Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis

2001 ◽  
Vol 114 (23) ◽  
pp. 4161-4172
Author(s):  
Justine Rudner ◽  
Albrecht Lepple-Wienhues ◽  
Wilfried Budach ◽  
Johannes Berschauer ◽  
Björn Friedrich ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Transmembrane Domain ◽  
Cytochrome B5 ◽  
Death Receptor ◽  
Jurkat Cells ◽  
Wild Type ◽  
Specific Sequence ◽  
Apoptotic Effect ◽  
Radiation Induced ◽  
Induced Apoptosis

The proto-oncogene Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different cellular organelles remains elusive. Here, we analyzed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/ΔTM) were employed. Apoptosis induced by ionizing radiation or by the death receptors for CD95L or TRAIL was analyzed by determination of the mitochondrial membrane potential (ΔΨm) and activation of different caspases. Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/ΔTM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial ΔΨm breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.

N-Acetylphytosphingosine Enhances the Radiosensitivity of Tumor Cells by Increasing Apoptosis

2005 ◽  
Vol 277-279 ◽  
pp. 536-541
Author(s):  
Young Soo Han ◽  
Yun Hwa Kim ◽  
Yeon Sook Yun ◽  
Soo Jin Jeon ◽  
Ki Sung Kim ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Mammalian Cells ◽  
Second Messengers ◽  
Clonogenic Survival ◽  
Jurkat Cells ◽  
Human Leukemia ◽  
Apoptotic Effect ◽  
Radiation Induced ◽  
Induced Apoptosis ◽  
Exogenous Treatment

Ceramides are well-known second messengers which mediate apoptosis, proliferation, differentiation in mammalian cells, but the physiological roles of phytosphingosines are poorly understood. We hypothesized that one of the phytosphingosine derivatives, N-acetylphytosphingosine (NAPS) can induce apoptosis in human leukemia Jurkat cell line and increase apoptosis in irradiated MDA-MB-231 cells. We first examined the effect of NAPS on apoptosis of Jurkat cells. NAPS had a more rapid and stronger apoptotic effect than C2-ceramide in Jurkat cells and significant increase of apoptosis was observed at 3 h after treatment. In contrast, the apoptosis induced by C2-ceramide was observed only after 16 h of treatment. NAPS induced apoptosis was mediated by caspase 3 and 8 activation and inhibited by z-VAD-fmk. Ceramide plays a pivotal role in radiation induced apoptosis. We postulated that exogenous treatment of NAPS sensitizes tumor cells to ionizing radiation, since NAPS might be used as a more effective alternative to C2-ceramide. As expected, NAPS decreased clonogenic survival of irradiated MDA-MB-231 cells dose dependently, and apoptosis of irradiated cells in the presence of NAPS was increased through the caspase activation. Taken together, NAPS is an effective apoptosis-inducing agent, which can be readily synthesized from yeast sources, and is a potent alternative to ceramide for the further study of ceramide associated signaling and the development of radiosensitizing agent.

Acquired Resistance to Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand (TRAIL): Histone Deacetylase Inhibitors Resensitize TRAIL-Resistant Jurkat Acute Lymphocytic Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5032-5032
Author(s):  
Pavel Klener ◽  
Jan Molinsky ◽  
Tereza Simonova ◽  
Emanuel Necas ◽  
Ladislav Andera ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Fas Ligand ◽  
Death Receptor ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Tnf Alpha ◽  
Apoptotic Pathway ◽  
Induced Apoptosis ◽  
Disc Formation

