scholarly journals Proton Sensor GPR68 Is Essential to Maintain Myeloid Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1353-1353
Author(s):  
Xiaofei He ◽  
Mark Wunderlich ◽  
Benjamin Mizukawa ◽  
James C. Mulloy ◽  
Saran Feng ◽  
...  
Keyword(s):  
Cell Growth ◽  
Molecular Mechanism ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Single Case ◽  
Leukemia Cell ◽  
Disease Free Survival ◽  
Myeloid Malignancies ◽  
Extracellular Acidosis ◽  
Induced Apoptosis

Abstract Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML. Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function. In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway. In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. Disclosures No relevant conflicts of interest to declare.

CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4174-4174
Author(s):  
Jiaying Tan ◽  
Jay L. Hess
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Polycomb Group ◽  
Human Leukemia ◽  
Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4469-4469
Author(s):  
Hilmar Quentmeier ◽  
Sonja Eberth ◽  
Julia Romani ◽  
Margarete Zaborski ◽  
Hans G. Drexler
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Leukemia Cell ◽  
Resistant Cell ◽  
Imatinib Resistance ◽  
Imatinib Resistant ◽  
Induced Apoptosis

Abstract Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.

Increased 14-3-3ζ Expression In The Multidrug-Resistant Leukemia Cell Line HL-60/Vcr As Compared To HL-60 Cell Line Mediates Cell Growth and Apoptosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5009-5009
Author(s):  
Rong Liang ◽  
Xiequn qun Chen ◽  
Qing xian Bai ◽  
Zhe Wang ◽  
Lan Yang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Protein Expression ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Subcellular Location ◽  
Leukemia Cell Line ◽  
Mobility Shift ◽  
Mrna And Protein Expression ◽  
Induced Apoptosis

Abstract Objectives Acute myeloid leukemia (AML) recurrence is largely a result of multidrug resistance (MDR). Because vincristine-resistant HL-60 cells (HL-60/VCR) express greater levels of 14-3-3¦Æ, which is associated with apoptosis and chemosensitivity in other cancers, this study sought to examine its role in AML chemosensitivity using HL-60 and HL-60/VCR cells. Methods The mRNA and protein expression of 14-3-3¦Æ, mdr1, Pgp, BCL-2 and MCL-1 were examined using semi-quantitative RT-PCR and Western blot analyses, respectively. The subcellular location of 14-3-3¦Æ protein in HL-60 and HL-60/VCR cells was determined using immunofluorescence and confocal microscopy. After siRNA-mediated silencing of 14-3-3¦Æ in HL-60 AND HL-60/VCR cells, cell growth and cell cycle progression were determined by direct counting and flow cytometry, respectively. The effect of 14-3-3¦Æ siRNA on topotecan (TPT)-induced apoptosis was evaluated using acridine orange/ethidium bromide and Annexin V staining as well as TUNEL analysis. NF-¦ÊB-DNA biding was also assessed using electrophoretic mobility shift assay. Results As compared to HL-60 cells, increased 14-3-3¦Æ mRNA and protein expression was observed in HL-60/VCR cells. In addition, increased mdr-1 mRNA as well as Pgp, Bcl-2, and Mcl-1 protein expression were observed in HL-60/VCR cells. In both HL-60 and HL-60/VCR cells, 14-3-3¦Æ was observed in the cytoplasm and nuclear compartments. 14-3-3¦Æ siRNA significantly reduced HL-60 and HL-60/VCR cell growth after 48 h and increased the proportion of cells in the G0/G1 phase. Moreover, 14-3-3¦Æ siRNA significantly increased the sensitivity of both HL-60 and HL-60/VCR cells to TPT possibly through inhibition of Bcl-2, Mcl-1 and Pgp protein expression. Conversely, increased Bad and Noxa protein expression was observed with 14-3-3¦Æ siRNA. NF-ĸB DNA binding was reduced with 14-3-3¦Æ siRNA. Conclusions Silencing of 14-3-3¦Æ increased the sensitivity of both sensitive and resistant HL-60 cells to TPT-induced apoptosis possibly through altering the expression of apoptosis-associated proteins, suggesting that it may be a potential target for MDR AML. Disclosures: No relevant conflicts of interest to declare.

