Nitric Oxide–Induced Apoptosis in Human Leukemic Lines Requires Mitochondrial Lipid Degradation and Cytochrome C Release

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2342-2352 ◽  
Author(s):  
Alexey Ushmorov ◽  
Frank Ratter ◽  
Volker Lehmann ◽  
Wulf Dröge ◽  
Volker Schirrmacher ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cytochrome C ◽  
Mitochondrial Protein ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Cytochrome C Release ◽  
Mitochondrial Functions ◽  
Mitochondrial Lipid ◽  
Induced Apoptosis

Abstract We have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.

Nitric Oxide–Induced Apoptosis in Human Leukemic Lines Requires Mitochondrial Lipid Degradation and Cytochrome C Release

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2342-2352 ◽  
Author(s):  
Alexey Ushmorov ◽  
Frank Ratter ◽  
Volker Lehmann ◽  
Wulf Dröge ◽  
Volker Schirrmacher ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cytochrome C ◽  
Mitochondrial Protein ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Cytochrome C Release ◽  
Mitochondrial Functions ◽  
Mitochondrial Lipid ◽  
Induced Apoptosis

We have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.

Resistance of leukemic cells to 2-chlorodeoxyadenosine is due to a lack of calcium-dependent cytochrome c release

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 655-663 ◽  
Author(s):  
Joya Chandra ◽  
Emma Mansson ◽  
Vladimir Gogvadze ◽  
Scott H. Kaufmann ◽  
Freidoun Albertioni ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Resistant Cell ◽  
Leukemic Cells ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Deoxyguanosine Kinase ◽  
Induced Apoptosis ◽  
Resistant Cells

Abstract The purine nucleoside 2-chlorodeoxyadenosine (CdA) is often used in leukemia therapy. Its efficacy, however, is compromised by the emergence of resistant cells. In the present study, 3 CdA-resistant cell lines were generated and characterized. Their ability to accumulate 2-chloroadenosine triphosphate (CdATP) varied, reflecting differences in activities of deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Nonetheless, the selected lines were uniformly resistant to CdA-induced apoptosis, as assessed by caspase activation and DNA fragmentation. In contrast, cytosols from resistant cells were capable of robust caspase activation when incubated in the presence of cytochrome c and dATP. Moreover, replacement of dATP with CdATP also resulted in caspase activation in the parental and some of the resistant cell lines. Strikingly, CdA-induced decreases in mitochondrial transmembrane potential and release of cytochrome c from mitochondria were observed in the parental cells but not in any resistant lines. The lack of cytochrome c release correlated with an increased ability of mitochondria from resistant cells to sequester free Ca2+. Consistent with this enhanced Ca2+buffering capacity, an early increase in cytosolic Ca2+after CdA treatment of parental cells but not resistant cells was detected. Furthermore, CdA-resistant cells were selectively cross-resistant to thapsigargin but not to staurosporine- or Fas-induced apoptosis. In addition, CdA-induced caspase-3 activation and DNA fragmentation were inhibited by the Ca2+ chelator BAPTA-AM in sensitive cells. Taken together, the data indicate that the mechanism of resistance to CdA may be dictated by changes in Ca2+-sensitive mitochondrial events.

Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhhibitor GX15-070 as a Novel Therapeutic Strategy in AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2584-2584
Author(s):  
Julie C. Watt ◽  
Marina Konopleva ◽  
Rooha Contractor ◽  
Ismael Samudio ◽  
David Harris ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Small Molecule ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Phase I Clinical Trials ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Mitochondrial Inner Membrane ◽  
Induced Apoptosis

Abstract GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in leukemias. In this study, we investigated the activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 cell lines at IC50’s of 0.1, 0.5, 0.5 and 2.5μM, respectively, at 72 hours. Neither overexpression of Bcl-2 or Bcl-XL nor loss of expression of Bax conferred resistance to GX15-070. GX15-070 inhibited Bim/Bcl-2 heterodimerization and induced association of activated Bak with Bax in OCI-AML3 cells, as demonstrated by co-immunoprecipitation studies using CHAPS buffer. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity, caspase activation and a decrease in mitochondrial inner membrane potential. Notably, GX15-070 induced cytochrome c release from isolated mitochondria of leukemic cells. GX15-070 synergized with both AraC and the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells, a notoriously chemoresistant cell line (GX15-070 and ABT-737 average CI value 0.3; GX15-070 and AraC average CI value 0.36). In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. GX15-070 potently inhibited clonogenic ability of AML blasts at sub-micromolar doses (58.5±10.6% CFU-Blast at 0.1μM and 38.1±10.5% at 0.25μM, n=7). In summary, BH3 inhibitor GX15-070 induces apoptosis in AML cells via inhibition of association of pro-survival Bcl-2 family proteins and BH3-only proteins, followed by Bax/Bak activation and initiation of the intrinsic apoptotic pathway. Hence, GX15-070 alone or in combination with chemotherapeutic agents may have utility in AML therapy.

