scholarly journals Bcr-Abl Exerts Its Antiapoptotic Effect Against Diverse Apoptotic Stimuli Through Blockage of Mitochondrial Release of Cytochrome C and Activation of Caspase-3

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1700-1705 ◽  
Author(s):  
Gustavo P. Amarante-Mendes ◽  
Caryn Naekyung Kim ◽  
Linda Liu ◽  
Yue Huang ◽  
Charles L. Perkins ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
High Dose ◽  
Antiapoptotic Effect ◽  
Endogenous Expression ◽  
Cyt C

Bcr-Abl expression in leukemic cells is known to exert a potent effect against apoptosis due to antileukemic drugs, but its mechanism has not been elucidated. Recent reports have indicated that a variety of apoptotic stimuli cause the preapoptotic mitochondrial release of cytochrome c (cyt c) into cytosol, which mediates the cleavage and activity of caspase-3 involved in the execution of apoptosis. Whether Bcr-Abl exerts its antiapoptotic effect upstream to the cleavage and activation of caspase-3 or acts downstream by blocking the ensuing degradation of substrates resulting in apoptosis, has been the focus of the present studies. In these, we used (1) the human acute myelogenous leukemia (AML) HL-60 cells that are stably transfected with thebcr-abl gene (HL-60/Bcr-Abl) and express p185 Bcr-Abl; and (2) the chronic myelogenous leukemia (CML)-blast crisis K562 cells, which have endogenous expression of p210 Bcr-Abl. Exposure of the control AML HL-60 cells to high-dose Ara-C (HIDAC), etoposide, or sphingoid bases (including C2 ceramide, sphingosine, or sphinganine) caused the accumulation of cyt c in the cytosol, loss of mitochondrial membrane potential (MMP), and increase in the reactive oxygen species (ROS). These preapoptotic events were associated with the cleavage and activity of caspase-3, resulting in the degradation of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) and DNA fragmentation factor (DFF), internucleosomal DNA fragmentation, and morphologic features of apoptosis. In contrast, in HL-60/Bcr-Abl and K562 cells, these apoptotic stimuli failed to cause the cytosolic accumulation of cyt c and other associated mitochondrial perturbations, as well as the failure to induce the activation of caspase-3 and apoptosis. While the control HL-60 cells showed high levels of Bcl-2 and barely detectable Bcl-xL, HL-60/Bcr-Abl cells expressed high levels of Bcl-xL and undetectable levels of Bcl-2, a pattern of expression similar to the one in K562 cells. Bax and caspase-3 expressions were not significantly different between HL-60/Bcr-Abl or K562 versus HL-60 cells. These findings indicate that Bcr-Abl expression blocks apoptosis due to diverse apoptotic stimuli upstream by preventing the cytosolic accumulation of cyt c and other preapoptotic mitochondrial perturbations, thereby inhibiting the activation of caspase-3 and execution of apoptosis.

scholarly journals Bcr-Abl Exerts Its Antiapoptotic Effect Against Diverse Apoptotic Stimuli Through Blockage of Mitochondrial Release of Cytochrome C and Activation of Caspase-3

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1700-1705 ◽  
Author(s):  
Gustavo P. Amarante-Mendes ◽  
Caryn Naekyung Kim ◽  
Linda Liu ◽  
Yue Huang ◽  
Charles L. Perkins ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
High Dose ◽  
Antiapoptotic Effect ◽  
Endogenous Expression ◽  
Cyt C