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a death-ligand from the TNF family. TRAIL induces programmed cell death by the cell-extrinsic p53-independent apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed and is preclinically and clinically tested. Development of TRAIL-resistant clones in the TRAIL-sensitive tumor cells may be a serious complication of TRAIL based cancer therapy. Jurkat acute lymphocytic leukemia cells are sensitive to TRAIL-induced apoptosis, as well as other apoptosis inducing ligands from TNF family, Fas and TNF-alpha. Jurkat cells express only one of the four receptors for TRAIL, death receptor 5 (DR5). Prolonged exposure of TRAIL-sensitive Jurkat cells to recombinant soluble TRAIL (1000 ng/mL) resulted in the establishment of three TRAIL-resistant (TR) Jurkat cell subclones, Jurkat TR1, TR2, and TR3. The Jurkat TR subclones were also resistant to TNF-alpha and Fas ligand, suggesting disruption of the extrinsic apoptotic pathway. TRAIL-resistant subclone TR1, but not TR2 and TR3, demonstrated decreased susceptibility to undergo apoptosis in response to histone-deacetylase inhibitors, valproic acid (VA), sodium butyrate (SB) and suberoylanilide hydroxamic acid (SAHA) and was resistant to fludarabine. Flow cytometry analysis showed Jurkat TR subclones had unchanged expression of cell surface death receptor DR5, Fas, and receptors for TNF-alpha, TNF-R1 and TNF-R2, compared to TRAIL-sensitive Jurkat cells. Analysis of death-inducing signaling complex (DISC) formation by immunoprecipitation (anti-TRAIL, anti-DR5) and subsequent western blotting (anti-caspase 8, anti-FADD) clearly demonstrated that the DISC formation in response to TRAIL binding to DR5 was significantly decreased in subclones TR2 and TR3, but remained unchanged in subclone TR1 compared to TRAIL-sensitive Jurkat cells. To gain further insight into potential molecular aletarations associated with acquired TRAIL resistance of Jurkat subclones, we measured gene expression of several key apoptotic regulators, including receptors for TRAIL, cFLIP, BCL2 family, IAP family, HSP family members in TRAIL-resistant and TRAIL-sensitive Jurkat cells and did not detect any significant (>2-fold) change. These results suggest acquired TRAIL resistance of Jurkat cells might be mediated by changes on the protein rather than mRNA level. We analyzed whether the TRAIL-resistant Jurkat cells could be resensitized to TRAIL-induced apoptosis by pretreatment with diverse inhibitors of important prosurvival pathways, including inhibitors of proteosynthesis (cycloheximid), inhibitors of transcription (actinomycin D), NFkB inhibitors (bortezomib, SN-50), PI3K-Akt-mTOR inhibitors (rapamycine, LY294002, Hsp90 inhibitor (17-AAG), cyclin-dependent kinase inhibitors (roscovitine), casein kinase II inhibitors (DRB), or histone deacetylase inhibitors (HDACi: SAHA, VA, SB). Pretreatment with HDAC inhibitors for 12 hour was able to resensitize all three TRAIL-resistant Jurkat subclones to TRAIL-induced apoptosis. The percentage of apoptotic cells of HDACi-pretreated subclones was 70–95% 24 h after the exposure to TRAIL compared to 5–15% apoptosis for HDACi-untreated TRAIL-exposed controls, and to 10–15% apoptosis for HDACi-treated TRAIL unexposed controls. We established TRAIL-resistant subclones from the original TRAIL-sensitive Jurkat cells. Acquired resistance to TRAIL was not mediated by downregulation of TRAIL death receptor DR5 and was associated with (cross)resistance to TNFa and Fas ligand, suggesting disruption of cell-extrinsic apoptotic pathway. We assume diverse molecular mechanisms were involved in the development of TRAIL-resistant subclones upon exposure to TRAIL, as exemplified by disrupted formation of DISC in case of subclones TR2 and TR3 and normal DISC formation and fludarabine resistance in subclone TR1, suggesting deregulated apoptotic pathway downstream of DISC. Finally, we observed that HDACi resensitized the TRAIL-resistant subclones to TRAIL. The results provide substantiation for combinatorial approaches in the potential TRAIL-based therapies of hematological malignancies.

scholarly journals 5-Aminoimidazole-4-carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved

2003 ◽  
Vol 370 (3) ◽  
pp. 1027-1032 ◽  
Author(s):  
José M. LÓPEZ ◽  
Antonio F. SANTIDRIÁN ◽  
Clara CAMPÀS ◽  
Joan GIL
Keyword(s):  
Protein Kinase ◽  
Cell Types ◽  
Jurkat Cells ◽  
Nucleoside Transport ◽  
Cytochrome C Release ◽  
Nucleotide Biosynthesis ◽  
Apoptotic Effect ◽  
Nucleoside Transport Inhibitor ◽  
Induced Apoptosis ◽  
Amp Activated Protein Kinase