Caspase-2 is activated at the CD95 death-inducing signaling complex in the course of CD95-induced apoptosis

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 559-565 ◽  
Author(s):  
Inna N. Lavrik ◽  
Alexander Golks ◽  
Simone Baumann ◽  
Peter H. Krammer
Keyword(s):  
B Cell ◽  
Molecular Mechanism ◽  
Cell Lines ◽  
Caspase 8 ◽  
Signaling Complex ◽  
Deficient Cell ◽  
Apoptotic Pathways ◽  
Caspase 2 ◽  
Induced Apoptosis

Caspase-2 was reported to be involved in a number of apoptotic pathways triggered by various stimuli. However, the molecular mechanism of procaspase-2 activation in the course of apoptosis remains poorly defined. In this report, we demonstrate that procaspase-2 is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex (DISC) in human T- and B-cell lines. We show that procaspase-2 is activated at the DISC on CD95 stimulation. Despite its presence at the DISC, caspase-2 does not initiate apoptosis on CD95 stimulation in caspase-8–deficient cell lines. Taken together, our data reveal that caspase-2 is activated at the DISC but does not play an initiating role in the CD95-induced apoptosis.

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

Arsenic Trioxide and Melarsoprol Induce Programmed Cell Death in Myeloid Leukemia Cell Lines and Function in a PML and PML-RAR Independent Manner

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1497-1504 ◽  
Author(s):  
Zhu-Gang Wang ◽  
Roberta Rivi ◽  
Laurent Delva ◽  
Andrea König ◽  
David A. Scheinberg ◽  
...  
Keyword(s):  
Cell Line ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Nuclear Bodies ◽  
Independent Manner ◽  
Leukemia Cell Lines ◽  
Induced Apoptosis ◽  
Myeloid Leukemia Cell

Abstract Inorganic arsenic trioxide (As2O3) and the organic arsenical, melarsoprol, were recently shown to inhibit growth and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and chronic B-cell leukemia cell lines, respectively. As2O3 has been proposed to principally target PML and PML-RAR proteins in APL cells. We investigated the activity of As2O3 and melarsoprol in a broader context encompassing various myeloid leukemia cell lines, including the APL cell line NB4-306 (a retinoic acid–resistant cell line derived from NB4 that no longer expresses the intact PML-RAR fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To examine the role of PML in mediating arsenical activity, we also tested these agents using murine embryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, we found that both compounds inhibited cell growth, induced apoptosis, and downregulated bcl-2 protein in all cell lines tested. Melarsoprol was more potent than As2O3 at equimolar concentrations ranging from 10−7 to 10−5 mol/L. As2O3 relocalized PML and PML-RAR onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RAR nuclear localization. Moreover, both As2O3 and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML−/− MEFs, and inhibited colony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyte formation in BM cultures of PML+/+ and PML−/− progenitors. Together, these results show that As2O3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RAR expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL. © 1998 by The American Society of Hematology.

scholarly journals Isoquinolinamine FX-9 Exhibits Anti-Mitotic Activity in Human and Canine Prostate Carcinoma Cell Lines

2019 ◽  
Vol 20 (22) ◽  
pp. 5567
Author(s):  
Jan Torben Schille ◽  
Ingo Nolte ◽  
Eva-Maria Packeiser ◽  
Laura Wiesner ◽  
Jens Ingo Hein ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
In Vivo Studies ◽  
Mediated Effects ◽  
Multinucleated Cells ◽  
Prostate Carcinoma Cell ◽  
Castrate Resistant ◽  
Induced Apoptosis

Current therapies are insufficient for metastatic prostate cancer (PCa) in men and dogs. As human castrate-resistant PCa shares several characteristics with the canine disease, comparative evaluation of novel therapeutic agents is of considerable value for both species. Novel isoquinolinamine FX-9 exhibits antiproliferative activity in acute lymphoblastic leukemia cell lines but has not been tested yet on any solid neoplasia type. In this study, FX-9′s mediated effects were characterized on two human (PC-3, LNCaP) and two canine (CT1258, 0846) PCa cell lines, as well as benign solid tissue cells. FX-9 significantly inhibited cell viability and induced apoptosis with concentrations in the low micromolar range. Mediated effects were highly comparable between the PCa cell lines of both species, but less pronounced on non-malignant chondrocytes and fibroblasts. Interestingly, FX-9 exposure also leads to the formation and survival of enlarged multinucleated cells through mitotic slippage. Based on the results, FX-9 acts as an anti-mitotic agent with reduced cytotoxic activity in benign cells. The characterization of FX-9-induced effects on PCa cells provides a basis for in vivo studies with the potential of valuable transferable findings to the benefit of men and dogs.