Abstract Bcr-Abl expression in leukemic cells is known to exert a potent effect against apoptosis due to antileukemic drugs, but its mechanism has not been elucidated. Recent reports have indicated that a variety of apoptotic stimuli cause the preapoptotic mitochondrial release of cytochrome c (cyt c) into cytosol, which mediates the cleavage and activity of caspase-3 involved in the execution of apoptosis. Whether Bcr-Abl exerts its antiapoptotic effect upstream to the cleavage and activation of caspase-3 or acts downstream by blocking the ensuing degradation of substrates resulting in apoptosis, has been the focus of the present studies. In these, we used (1) the human acute myelogenous leukemia (AML) HL-60 cells that are stably transfected with thebcr-abl gene (HL-60/Bcr-Abl) and express p185 Bcr-Abl; and (2) the chronic myelogenous leukemia (CML)-blast crisis K562 cells, which have endogenous expression of p210 Bcr-Abl. Exposure of the control AML HL-60 cells to high-dose Ara-C (HIDAC), etoposide, or sphingoid bases (including C2 ceramide, sphingosine, or sphinganine) caused the accumulation of cyt c in the cytosol, loss of mitochondrial membrane potential (MMP), and increase in the reactive oxygen species (ROS). These preapoptotic events were associated with the cleavage and activity of caspase-3, resulting in the degradation of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) and DNA fragmentation factor (DFF), internucleosomal DNA fragmentation, and morphologic features of apoptosis. In contrast, in HL-60/Bcr-Abl and K562 cells, these apoptotic stimuli failed to cause the cytosolic accumulation of cyt c and other associated mitochondrial perturbations, as well as the failure to induce the activation of caspase-3 and apoptosis. While the control HL-60 cells showed high levels of Bcl-2 and barely detectable Bcl-xL, HL-60/Bcr-Abl cells expressed high levels of Bcl-xL and undetectable levels of Bcl-2, a pattern of expression similar to the one in K562 cells. Bax and caspase-3 expressions were not significantly different between HL-60/Bcr-Abl or K562 versus HL-60 cells. These findings indicate that Bcr-Abl expression blocks apoptosis due to diverse apoptotic stimuli upstream by preventing the cytosolic accumulation of cyt c and other preapoptotic mitochondrial perturbations, thereby inhibiting the activation of caspase-3 and execution of apoptosis.

Arsenic Trioxide Enhances STI571-Induced Apoptosis of K562 Cells through Downregulating Bcl-XL and Bcr-Abl.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4449-4449
Author(s):  
Ri Zhang ◽  
Xuhui Zhang ◽  
YaJun Zhi ◽  
ZiLing Zhu ◽  
De Pei Wu
Keyword(s):  
Cytochrome C ◽  
Cell Line ◽  
Arsenic Trioxide ◽  
Drug Administration ◽  
Caspase 3 ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Mrna Levels ◽  
Down Regulation ◽  
Induced Apoptosis

Abstract We aimed to evaluate the effects of arsenic trioxide (ATO, Trisenox) on STI571 (Gleevec, imatinib mesylate)-induced apoptosis of a chronic myelogenous leukemia (CML) cell line, K562 cells. Cell prolifration and colony-forming assays were performed to determine the cytotoxicity of ATO alone and in combination with STI571. Apoptosis was analyzed by morphological changes, apoptosis rate and cell cycles. An Elisa assay was used to detect the levels of cytosolic cytochrome c (cyt c) and caspase-3 in K562 cells exposed to ATO and STI571 at graded concentrations. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assay the transcriptional levels of Bcl-XL and Bcr-Abl genes in K562 cells. Results showed that both the colony-forming assay and cell proliferation assay demonstrated additive to synergistic effects of ATO on STI571-induced apoptosis in K562 cells, a Bcr-Abl positive cell line. Caspase-3 was activated during apoptosis and there was an increase in cytosolic accumulation of cytochrome c. Treatment of K562 cells with STI571 alone led to down-regulation of transcriptional levels of Bcl-XL at 12 hours and Bcr-Abl at 96 hours after drug administration. Treatment with ATO alone only led to reduce the mRNA levels of Bcl-XL, but not Bcr-Abl. Combined treatment with ATO and STI571 down regulated the transcripts of Bcl-XL at 12 hours and Bcr-Abl 72 hours after drug administration. We conclude that ATO enhanced cytotoxic and proapoptotic actions of STI571 could be mediated by the down-regulation of Bcr-Abl and Bcl-XL genes in K562 cells. Therefore ATO in combination with STI571 could be a promising therapy for CML.

Higher Expression of Cytochrome-c and Caspase-3 in the Primary Acute Lymphoblastic Leukemic Cells with Negative Expression of VEGF and Its Receptors Than with Positive Expressed Cells under VP16 Induction.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4448-4448 ◽  
Author(s):  
Aibin Liang ◽  
Long-Jun Gu
Keyword(s):  
Flow Cytometry ◽  
Cytochrome C ◽  
Caspase 3 ◽  
De Novo ◽  
Leukemic Cells ◽  
Immunofluorescent Staining ◽  
Leukemia Cells ◽  
Cyt C ◽  
Primary Leukemia