5-Aminoimidazole-4-carboxamide (AICA) riboside, a precursor of purine nucleotide biosynthesis, induces apoptosis in Jurkat cells. Incorporation of AICAriboside into the cells is necessary for this effect since addition of nitrobenzylthioinosine, a nucleoside-transport inhibitor, completely protects Jurkat cells from apoptosis. Adenosine, but not other nucleosides, also protects Jurkat cells from AICAriboside-induced apoptosis. The apoptotic effect is caspase-dependent since caspases 9 and 3 are activated and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) blocks apoptosis. Furthermore, AICAriboside induces mitochondrial cytochrome c release. AICAriboside, when phosphorylated to AICAribotide (ZMP), is a specific activator of the AMP-activated protein kinase (AMPK) in certain cell types. However, AICAriboside does not activate AMPK in Jurkat cells. Moreover, 5-iodotubercidin, an inhibitor of AICAriboside phosphorylation, does not inhibit apoptosis in Jurkat cells. These results indicate that AICAriboside induces apoptosis independently of ZMP synthesis and AMPK activation in Jurkat cells.

Daxx overexpression in T-lymphoblastic Jurkat cells enhances caspase-dependent death receptor- and drug-induced apoptosis in distinct ways

2005 ◽  
Vol 17 (5) ◽  
pp. 581-595 ◽  
Author(s):  
Simone Boehrer ◽  
Daniel Nowak ◽  
Simone Hochmuth ◽  
Soo-Zin Kim ◽  
Bettina Trepohl ◽  
...  
Keyword(s):  
Death Receptor ◽  
Jurkat Cells ◽  
Drug Induced ◽  
Induced Apoptosis

scholarly journals NF-κB Inducers Upregulate cFLIP, a Cycloheximide-Sensitive Inhibitor of Death Receptor Signaling

2001 ◽  
Vol 21 (12) ◽  
pp. 3964-3973 ◽  
Author(s):  
Sebastian Kreuz ◽  
Daniela Siegmund ◽  
Peter Scheurich ◽  
Harald Wajant
Keyword(s):  
Cell Lines ◽  
Death Receptor ◽  
Jurkat Cells ◽  
Negative Regulator ◽  
Inhibitory Protein ◽  
Iκb Kinase ◽  
Low Concentrations ◽  
Death Receptor Signaling ◽  
Induced Apoptosis ◽  
Necrosis Factor

ABSTRACT The caspase 8 homologue FLICE-inhibitory protein (cFLIP) is a potent negative regulator of death receptor-induced apoptosis. We found that cFLIP can be upregulated in some cell lines under critical involvement of the NF-κB pathway, but NF-κB activation was clearly not sufficient for cFLIP induction in all cell lines. Treatment of SV80 cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) or geldanamycin, a drug interfering with tumor necrosis factor (TNF)-induced NF-κB activation, inhibited TNF-induced upregulation of cFLIP. Overexpression of a nondegradable IκBα mutant (IκBα-SR) or lack of IκB kinase γ expression completely prevented phorbol myristate acetate-induced upregulation of cFLIP mRNA in Jurkat cells. These data point to an important role for NF-κB in the regulation of the cFLIP gene. SV80 cells normally show resistance to TNF-related apoptosis-inducing ligand (TRAIL) and TNF, as apoptosis can be induced only in the presence of low concentrations of cycloheximide (CHX). However, overexpression of IκBα-SR rendered SV80 cells sensitive to TRAIL-induced apoptosis in the absence of CHX, and cFLIP expression was able to reverse the proapoptotic effect of NF-κB inhibition. Western blot analysis further revealed that cFLIP, but not TRAF1, A20, and cIAP2, expression levels rapidly decrease upon CHX treatment. In conclusion, these data suggest a key role for cFLIP in the antiapoptotic response of NF-κB activation.

scholarly journals Identification of Proteins that Regulate Radiation-induced Apoptosis in Murine Tumors with Wild Type p53

2007 ◽  
Vol 48 (5) ◽  
pp. 435-441 ◽  
Author(s):  
Jinsil SEONG ◽  
Hae Jin OH ◽  
Jiyoung KIM ◽  
Jeung Hee AN ◽  
Wonwoo KIM
Keyword(s):  
Wild Type ◽  
Murine Tumors ◽  
Radiation Induced ◽  
Wild Type P53 ◽  
Induced Apoptosis

scholarly journals Leukemic HRX Fusion Proteins Inhibit GADD34-Induced Apoptosis and Associate with the GADD34 and hSNF5/INI1 Proteins

1999 ◽  
Vol 19 (10) ◽  
pp. 7050-7060 ◽  
Author(s):  
Haskell T. Adler ◽  
Rebecca Chinery ◽  
Daniel Y. Wu ◽  
Steven J. Kussick ◽  
John M. Payne ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Chromosomal Abnormalities ◽  
Protein A ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Amino Terminal ◽  
Apoptotic Effect ◽  
The Difference ◽  
Induced Apoptosis ◽  
First Time

