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.

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.

scholarly journals Corrigendum to: Cytochrome c release and caspase activation in hydrogen peroxide- and tributyltin-induced apoptosis (FEBS 20394)

FEBS Letters ◽  
1998 ◽  
Vol 437 (1-2) ◽  
pp. 163-163 ◽  
Author(s):  
Hélène Stridh ◽  
Monica Kimland ◽  
Dean P. Jones ◽  
Sten Orrenius ◽  
Mark B. Hampton
Keyword(s):  
Hydrogen Peroxide ◽  
Cytochrome C ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Induced Apoptosis

scholarly journals BCL-2 expression and mitochondrial activity in leukemic cells with different sensitivity to glucocorticoid-induced apoptosis

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1613-1619 ◽  
Author(s):  
LA Smets ◽  
J Van den Berg ◽  
D Acton ◽  
B Top ◽  
H Van Rooij ◽  
...  
Keyword(s):  
Cell Lines ◽  
Glucocorticoid Receptors ◽  
Adenosine Diphosphate ◽  
Resistant Cell ◽  
Leukemic Cells ◽  
Mitochondrial Activity ◽  
Atp Content ◽  
Cellular Targets ◽  
Mitochondrial Inhibitors ◽  
Induced Apoptosis

The present study investigates the relationship between mitochondrial activity and the expression of the BCL-2 gene in a panel of six human and murine leukemia/lymphoma cell lines. The cell lines all contained normal glucocorticoid receptors but differed widely in sensitivity to dexamethasone, ranging from very sensitive S49 lymphoma to completely resistant HL-60 acute leukemia cells. In this panel, 10- to 15-fold differences in basal adenosine triphosphate (ATP) content and adenosine diphosphate (ADP)/ATP ratio were correlated with up to fivefold differences in bcl-2 protein (in human cells) and approximately 25-fold difference in bcl-2 mRNA content (all cell lines). Moreover, ATP content and BCL-2 gene expression were inversely correlated with glucocorticoid sensitivity and cell cycle length. In resistant cell lines, sensitivity to dexamethasone was restored by the mitochondrial inhibitors rotenone and meta-iodobenzylguanidine. This sensitization was not accompanied by detectable reductions in bcl-2 mRNA or protein content, suggesting that the inhibitors were capable of overriding BCL- 2-mediated inhibition of apoptosis. Increased mitochondrial activity and (overexpressed) BCL-2 appeared closely related properties of glucocorticoid-resistant cells, sharing common cellular targets in hormone-induced apoptosis.

Cytochrome c release into cytosol with subsequent caspase activation during warm ischemia in rat liver

2001 ◽  
Vol 281 (4) ◽  
pp. G1115-G1123 ◽  
Author(s):  
Junpei Soeda ◽  
Shinichi Miyagawa ◽  
Kenji Sano ◽  
Junya Masumoto ◽  
Shun'Ichiro Taniguchi ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Dna Fragmentation ◽  
Warm Ischemia ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dependent Manner ◽  
Mitochondrial Cytochrome ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Mitochondrial Cytochrome C

Apoptosis plays an important role in liver ischemia and reperfusion (I/R) injury. However, the molecular basis of apoptosis in I/R injury is poorly understood. The aims of this study were to ascertain when and how apoptotic signal transduction occurs in I/R injury. The apoptotic pathway in rats undergoing 90 min of warm ischemia with reperfusion was compared with that of rats undergoing prolonged ischemia alone. During ischemia, mitochondrial cytochrome c was released into the cytosol in a time-dependent manner in hepatocytes and sinusoidal endothelial cells, and caspase-3 and an inhibitor of caspase-activated DNase were cleaved. However, apoptotic manifestation and DNA fragmentation were not observed. After reperfusion, nuclear condensation, cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, and DNA fragmentation were observed and caspase-8 and Bid cleavage occurred. In contrast, prolonged ischemia alone induced necrosis rather than apoptosis. In summary, our results show that release of mitochondrial cytochrome c and caspase activation proceed during ischemia, although apoptosis is manifested after reperfusion.

Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-α in IEC-6 cells

2003 ◽  
Vol 285 (5) ◽  
pp. G980-G991 ◽  
Author(s):  
Sujoy Bhattacharya ◽  
Ramesh M. Ray ◽  
Mary Jane Viar ◽  
Leonard R. Johnson
Keyword(s):  
Cytochrome C ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Tnf Α ◽  
Induced Apoptosis ◽  
Jnk Activation

Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-α (TNF-α). TNF-α or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-α, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with α-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-α/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-α-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.

Cytochrome c release and caspase activation during menadione-induced apoptosis in plants

FEBS Letters ◽  
1999 ◽  
Vol 462 (3) ◽  
pp. 317-321 ◽  
Author(s):  
Ying-Li Sun ◽  
Yun Zhao ◽  
Xia Hong ◽  
Zhong-He Zhai
Keyword(s):  
Cytochrome C ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Induced Apoptosis

Morphine mimics the antiapoptotic effect of preconditioning via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes

2005 ◽  
Vol 288 (1) ◽  
pp. H83-H88 ◽  
Author(s):  
Stéphanie Barrère-Lemaire ◽  
Nicolas Combes ◽  
Catherine Sportouch-Dukhan ◽  
Sylvain Richard ◽  
Joël Nargeot ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Signaling Pathway ◽  
Dna Fragmentation ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Cytochrome C Release ◽  
Rat Cardiomyocytes ◽  
Rat Ventricular Myocytes ◽  
Antiapoptotic Effect ◽  
Induced Apoptosis

Morphine has cardioprotective effects against ischemic-reperfusion injuries. This study investigates whether morphine could mimic the antiapoptotic effect of preconditioning using a model of cultured neonatal rat cardiomyocytes subjected to metabolic inhibition (MI). To quantify MI-induced apoptosis, DNA fragmentation and mitochondrial cytochrome c release levels were measured by ELISA. MI-dependent DNA fragmentation was prevented by both Z-VAD-fmk (20 μM), a pan-caspase inhibitor, and cyclosporine A (CsA; 5 μM), a mitochondrial pore transition blocker, added during MI (36% and 54% decrease, respectively). MI-dependent cytochrome c release was not blocked by Z-VAD-fmk but was decreased (38%) by CsA during MI. Metabolic preconditioning (MIP) and preconditioning with morphine (1 μM) were also assessed. MI-dependent DNA fragmentation and cytochrome c release were prevented by MIP (40% and 45% decrease, respectively) and morphine (34% and 45%, respectively). The antiapoptotic effect of morphine was abolished by naloxone (10 nM), a nonselective opioid receptor antagonist, or xestospongin C (XeC, 400 nM), an inhibitor of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ release. Ca2+ preconditioning, induced by increasing extracellular Ca2+ from 1.8 to 3.3 mM, mimicked the antiapoptotic effect of morphine on DNA fragmentation (24% decrease) and cytochrome c release (57% decrease). This effect mediated by extracellular Ca2+ was also abolished by XeC. Measurements of intracellular Ca2+ concentration using fura-2 microspectrofluorimetry showed that morphine induces Ins(1,4,5)P3-dependent Ca2+ transients abolished by 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable Ins(1,4,5)P3 antagonist. These results suggest that morphine preconditioning prevents simulated ischemia-reperfusion-induced apoptosis via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes.

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