TPCK-induced apoptosis in EBV-transformed B cell lines and prevented the down-regulation or cleavage of anti-apoptotic proteins. Taken together, our studies demonstrate that TPCK induces apoptosis in human B cell lines and exerts multiple effects on pro- and anti-apoptotic factors.

FIGURE 1.

TPCK induces time- and dose-dependent apoptosis in EBV-transformed human B cells. B cells were incubated with different concentrations of TPCK for 6 h. Apoptosis was determined by measurement of PS exposure (A) as well as caspase-3 activation (B) using the annexin V-FITC/PI staining assay and DEVD-AMC cleavage assay, respectively. Data shown are mean values ± S.D. of at least three independent experiments. C, cleavage of PARP was monitored using specific anti-PARP antibodies. Membranes were stripped and reprobed with antibodies to β-actin to control for equal loading of protein. D, B cells were incubated with TPCK (50 μm) for the indicated time points, and apoptosis was monitored by flow cytometry using the annexin V-FITC/PI staining assay. Data are reported as mean values ± S.D. of at least three independent experiments. E, B cells were incubated with TPCK (50 μm) for the indicated time points and then harvested, permeabilized, and stained with PI. DNA content was determined using flow cytometry. Data are reported as mean values ± S.D. of at least three independent experiments.

FIGURE 2.

TPCK triggers caspase-dependent apoptosis in EBV-transformed B cells. A, B cells were incubated with TPCK (50 μm) for the indicated time points, and caspase activation was determined using the flurorogenic caspase substrate, DEVD-AMC. Data are shown as mean values ± S.D. of at least three independent experiments. B, Western blotting of B cells treated with TPCK. Total cell lysates were probed with antibodies to pro-caspase-3 (32 kDa) and its active subunit (17 kDa), and membranes were then stripped and reprobed with specific antibodies to PARP, a well known nuclear caspase substrate and to β-actin to control for equal loading of protein. C–E, B cells were pre-treated with the pan-caspase inhibitor z-VAD-fmk (25 μm) for 30 min and then subjected to treatment with TPCK (50 μm) for 6 or 12 h. Apoptosis was determined by measurement of caspase activation (C), hypodiploid DNA content (D), and PS exposure (E). Data are shown as mean values ± S.D. of at least three independent experiments. *, p < 0.001 TPCK versus TPCK plus z-VAD-fmk (C and E); #, p < 0.01 TPCK versus TPCK plus z-VAD-fmk (D).

FIGURE 3.

Loss of mitochondrial membrane potential (Δψ_m) and release of mitochondrial proteins in TPCK-treated human B cells. EBV-transformed B cells were incubated with 50 μm TPCK for 1, 2, 4, 6, 8, and 12 h (A) or with 10, 25, 50, and 75 μm TPCK for 6 h (B). Cells were then harvested for flow cytometric detection of Δψ_m using the fluorescent cationic probe, TMRE. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 100 μm) was included as a positive control. Data shown are mean values ± S.D. of at least three independent experiments. C, EBV-transformed B cells were incubated with TPCK (50 μm) for the indicated time points. Cytosolic (supernatant) and mitochondrial (pellet) fractions were prepared and subjected to Western blot analysis using specific antibodies against cytochrome c and AIF. Expression of β-actin and COXIV were also monitored to determine the purity of cytosolic and mitochondrial fractions, respectively. D, total cell lysates were probed with antibodies directed toward pro-caspase-9 and its cleavage products (active caspase-9). The membrane was stripped and reprobed with anti-β-actin antibodies to control for equal loading of protein.

FIGURE 4.

TPCK induces the production of ROS in EBV-transformed B cells. B cells were incubated with TPCK (50 μm) for the indicated time points and the oxidation of DHE to ethidium was then monitored by flow cytometry (A). B cells were treated with TPCK (50 μm) or anti-Fas antibody (250 ng/ml) for 6 h in the presence or absence of the pan-caspase inhibitor, z-VAD-fmk (25 μm). Loss in mitochondrial membrane potential (B) and superoxide production (C) was then assessed by flow cytometry. Data are shown as mean values ± S.D. of at least three independent experiments. *, p < 0.03 TPCK versus TPCK plus z-VAD-fmk (B); #, p < 0.01 TPCK versus TPCK plus z-VAD-fmk (b); §, p < 0.02 anti-Fas versus anti-Fas plus z-VAD-fmk (B); *, p < 0.03 TPCK versus TPCK plus z-VAD-fmk (C); and #, p < 0.04 TPCK versus TPCK plus z-VAD-fmk (C).

FIGURE 5.

Effects of TPCK on anti-apoptotic proteins Bcl-2, HAX-1, and IAPs in EBV-transformed B cells. B cells were treated with the indicated concentration of TPCK for 6 h (A) or treated with 50 μm TPCK for 1, 2, 4, 6, 8, and 12 h (B) in the presence or absence of 25 μm z-VAD-fmk. Total cell extracts were prepared and subjected to Western blot analysis using antibodies against Bcl-2, XIAP, cIAP1, cIAP2, and HAX-1. Membranes were subsequently reprobed with antibodies against β-actin to control for equivalent protein loading. C and D, HAX-1-deficient cells show increased susceptibility to TPCK-induced apoptosis. C, HAX-1 protein expression was monitored in total cell extracts of EBV-transformed B cell lines derived from two SCN patients and from normal adult blood donors using specific anti-HAX-1 antibodies. The membrane was stripped and reprobed with anti-β-actin antibodies to control for equal loading of protein. D, B cell lines derived from SCN patients 1 and 2 and one healthy control were incubated with 50 μm TPCK for the indicated time points, and caspase activity was monitored using the DEVD-AMC assay. Data shown are mean values of triplicate determinations ± S.D.