Heat-shock protein 70 attenuates nitric oxide-induced apoptosis in RAW macrophages by preventing cytochrome c release

2002 ◽  
Vol 362 (3) ◽  
pp. 635-641 ◽  
Author(s):  
Sabine D. KLEIN ◽  
Bernhard BRÜNE
Keyword(s):  
Nitric Oxide ◽  
Cell Cycle ◽  
Heat Shock ◽  
Cytochrome C ◽  
Heat Shock Protein ◽  
Cytochrome C Release ◽  
Interacting Protein ◽  
P53 Expression ◽  
Induced Apoptosis ◽  
Hoechst Staining

Heat-shock protein (Hsp) 70 is an inhibitor of apoptosis and has been shown to protect against nitric oxide-mediated toxicity. To gain mechanistic insights into the actions of Hsp70, we stably transfected RAW 264.7 mouse macrophages with the human Hsp70 gene and investigated critical steps in the progression towards cell demise. Incubation of control and Hsp70-transfected macrophages with S-nitrosoglutathione induced accumulation of the tumour suppressor p53, expression of p21WAF1/CIP1 (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1) and G1 cell-cycle arrest. However, cytochrome c translocation to the cytosol and activation of caspase 9 and caspase 3 were markedly reduced in Hsp70-overexpressing cells. In addition, changes in nuclear morphology, as determined by Hoechst staining, and the appearance of cells in the sub-G1 phase were diminished in Hsp70-overexpressing cells compared with controls. We conclude that, in macrophages, Hsp70 interferes with cytochrome c release from mitochondria and, thereby, prevents nitric oxide-induced apoptosis, but leaves p53 accumulation and interference in the cell cycle intact.

Subcellular Distribution and Redistribution of Bcl-2 Family Proteins in Human Leukemia Cells Undergoing Apoptosis

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2353-2359 ◽  
Author(s):  
Li Jia ◽  
Marion G. Macey ◽  
Yuzhi Yin ◽  
Adrian C. Newland ◽  
Stephen M. Kelsey
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Western Blotting ◽  
Leukemic Cells ◽  
Mitochondrial Outer Membrane ◽  
Human Leukemia ◽  
Cell Level ◽  
Cytochrome C Release ◽  
Free System ◽  
Whole Cell

It has been suggested that the ratio of Bcl-2 family proapoptotic proteins to antiapoptotic proteins determines the sensitivity of leukemic cells to apoptosis. However, it is believed that Bcl-2 family proteins exert their function on apoptosis only when they target to the mitochondrial outer membrane. The vinblastine-resistant T-lymphoblastic leukemic cell line CEM/VLB100 has increased sensitivity to tumor necrosis factor- (TNF-)–induced cytochrome crelease, mitochondrial respiratory inhibition, and consequently apoptosis, compared with parental CEM cells. However, there was no difference between the two cell lines in the expression of Bcl-2 family proteins Bcl-2, Bcl-XL, Bcl-XS, Bad, and Bax at the whole cell level, as analyzed by Western blotting. Bcl-2 mainly located to mitochondria and light membrane as a membrane-bound protein, whereas Bcl-XL was located in both mitochondria and cytosol. Similar levels of both Bcl-2 and Bcl-XL were present in the resting mitochondria of the two cell lines. Although the proapoptotic proteins Bcl-XS, Bad, and Bax were mainly located in the cytosol, CEM/VLB100 mitochondria expressed higher levels of these proapoptotic proteins. Subcellular redistribution of the Bcl-2 family proteins was detected in a cell-free system by both Western blotting and flow cytometry after exposure to TNF-. The levels of Bcl-2 family proteins were not altered at the whole cell level by TNF-. However, after exposure to TNF-, Bax, Bad, and Bcl-XS translocated from the cytosol to the mitochondria of both cell lines. An increase in Bcl-2 levels was observed in CEM mitochondria, which showed resistance to TNF-–induced cytochrome c release. By contrast, decreased mitochondrial Bcl-2 was observed in CEM/VLB100 cells, which released cytochrome c from the mitochondria and underwent apoptosis as detected by fluorescence microscopy. We conclude that mitochondrial levels of Bcl-2 family proteins may determine the sensitivity of leukemic cells to apoptosis and that, furthermore, these levels may change rapidly after exposure of cells to toxic stimuli.

