DIGE-Based Proteomic Analysis Identifies NPM/B23 and Nucleolin C23 as Overexpressed Proteins In Relapsed /Refractory Acute Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1711-1711
Author(s):  
Jian Da Hu ◽  
MinHui Lin ◽  
TingBo Liu ◽  
Jing Li ◽  
XinJi Chen ◽  
...  
Keyword(s):  
Treatment Outcome ◽  
Multidrug Resistance ◽  
Acute Leukemia ◽  
Western Blot ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemic Cell Lines

Abstract Abstract 1711 Resistance to chemotherapy is a challenge in treatment of acute leukemia. Although the classic multidrug resistance (MDR) phenotype is often characterized by expression of drug efflux pump P-glycoprotein or by multidrug resistance-associated proteins, precise molecular mechanisms are largely unknown. To investigate novel protein changes involved in resistance mechanism, protein expression profiles between human myeloid leukemia HL-60 cell lines and adriamycin- resistant HL-60 cell lines (HL-60/ADR) was compared, which was based on a differential proteomic approach — 2 dimensional difference in gel Electrophoresis(2D-DIGE) followed by mass spectrometry (MALDI-TOF-MS) and complemented by western blot validation. 16 protein spots were identified as being differentially expressed (> 1.2 fold change and p≤ 0.05) between above two cell lines, among which 13 protein spots were identified as up-regulated and 3 as down-regulated in the HL-60/ ADR cell line. Proteins found to have higher abundance levels in the resistant HL-60/ADR cells included enzymes, proteins and oncogenes related to signal transduction, protein synthesis, cell growth regulation and metabolism. 3 lower abundance proteins are related to transcription. From 16 proteins, 2 proteins, nucleophosmin B23 (NPM B23)and nucleolin C23, were selected and verified in leukemia cell lines and primary leukemia samples by western blot. Compared to healthy control samples, which showed no expressions of these 2 proteins, leukemic cell lines revealed an obvious up-regulation of B23 and C23. Moreover, significantly higher expressions of B23 and C23 were found in 3 resistant leukemic cell lines, HL-60/ADR, K562/ADR and KG01 cells, compared to the parent HL-60 and K562 cells, and other leukemic cell lines. In de novo leukemia samples, 43.8%(35/80) expressed B23 and C23 proteins, 37.9% (22/58) AML and 59.1% (13/22) ALL respectively. Meanwhile, concomitant expression of B23 and C23, both positive or negative, was noted in 97%(79/80)patients. Over-expressions of B23 and C23 were observed in 68.8% relapased/refractory leukemia patients. With regard to treatment outcome,among those patients who achieved ongoing CR, fewer patients expressed 2 proteins, only 13.35% (7/52) AML and 46%(7/15) ALL respectively. It implicated that B23 and C23 may be involved in drug resistance and be useful in assessing treatment outcome and prognosis of leukemia. To a conclusion, these results provide a novel clue for the molecular mechanism of MDR and suggest that B23 and C23 are prognostic indicators for leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The chromosome 4q21 gene (AF-4/FEL) is widely expressed in normal tissues and shows breakpoint diversity in t(4;11)(q21;q23) acute leukemia

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1080-1085 ◽  
Author(s):  
CS Chen ◽  
JM Hilden ◽  
J Frestedt ◽  
PH Domer ◽  
R Moore ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Chromosomal Translocation ◽  
Leukemic Cell ◽  
Genomic Region ◽  
Acute Leukemias ◽  
Normal Tissues ◽  
Leukemic Cell Lines ◽  
A Minor ◽  
Chromosome 4Q21

The chromosomal translocation, t(4;11)(q21;q23), is the most common type of 11q23 chromosomal abnormality, being highly prevalent in infant acute leukemias and associated with a poor prognosis. The t(4;11) results in the fusion of an 11q23 gene (MLL, HRX, Htrx-1, or ALL-1) and a 4q21 gene (AF-4 or FEL). To further evaluate the 4q21 gene and its role in t(4;11) acute leukemia, we have cloned a 38-kb genomic region and mapped exons of the AF-4 gene. The 4q21 breakpoints in 19 cases of t(4;11) acute leukemia were analyzed by Southern analysis and pulsed- field gels. Seventeen of the 19 cases had breakpoints on chromosome 4q21 that were scattered in this 38 kb region. Expression of the AF-4 gene was studied in a total of 28 various nonhematopoietic, hematopoietic, and t(4;11) leukemic cell lines. The AF-4 gene was expressed in all cell lines as a major and a minor transcript. In addition to the normal transcripts, two fusion transcripts from the derivative 11 and derivative 4 chromosomes were identified in all t(4;11) cell lines except B1, which had only the der(11) transcript. These findings suggest that the breakpoints on 4q21 cluster over a broader area than do the breakpoints in the 11q23 gene, and that der(11) encodes the fusion RNA found consistently in leukemia cells.