Abstract Flow cytometry and immunofluorescent staining were employed to examine the apoptosis signals, cytochrome-c and Caspase-3, in the primary leukemia cells with or without expression of VEGF and its receptors and normal healthy donor’s lymphocytes. We found that primary leukemic cells and normal lymphocytes didn’t secrete Cyt-c from mitochondria and no caspase-3 activation without the treatment of VP16. Instead, after incubation with VP16 (30mg/L) for 8 hours, normal lymphocytes and most of de novo leukemia cells secreted Cyt-c into cytosol from mitochondria and had the activation of caspase-3 in the cellular plasma. However, primary leukemia blasts with higher expression of VEGF and its receptors revealed lower secretion of Cyt-C and down-regulated activation of Caspase-3. Results showed that Cyt-c secretion and Caspase-3 activation were prone to occur in the VEGF negatively expressed blasts than positively expressed cells. This indicates that lower secretion of Cyt-c from mitochondria and inactivation of Caspase-3 are two of the pivotal mechanisms of anti-apoptosis for refractory leukemia clone with highly expressed VEGF and its receptors.

scholarly journals Possible Role of Peroxynitrite in the Responses Induced by Fusicoccin in Plant Cultured Cells

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 182 ◽  
Author(s):  
Massimo Malerba ◽  
Raffaella Cerana
Keyword(s):  
Cytochrome C ◽  
Dna Fragmentation ◽  
Stress Responses ◽  
Molecular Chaperones ◽  
Caspase 3 ◽  
Cultured Cells ◽  
Acer Pseudoplatanus ◽  
Cytochrome C Release ◽  
Induced Stress

Fusicoccin (FC) is a well-known phytotoxin able to induce in Acer pseudoplatanus L. (sycamore) cultured cells, a set of responses similar to those induced by stress conditions. In this work, the possible involvement of peroxynitrite (ONOO−) in FC-induced stress responses was studied measuring both in the presence and in the absence of 2,6,8-trihydroxypurine (urate), a specific ONOO− scavenger: (1) cell death; (2) specific DNA fragmentation; (3) lipid peroxidation; (4) production of RNS and ROS; (5) activity of caspase-3-like proteases; and (6) release of cytochrome c from mitochondria, variations in the levels of molecular chaperones Hsp90 in the mitochondria and Hsp70 BiP in the endoplasmic reticulum (ER), and of regulatory 14-3-3 proteins in the cytosol. The obtained results indicate a role for ONOO− in the FC-induced responses. In particular, ONOO− seems involved in a PCD form showing apoptotic features such as specific DNA fragmentation, caspase-3-like protease activity, and cytochrome c release from mitochondria.

Role of F-actin organization in p38 MAP kinase-mediated apoptosis and necrosis in neonatal rat cardiomyocytes subjected to simulated ischemia and reoxygenation

2005 ◽  
Vol 289 (6) ◽  
pp. H2310-H2318 ◽  
Author(s):  
Takayuki Okada ◽  
Hajime Otani ◽  
Yue Wu ◽  
Shiori Kyoi ◽  
Chiharu Enoki ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Map Kinase ◽  
Dna Fragmentation ◽  
P38 Mapk ◽  
Caspase 3 ◽  
Neonatal Rat ◽  
Cytochrome C Release ◽  
Actin Reorganization ◽  
Ldh Release ◽  
Sb 203580

Activation of p38 mitogen-activated protein (MAP) kinase (MAPK) has been implicated in the mechanism of cardiomyocyte (CMC) protection and injury. The p38 MAPK controversy may be related to differential effects of this kinase on apoptosis and necrosis. We have hypothesized that p38 MAPK-mediated F-actin reorganization promotes apoptotic cell death, whereas it protects from osmotic stress-induced necrotic cell death. Cultured neonatal rat CMCs were subjected to 2 h of simulated ischemia followed by reoxygenation. p38 MAPK activity measured by phosphorylation of MAP kinase-activated protein (MAPKAP) kinase 2 was increased during simulated ischemia and reoxygenation. This was associated with translocation of heat shock protein 27 (HSP27) from the cytosolic to the cytoskeletal fraction and F-actin reorganization. Cytochrome c release from mitochondria, caspase-3 activation, and DNA fragmentation were increased during reoxygenation. Robust lactate dehydrogenase (LDH) release was observed under hyposmotic (140 mosM) reoxygenation. The p38 MAPK inhibitor SB-203580 abrogated activation of p38 MAPK, translocation of HSP27, and F-actin reorganization and prevented cytochrome c release, caspase-3 activation, and DNA fragmentation. Conversely, SB-203580 enhanced LDH release during hyposmotic reoxygenation. The F-actin disrupting agent cytochalasin D inhibited F-actin reorganization and prevented cytochrome c release, caspase-3 activation, and DNA fragmentation, whereas it enhanced LDH release during hyposmotic reoxygenation. When CMCs were incubated under the isosmotic condition for the first 15 min of reoxygenation, SB-203580 and cytochalasin D increased ATP content of CMCs and prevented LDH release after the conversion to the hyposmotic condition. These results suggest that F-actin reorganization mediated by activation of p38 MAPK plays a differential role in apoptosis and protection against osmotic stress-induced necrosis during reoxygenation in neonatal rat CMCs; however, the sarcolemmal fragility caused by p38 MAPK inhibition can be reversed during temporary blockade of physical stress during reoxygenation.