Combination of a Spicamycin Analogue KRN5500 and Chemotherapeutic Agents Synergistically Enhances Their Cytotoxicity in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4783-4783
Author(s):  
Hirokazu Miki ◽  
Shuji Ozaki ◽  
Osamu Tanaka ◽  
Shiro Fujii ◽  
Shingen Nakamura ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Growth ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Flow Cytometric Analysis ◽  
Human Tumor ◽  
Chemotherapeutic Agents ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. Although new classes of agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains an incurable disease due to increased resistance to these agents. Therefore, alternative approaches are necessary to overcome drug resistance in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan). This drug potently decreases protein synthesis and inhibits cell growth in human tumor cell lines both in vitro and in vivo. Several phase I studies of KRN5500 were conducted in patients with solid tumors, which showed Cmax values of 1000–3000 nM at the maximum tolerated doses. However, no objective anti-tumor response to KRN5500 alone was observed in these patients. In this study, we examined the anti-tumor activity of KRN5500 against MM cells and evaluated its therapeutic potential in combination with other anti-MM agents. MM cell lines and freshly-isolated MM cells were incubated with various concentrations of KRN5500 for 24 hours. Cell proliferation assay showed marked inhibition of cell growth in MM cells such as RPMI 8226, KMS12-BM, and UTMC-2 (IC50 = 10–40 nM), and U266, MM.1S, and primary MM cells (IC50 = 500–1000 nM). Importantly, a chemotherapy-resistant subclone of RPMI 8226 had a similar sensitivity to KRN5500. Annexin V/propidium iodide staining confirmed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Moreover, cleavage of poly (ADP-ribose) polymerase (PARP) was detected after 24 hours with only modest activation of caspase-8, -9, and -3 by immunoblotting. Flow cytometric analysis of anti-apoptotic proteins revealed that apoptosis induced by KRN5500 was associated with down-regulation of Mcl-1 and Bcl-2 expression. To determine the effect of KRN5500 on the unfolded protein response (UPR), splicing of XBP-1 mRNA was analyzed by reverse transcription-polymerase chain reaction. In response to stimulation with KRN5500, splicing of XBP-1 mRNA occurred after 24 hours in RPMI 8226 cells, suggesting that KRN5500-induced apoptosis is mediated in part by the inhibition of UPR. Furthermore, synergistic effects on MM cells were observed when KRN5500 was combined with anti-MM agents including melphalan, dexamethasone, and bortezomib. These results suggest that KRN5500 induces apoptosis in MM cells mainly by the caspase-independent pathway and that its unique mechanism of action provides a valuable therapeutic option to overcome drug resistance in patients with MM.

Aspirin Induces Apoptosis in Human Leukemia Cells Independently of NF-κB and MAPKs through Alteration of the Mcl-1/Noxa Balance.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4825-4825
Author(s):  
Ana M Cosialls ◽  
Daniel Iglesias-Serret ◽  
Maria Piqué ◽  
Montserrat Barragán ◽  
Antonio F Santidrián ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Protein Levels ◽  
Human Leukemia Cells ◽  
Induced Apoptosis