FIGURE 6.

TPCK induces prominent transcriptional down-regulation of HAX-1. A, EBV-transformed B cells were treated with TPCK (50 μm) for the indicated time points. Total RNA was then extracted, and expression of HAX-1 mRNA was assessed. The relative HAX-1 mRNA level was calculated using the comparative cycle threshold method and normalized against β-actin. Data shown are mean ± S.D. from three independent measurements and with triplicate samples for each condition. B, B cells were incubated with the NF-κB inhibitor, Bay 11-7082, or TPCK at the indicated concentrations for 6 h. HAX-1 mRNA expression was then determined as above. C, cells treated with Bay 11-7082 or TPCK as indicated above were harvested, and total cell extracts were subjected to Western blot using specific antibodies against HAX-1, XIAP, cIAP1, and cIAP2. Membranes were stripped and reprobed with antibodies to β-actin as a loading control. D and E, TPCK induces down-regulation of HAX-1 in Jurkat cells. D, Jurkat leukemic T cells were pretreated or not with z-VAD-fmk (25 μm) for 30 min followed by incubation with different concentrations of TPCK for 6 h. Western blotting was then performed on total cell lysates to monitor the expression of HAX-1. The membrane was stripped and reprobed with antibodies to β-actin to control for equal loading of protein. E, parallel samples of TPCK-treated cells were harvested for apoptosis detection using the annexin V/PI assay, and the percentages of apoptotic cells for each sample are shown. Data are shown as mean values ± S.D. of at least three independent experiments.

FIGURE 7.

NAC blocks TPCK-induced apoptosis in EBV-transformed B cells. B cells were pretreated with NAC (5 mm) for 30 min followed by incubation with TPCK (50 μm) for 6 h. PS exposure (A), hypodiploid DNA content (B), caspase activation (C), and PARP cleavage (D) were then determined using flow cytometry and Western blotting, respectively. Data reported in A–C are mean values ± S.D. of at least three independent experiments. *, p < 0.001 TPCK versus TPCK plus NAC (A and B); #, p < 0.05 TPCK versus TPCK plus NAC (C).

FIGURE 8.

Effect of NAC on TPCK-induced mitochondrial events in EBV-transformed B cells. A, B cells were pretreated or not with NAC (5 mm) for 30 min followed by TPCK (50 μm) for 12 h, and ROS production and drop of Δψ_m were determined using the DHE assay and TMRE staining assay, respectively. Data shown are mean values ± S.D. of at least three independent experiments. *, p < 0.001 TPCK versus TPCK plus NAC (DHE assay); #, p < 0.01 TPCK versus TPCK plus NAC (TMRE assay). B, cytosolic (supernatant) and mitochondrial (pellet) fractions were prepared from B cells treated with TPCK (50 μm) for 6 h in the presence or absence of z-VAD-fmk (25 μm) or NAC (5 mm) and subjected to Western blotting using antibodies to cytochrome c and AIF. Membranes were reprobed with antibodies to β-actin and COXIV to determine the purity of cytosolic and mitochondrial fractions, respectively. C, B cells were pretreated or not with NAC (5 mm) for 30 min followed by TPCK (50 μm) for 6 h, and total cell extracts were subjected to Western blot analysis using antibodies against Bcl-2, XIAP, cIAP1, cIAP2, and HAX-1. Antibodies directed to β-actin were used to control for equal loading of protein.

FIGURE 9.

l-Cysteine and d-cysteine block TPCK-induced apoptosis in EBV-transformed B cells. A, B cells were pre-treated with l-cysteine (3 mm) or d-cysteine (3 mm) for 30 min followed by incubation with TPCK (50 μm) for 6 h. Apoptosis was determined based on flow cytometric detection of annexin V-FITC labeling. Representative histograms are shown. B, TPCK blocks the formation of fluorescent NAC-ThioGlo^TM3 adducts. TPCK at concentrations 0, 10, 25, 50, 100, and 200 μm was incubated with 10 μm NAC at 37 °C for 1, 3 or 6 h in PBS; pH 7.2. ThioGlo^TM3 at a final concentration of 1 μm was added to each reaction mixture. Samples were incubated at 25 °C for 30 min and fluorescence of ThioGlo^TM3-NAC adducts was measured at 465 nm. TPCK alone at the indicated concentrations was used as a negative control. Results are expressed as mean ± S.D. of three independent measurements.

FIGURE 10.

TPCK-induced apoptosis in transformed human B cells. A schematic representation of the experimental findings in the current study. Mitochondrial generation of ROS, and the dissipation of mitochondrial transmembrane potential is partially caspase-dependent in this model, suggesting that effector caspases that are activated downstream of mitochondria may act on these organelles in a feedback loop to potentiate the initial apoptotic insult. In addition, the down-regulation and/or inactivation of endogenous caspase inhibitors may unleash further caspase activation in the cell. However, our experimental data suggest that the protective effect of NAC in this model is likely due to a direct interaction between NAC and TPCK. Consult text for abbreviations and a more detailed discussion.