scholarly journals Activation of caspase-3 may not contribute to postresuscitation myocardial dysfunction

2009 ◽  
Vol 296 (4) ◽  
pp. H1164-H1174 ◽  
Author(s):  
Jeejabai Radhakrishnan ◽  
Iyad M. Ayoub ◽  
Raúl J. Gazmuri
Keyword(s):  
Cytochrome C ◽  
Dna Fragmentation ◽  
Rat Model ◽  
Caspase 3 ◽  
Myocardial Dysfunction ◽  
Left Ventricular ◽  
Stroke Work ◽  
Protective Effects ◽  
Apoptotic Pathway ◽  
Apoptosis Protein

We have previously reported that postresuscitation myocardial dysfunction is accompanied by the release of cytochrome c and caspase-3 activation. We now investigated the role of caspase-3 activation by examining whether such process prompts apoptotic DNA fragmentation, whether caspase-3 inhibition attenuates myocardial dysfunction, and whether myocardial protective effects of sodium-hydrogen exchanger isoform-1 (NHE-1) inhibition involve caspase-3 inhibition using a rat model of ventricular fibrillation (VF) of closed-chest resuscitation. Resuscitation after 4 or 8 min of untreated VF caused significant reductions in left ventricular stroke work index averaging 23% of sham control rats at 4 h postresuscitation. Left ventricular dysfunction was accompanied by increases in cytosolic cytochrome c, decreases in pro- and cleaved caspase-9 fragments, increases in 17-kDa caspase-3 fragments, and increases in caspase-3 activity indicating the activation of the mitochondrial apoptotic pathway but without evidence of apoptotic DNA fragmentation. In addition, levels of heat shock protein 70 were increased and levels of X-linked inhibitor of apoptosis protein and αβ-crystallin were preserved, all of which can exert antiapoptotic effects. In a separate series, the caspase-3 inhibitor z-Asp-Glu-Val-Asp chloromethyl ketone given before the induction of VF failed to prevent postresuscitation myocardial dysfunction despite reductions in caspase-3 activity (2.3 ± 0.5 vs. 1.3 ± 0.5 pmol fluorophore AFC released·mg protein−1·min−1; P < 0.03). Treatment with the NHE-1 inhibitor cariporide had no effect on caspase-3 activity. Accordingly, in this rat model of VF and severe postresuscitation myocardial dysfunction, activation of caspase-3 did not lead to DNA fragmentation or contribute to myocardial dysfunction. Concomitant activation of intrinsic antiapoptotic mechanisms could play a protective role downstream to caspase-3 activation.

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.

JS-K, a Novel Nitric Oxide (NO) Generator, Induces Cytochrome c Release and Caspase Activation in HL-60 Myeloid Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3415-3415
Author(s):  
Paul J. Shami ◽  
Vidya Udupi ◽  
Margaret Yu ◽  
Swati Malaviya ◽  
Joseph E. Saavedra ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Caspase 3 ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Dose Dependent Fashion ◽  
Acute Myelogenous ◽  
Dose Dependent

Abstract NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity. We have previously shown that JS-K induces apoptosis in HL-60 cells by a caspase dependent mechanism (Molecular Cancer Therapeutics2:409-417,2003). The purpose of this study was to determine the pathway through which JS-K induces apoptosis. Western blot analysis showed that treatment of HL-60 cells with JS-K (0 – 1 μM) for 6 hours results in release of Cytochrome c from mitochondria in a dose dependent fashion. Treatment with JS-K resulted in a dose dependent activation of Caspase 9. Sixteen and 24 hours after exposure to 1 μM JS-K, Caspase 9 activity was induced by 393 ± 93% and 237 ± 13% of control, respectively (p = 0.03 at the 24 hours time point). Treatment with JS-K resulted in a dose dependent activation of Caspase 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 3 activity was 208 ± 3.4 % of control (p = 0.02). Treatment with JS-K also resulted in a dose dependent activation of Caspase 8, but to a lesser extent than Caspase 9 and 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 8 activity was 144 ± 5.3 % of control (p = 0.04). We conclude that JS-K activates the intrinsic pathway of apoptosis in leukemia cells by inducing the release of Cytochrome c from mitochondria. (NO1-CO-12400).