Abstract Abstract 4825 Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in most cell types. We examined the mechanism of aspirin-induced apoptosis in human leukemia cells. Our results show that aspirin induced apoptosis in leukemia Jurkat T cells independently of NF-κB. Although aspirin induced p38 MAPK and c-Jun N-terminal kinase (JNK) activation, selective inhibitors of these kinases did not inhibit aspirin-induced apoptosis. We studied the regulation of Bcl-2 family members in aspirin-induced apoptosis. The mRNA levels of some pro-apoptotic members, such as BIM, NOXA, BMF or PUMA, were induced by aspirin. However, none of these pro-apoptotic proteins increased and the levels of Mcl-1 protein were reduced. Interestingly, in the presence of aspirin the protein levels of Noxa remained high. This alteration of the Mcl-1/Noxa balance was also found in other leukemia cell lines and primary chronic lymphocytic leukemia cells (CLL). Furthermore, in CLL cells aspirin induced an increase in the protein levels of Noxa. Knockdown of Noxa or Puma significantly attenuated aspirin-induced apoptosis. These results indicate that aspirin induces apoptosis through alteration of the Mcl-1/Noxa balance. Disclosures No relevant conflicts of interest to declare.

Establishment and Characterization of a Novel Human Myeloid Leukemia Cell Line, AMU-AML1, Carrying t(12;22)(p13;q11) with No Fusion of MN1-TEL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4375-4375
Author(s):  
Mayuko Goto ◽  
Ichiro Hanamura ◽  
Motohiro Wakabayashi ◽  
Hisao Nagoshi ◽  
Tomohiko Taki ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Chromosome Band ◽  
Structural Abnormality ◽  
Myeloid Leukemia Cell

Abstract Abstract 4375 Leukemia cell lines are ubiquitous powerful research tools that are available to many investigators. In balanced chromosomal aberration in leukemia, a chimeric fusion gene formed by genes existing on breakpoints is frequently related to leukemogenesis. Cytogenetic abnormalities of chromosome band 12p13 are detected non-randomly in various hematological malignancies and usually involved TEL, which encodes a protein of the ETS transcription factor family. Chromosome band 22q11-12 is one of partners of translocation 12p13 and t(12;22)(p13;q11-12) results in fusion of TEL and MN1 or in just the partial inactivation of TEL. It is important to analyze precisely the breakpoint in a non-random translocation such as t(12;22)(p13;q11-12) and in addition it contributes to the better understanding of the molecular pathogenesis of leukemogenesis. In this study, we established a novel human myeloid leukemia cell line, AMU-AML1, having t(12;22) from a patient with acute myeloid leukemia with multilineage dysplasia and analyzed its characters. Mononuclear cells were isolated by Ficoll-Hypaque sedimentation from patient's bone marrow before initiation of chemotherapy and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS). After 3 months, cell proliferation became continuous. The cell line, named AMU-AML1, was established. In AMU-AML1, the following pathogens were negative for EBV, CMV, HBV, HCV, HIV-1, HTLV-1 and mycoplasma. A doubling time of AMU-AML1 cells was about 96 hours. Proliferation of the cells was stimulated by rhG-CSF (10 ng/ml), rhGM-CSF (10 ng/ml), M-CSF (50 ng/ml), rhIL-3 (10 ng/ml) and rhSCF (100 ng/ml) but not by IL-5 (10 ng/ml), rhIL-6 (10 ng/ml), and rhEPO (5 U/ml). AMU-AML1 was positive for CD13, CD33, CD117 and HLA-DR, negative for CD3, CD4, CD8 and CD56 by flow cytometry analysis. G-banding combined with SKY analysis of AMU-AML1 cells showed single structural abnormality; 46, XY, t(12;22)(p13;q11.2). Double-color FISH using PAC/BAC clones listed in NCBI website and array CGH analyses indicated that the breakpoint in 12p13 was within TEL or telomeric to TEL and it of 22q11 was centromeric to MN1. A chimeric MN1-TEL transcript and fusion protein of MN1-TEL could not be detected by RT-PCR and western blot analysis. The wild type of MN1 protein was strongly expressed in AMU-AML1 compared with other leukemic cell lines with t(12;22), MUTZ-3 and UCSD/AML1. Our data suggest that AMU-AML1 had a t(12;22)(p13;q11.2) without fusion of MN1-TEL and the expression level of MN1 protein was relatively high, which might have some effects on leukemogenesis. In conclusion, AMU-AML1 is a useful cell line to analyze the biological consequences of the leukemic cells with t(12;22)(p13;q11.2) but no fusion of MN1-TEL. Disclosures: No relevant conflicts of interest to declare.

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