scholarly journals Bcr-abl translocation can occur during the induction of multidrug resistance and confers apoptosis resistance on myeloid leukemic cell lines

1997 ◽  
Vol 4 (8) ◽  
pp. 806-814 ◽  
Author(s):  
Francis Belloc ◽  
Sophie Cotteret ◽  
Gilles Labroille ◽  
Valérie Schmit ◽  
Claudine Jaloustre ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Apoptosis Resistance ◽  
Leukemic Cell Lines ◽  
Myeloid Leukemic Cell

Metformin Exerts Anti-Leukemic Effects Via Direct Inhibition Of Oncogenic Kinase Activity In Leukemia Cells Derived From Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2853-2853 ◽  
Author(s):  
Ichiro Kawashima ◽  
Keita Kirito
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Myeloproliferative Neoplasms ◽  
Leukemic Cell ◽  
Leukemia Cells ◽  
Signal Molecules ◽  
Dependent Manner ◽  
Direct Inhibition ◽  
Leukemic Cell Lines ◽  
Ampk Activity

Abstract Metformin, a biguanide family member of a group of anti-hyperglycemic reagents, is one of the most widely used anti-diabetic drugs. Recent studies have revealed that metformin can block growth and induce apoptosis in several leukemia cells types. These studies also demonstrated that AMP-activated protein kinase (AMPK), a sensor for cellular energy status, is an important effector that mediates the anti-leukemic effects of metformin. Activation of AMPK leads to the inhibition of p70S6 kinase(p70S6K), an important regulator of protein synthesis, which consequently causes cell death. In contrast to these findings, other studies reported that metformin exerts anti-leukemic effects independent of AMPK. To clarify this discrepancy and determine the anti-leukemic function of metformin, we analyzed the molecular events induced by metformin with a series of leukemic cell lines: JAK2V617F positive cells (SET2), BCR-ABL expressing cells (KU812), and Flt-3-ITD mutant cells (PL21 and MV4-11). Treatment with metformin inhibited growth and induced apoptosis in all of the tested leukemic cell lines. However, metformin exerted differing effects on AMPK activity. In the Flt-3-ITD positive cells, metformin clearly induced the phosphorylation of AMPK at Thr172, which is required for the activation of the enzyme. Concomitant with this finding, the inhibition of p70S6K and activation of ACC (acetyl-CoA carboxylase), both of which are important downstream mediators of AMPK, were also observed in these cell lines. However, metformin did not enhance AMPK activity in either the SET-2 or KU812 cell lines. Furthermore, neither inhibition of p70S6K nor activation of ACC was induced by metformin in these cells. These results suggested that metformin exerts anti-leukemic action independent of AMPK in these cells. To investigate the molecular mechanisms of the AMPK-independent action of metformin, we investigated whether metformin affects any signal transduction pathways in these cells. We discovered that metformin inhibited the tyrosine phosphorylation of JAK2V61F and BCR-ABL in a dose and time dependent manner in SET2 and KU812 cells, respectively. This inhibitory function was confirmed with the HEL and K562 cells lines, which express JAK2V617F and BCR-ABL, respectively. Metformin also prevented the phosphorylation of targets downstream of JAK2V617F and BCR-ABL, including STAT5, ERK and AKT, in these cells. Compared to JAK2V617F and BCR-ABL, the phosphorylation of Flt-3-ITD was not inhibited by metformin, and downstream signal molecules were instead activated by metformin in Flt-3-ITD expressing cells. It is well known that both JAK2V617F and BCR-ABL induce overproduction of reactive oxygen species, and anti-oxidants (e.g., N-acetyl-L-cysteine) block the action of both oncogenic proteins. Interestingly, recent studies revealed that metformin modulates the intracellular oxidative status by inhibiting mitochondrial electron transport complex I. Together with our observations, this suggests that metformin might block the activity of JAK2V617F and BCR-ABL by modulating ROS levels. In conclusion, we elucidated a new anti-leukemic action of metformin: direct inhibition of oncogenic tyrosine kinases. These observations also provide a potential new treatment strategy for myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
M. R. Ricciardi ◽  
R. Licchetta ◽  
S. Mirabilii ◽  
M. Scarpari ◽  
A. Parroni ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Leukemic Cell ◽  
Bioactive Compound ◽  
Primary Cells ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Leukemic Cell Lines

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

scholarly journals The chromosome 4q21 gene (AF-4/FEL) is widely expressed in normal tissues and shows breakpoint diversity in t(4;11)(q21;q23) acute leukemia

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1080-1085 ◽  
Author(s):  
CS Chen ◽  
JM Hilden ◽  
J Frestedt ◽  
PH Domer ◽  
R Moore ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Chromosomal Translocation ◽  
Leukemic Cell ◽  
Genomic Region ◽  
Acute Leukemias ◽  
Normal Tissues ◽  
Leukemic Cell Lines ◽  
A Minor ◽  
Chromosome 4Q21