Identification of the Epigenetic Reader BRD4 As a Novel Potential Target in Ph+ CML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1571-1571
Author(s):  
Barbara Peter ◽  
Gregor Eisenwort ◽  
Gabriele Stefanzl ◽  
Daniela Berger ◽  
Wolfgang R Sperr ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Drug Target ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Potential Drug ◽  
Ku812 Cells

Abstract Chronic myelogenous leukemia (CML) is a bone marrow-derived hematopoietic neoplasm in which BCR/ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients with CML, leukemic cells can be kept under control by BCR/ABL1 tyrosine kinase inhibitors (TKI), including imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. Nevertheless, resistance or intolerance against one or more of these TKI may occur. Therefore, current research is focusing on novel potential drug targets in CML. A promising class of targets may be epigenetic regulators of cell growth, such as members of the bromodomain and extra-terminal domain (BET) family. The epigenetic reader and BET family member BRD4 has recently been identified as a novel potential drug target in acute myeloid leukemia (AML). However, so far, little is known about the expression and function of BRD4 in CML cells. The aims of the present study were to determine the expression of BRD4 and its downstream target MYC in CML cells and to explore whether BRD4 can serve as a novel drug target in this disease. As determined by qPCR, primary CML cells (chronic phase patients, n=7) as well as the CML cell lines KU812 and K562 expressed BRD4 mRNA. In addition, both CML cell lines stained positive for BRD4 in our immunocytochemistry staining experiments. In one patient with accelerated phase CML, putative leukemic (CD34+/CD38-) stem cells were sorted to near homogeneity and found to express BRD4 mRNA by qPCR. In order to examine the functional role of BRD4 in CML cells, a BRD4-specific shRNA was applied. In these experiments, the shRNA-induced knockdown of BRD4 in KU812 cells and K562 resulted in reduced growth compared to a control shRNA. Furthermore, the BRD4-targeting drug JQ1 was found to inhibit 3H-thymidine uptake and thus proliferation in KU812 cells in a dose-dependent manner (IC50: 0.25-0.75 µM). In addition, we were able to show that JQ1 inhibits growth of primary CML cells with variable IC50 values (0.1-5 µM). However, no substantial growth-inhibitory effects of JQ1 were seen in K562 cells (IC50: >5 µM). As determined by Annexin V/PI staining, JQ1 induced apoptosis in KU812 cells whereas no apoptosis-inducing effect of JQ1 was observed in K562 cells. Nevertheless, we were able to show that both CML cell lines as well as primary CML cells express MYC mRNA, and treatment of KU812 cells or K562 cells with JQ1 resulted in a decreased expression of MYC mRNA and MYC protein. Next, we analyzed whether MYC expression in CML cells can be blocked by BCR/ABL1 TKI. We found that imatinib, nilotinib, dasatinib, and ponatinib decrease MYC mRNA- and MYC protein expression in KU812 and K562 cells. Finally, we found that JQ1 cooperates with imatinib, nilotinib, ponatinib and dasatinib in inhibiting the proliferation of KU812 and K562 cells. Together, our data show that BRD4 serves as a potential new target in CML cells, and that the BRD4 blocker JQ1 cooperates with BCR/ABL1 TKI in inducing growth-inhibition. Whether BRD4 inhibition is a pharmacologically meaningful approach in patients with TKI-resistant CML remains to be determined in clinical trials. Disclosures Sperr: Ariad: Consultancy; Celgene: Consultancy. Zuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

scholarly journals Hyperglycemia Enhances DNA Fragmentation after Transient Cerebral Ischemia

2001 ◽  
Vol 21 (5) ◽  
pp. 568-576 ◽  
Author(s):  
Ping-An Li ◽  
Ingrid Rasquinha ◽  
Qing Ping He ◽  
Bo K. Siesjö ◽  
Katalin Csiszár ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cell Death ◽  
Cytochrome C ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Cingulate Cortex ◽  
Tunel Staining ◽  
Dna Fragments ◽  
Cytochrome C Release ◽  
Klenow Polymerase

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3′-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.

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