ABSTRACT One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.

scholarly journals Apparently Normal Tumor Necrosis Factor Receptor 1 Signaling in the Absence of the Silencer of Death Domains

2003 ◽  
Vol 23 (18) ◽  
pp. 6609-6617 ◽  
Author(s):  
Robert Endres ◽  
Georg Häcker ◽  
Inge Brosch ◽  
Klaus Pfeffer
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Receptor ◽  
Death Receptor ◽  
High Dose ◽  
Wild Type ◽  
Induced Apoptosis ◽  
Necrosis Factor

ABSTRACT The silencer of death domains (SODD) has been proposed to prevent constitutive signaling of tumor necrosis factor receptor 1 (TNFR1) in the absence of ligand. Besides TNFR1, death receptor 3 (DR3), Hsp70/Hsc70, and Bcl-2 have been characterized as binding partners of SODD. In order to investigate the in vivo role of SODD, we generated mice congenitally deficient in expression of the sodd gene. No spontaneous inflammatory infiltrations were observed in any organ of these mice. Consistent with this finding, in the absence of SODD no alteration in the activation patterns of nuclear factor κB (NF-κB), stress kinases, or ERK1 or -2 was observed after stimulation with tumor necrosis factor (TNF). Activation of NF-κB by DR3 was also unchanged. The extents of DR3- and TNF-induced apoptosis were comparable in gene-deficient and wild-type cells. Protection of cells against heat shock as mediated by the Hsp70 system and against staurosporine-induced apoptosis was independent of SODD. Furthermore, resistance to high-dose lipopolysaccharide (LPS) injections, LPS-d-GalN injections, and infection with listeriae was similar in wild-type and gene-deficient mice. In conclusion, our data do not support the concept of a unique, nonredundant role of SODD for the functions of TNFR1, Hsp70, and DR3.

scholarly journals Comparison of Apoptosis in Wild-Type and Fas-Resistant Cells: Chemotherapy-Induced Apoptosis Is Not Dependent on Fas/Fas Ligand Interactions

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 935-943 ◽  
Author(s):  
Christine M. Eischen ◽  
Timothy J. Kottke ◽  
Luis M. Martins ◽  
Guriqbal S. Basi ◽  
Jay S. Tung ◽  
...  
Keyword(s):  
Cell Death ◽  
Fas Ligand ◽  
Apoptotic Cell ◽  
Chemotherapeutic Agents ◽  
Jurkat Cells ◽  
Wild Type ◽  
Γ Irradiation ◽  
Human T Cell ◽  
Apoptotic Pathways ◽  
Induced Apoptosis

Abstract The Fas/Fas ligand (FasL) pathway is widely involved in apoptotic cell death in lymphoid and nonlymphoid cells. It has recently been postulated that many chemotherapeutic agents also induce cell death by activating the Fas/FasL pathway. In the present study we compared apoptotic pathways induced by anti-Fas or chemotherapeutic agents in the Jurkat human T-cell leukemia line. Immunoblotting showed that treatment of wild-type Jurkat cells with anti-Fas or the topoisomerase II-directed agent etoposide resulted in proteolytic cleavage of precursors for the cysteine-dependent aspartate-directed proteases caspase-3 and caspase-7 and degradation of the caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B1 . Likewise, affinity labeling with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl)aspartic acid [(2,6-dimethyl-benzoyl)oxy]methyl ketone [Z-EK (bio)D-amok] labeled the same five active caspase species after each treatment, suggesting that the same downstream apoptotic pathways have been activated by anti-Fas and etoposide. Treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to block etoposide-induced apoptosis, raising the possibility that etoposide does not initiate apoptosis through Fas/FasL interactions. To further explore the relationship between Fas- and chemotherapy-induced apoptosis, Fas-resistant Jurkat cells were treated with various chemotherapeutic agents. Multiple independently derived Fas-resistant Jurkat lines underwent apoptosis that was indistinguishable from that of the Fas-sensitive parental cells after treatment with etoposide, doxorubicin, topotecan, cisplatin, methotrexate, staurosporine, or γ-irradiation. These results indicate that antineoplastic treatments induce apoptosis through a Fas-independent pathway even though Fas- and chemotherapy-induced pathways converge on common downstream apoptotic effector molecules.

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