scholarly journals Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

2007 ◽  
Vol 405 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Emily E. Franklin ◽  
John D. Robertson
Keyword(s):  
Cytochrome C ◽  
Genotoxic Stress ◽  
Jurkat Cells ◽  
Mitochondrial Outer Membrane ◽  
Cytochrome C Release ◽  
Drug Induced ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Caspase 2 ◽  
Induced Apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.

Apaf-1 protein deficiency confers resistance to cytochromec–dependent apoptosis in human leukemic cells

Blood ◽  
2001 ◽  
Vol 98 (2) ◽  
pp. 414-421 ◽  
Author(s):  
Li Jia ◽  
Srinivasa M. Srinivasula ◽  
Feng-Ting Liu ◽  
Adrian C. Newland ◽  
Teresa Fernandes-Alnemri ◽  
...  
Keyword(s):  
Uv Light ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Leukemia Cell Lines ◽  
Inhibitors Of Apoptosis Proteins ◽  
Human Leukemic Cells ◽  
Induced Apoptosis

The human leukemia cell lines K562, CEM, CEM/VLB100, human leukemic blasts, and the bladder cancer J82 cell line have different sensitivities to UV light–induced apoptosis. It is reported that resistance to UV light–induced apoptosis occurs at a point in the apoptotic pathway upstream of caspase-3 but downstream of mitochondrial cytochrome c release. It is demonstrated that the block is due to deficiency of Apaf-1, a critical member of the apoptosome. Sensitivity to apoptosis was independent of caspase-9b or XIAP (inhibitors of apoptosis proteins) expression or levels of procaspase-9. Transfection of Apaf-1 conferred sensitivity to apoptosis in resistant cells. Apaf-1 deficiency may constitute a significant mode of resistance to apoptosis in human leukemia.

scholarly journals d,l-Methadone causes leukemic cell apoptosis via an OPRM1-triggered increase in IP3R-mediated ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+]i

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
JungKwon Lee ◽  
Jesusa L. Rosales ◽  
Hee-Guk Byun ◽  
Ki-Young Lee
Keyword(s):  
Cytochrome C ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Specific Target ◽  
Induced Apoptosis

AbstractThe search continues for improved therapy for acute lymphoblastic leukemia (aLL), the most common malignancy in children. Recently, d,l-methadone was put forth as sensitizer for aLL chemotherapy. However, the specific target of d,l-methadone in leukemic cells and the mechanism by which it induces leukemic cell apoptosis remain to be defined. Here, we demonstrate that d,l-methadone induces leukemic cell apoptosis through activation of the mu1 subtype of opioid receptors (OPRM1). d,l-Methadone evokes IP3R-mediated ER Ca2+ release that is inhibited by OPRM1 loss. In addition, the rate of Ca2+ extrusion following d,l-methadone treatment is reduced, but is accelerated by loss of OPRM1. These d,l-methadone effects cause a lethal rise in [Ca2+]i that is again inhibited by OPRM1 loss, which then prevents d,l-methadone-induced apoptosis that is associated with activation of calpain-1, truncation of Bid, cytochrome C release, and proteolysis of caspase-3/12. Chelating intracellular Ca2+ with BAPTA-AM reverses d,l-methadone-induced apoptosis, establishing a link between the rise in [Ca2+]i and d,l-methadone-induced apoptosis. Altogether, our findings point to OPRM1 as a specific target of d,l-methadone in leukemic cells, and that OPRM1 activation by d,l-methadone disrupts IP3R-mediated ER Ca2+ release and rate of Ca2+ efflux, causing a rise in [Ca2+]i that upregulates the calpain-1-Bid-cytochrome C-caspase-3/12 apoptotic pathway.

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