Abstract The chromosomal translocation, t(4;11)(q21;q23), is the most common type of 11q23 chromosomal abnormality, being highly prevalent in infant acute leukemias and associated with a poor prognosis. The t(4;11) results in the fusion of an 11q23 gene (MLL, HRX, Htrx-1, or ALL-1) and a 4q21 gene (AF-4 or FEL). To further evaluate the 4q21 gene and its role in t(4;11) acute leukemia, we have cloned a 38-kb genomic region and mapped exons of the AF-4 gene. The 4q21 breakpoints in 19 cases of t(4;11) acute leukemia were analyzed by Southern analysis and pulsed- field gels. Seventeen of the 19 cases had breakpoints on chromosome 4q21 that were scattered in this 38 kb region. Expression of the AF-4 gene was studied in a total of 28 various nonhematopoietic, hematopoietic, and t(4;11) leukemic cell lines. The AF-4 gene was expressed in all cell lines as a major and a minor transcript. In addition to the normal transcripts, two fusion transcripts from the derivative 11 and derivative 4 chromosomes were identified in all t(4;11) cell lines except B1, which had only the der(11) transcript. These findings suggest that the breakpoints on 4q21 cluster over a broader area than do the breakpoints in the 11q23 gene, and that der(11) encodes the fusion RNA found consistently in leukemia cells.

Charcterization of a Newly Established Megakaryo/Erythroid Leukemia Cell Line, JAS-R.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4327-4327
Author(s):  
Hisashi Yamada ◽  
Junko Horiguchi-Yamada ◽  
Tetsuaki Sekikawa
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Neutralizing Antibody ◽  
Chromosome Analysis ◽  
Erythropoietin Receptor ◽  
Leukemia Cell Line ◽  
Erythroid Cell ◽  
Leukemic Cell Lines

Abstract A few leukemic cell lines which express megakaryo/erythroid markers are available. We recently established a new cell line, designated JAS-R, from a 64-year-old patient with acute megakaryocytic leukemia (AML M7). Immunophenotyping showed that JAS-R cells were positive for CD4, CD7, CD13, CD33, CD41, CD61 and glycophorin A. Chromosome analysis was composite karyotype, but no major translocation abnormalities were observed. Electron-microscope examination disclosed that JAS-R had bleb like surface margin and a-granules in cytoplasm. Major four proteins which exist in a-granule were expressed high levels in JAS-R by RT-PCR. To further characterize JAS-R from four other megakaryo/erythroid cell lines (MEG-01, CMK, K562 and KU812), the comparison of gene expression profiling was studied by using oligo-DNA array. JAS-R was categorized as most different cell line among them. Of note, JAS-R secreted erythropoietin and expressed erythropoietin-receptor. But erythropoietin-neutralizing antibody failed to inhibit the growth of JAS-R cells. JAS-R may be useful for the further understanding of megakaryo and erythroid regulation and for the study of megakaryo/erythroid leukemogenesis.

Targeting Aberrant Self-Renewal of Leukemic Cells with a Novel CBP/p300 Bromodomain Inhibitor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3750-3750
Author(s):  
Angeliki Thanasopoulou ◽  
Katharina Dumrese ◽  
Sarah Picaud ◽  
Oleg Fedorov ◽  
Stefan Knapp ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines

Abstract The CBP/p300 histone acetyltransferases are key transcriptional regulators of hematopoiesis that have been found to be involved in AML-associated recurrent chromosomal translocations and shown to function as co-activators of leukemogenic fusion oncogenes, suggesting that specific targeting of CBP/p300 may be beneficial for therapy. We characterized the anti-leukemic potential of I-CBP112, a novel chemical inhibitory probe targeting the CBP/p300 bromodomain (BRD). BRDs belong to a diverse family of evolutionary conserved protein-interaction modules recognizing acetylated lysine residues and thereby mediating recruitment of proteins to macromolecular complexes. I-CBP112 represents a new, potent and selective class of BRD inhibitors (oxazepines) binding to recombinant CBP/p300 BRDs with a KD of 151nM and 157nM respectively. Initial characterization by FRAP and BRET assays revealed that I-CBP112 displaced the isolated BRD construct from chromatin but not the full length CBP. I-CBP112 also impaired the interaction of CBP/p300 with p53, resulting in reduced p53-K382 acetylation, reduced p21 expression, and high sensitivity to Doxorubicin-induced DNA damage. We started to explore the effects of the compound on leukemic cells by exposing a series of murine cell lines immortalized by the MLL-CBP fusion and other potent leukemia-associated oncogenes including the MLL-AF9, MLL-ENL, or the NUP98-HOXA9 fusion to increasing doses of I-CBP112. Interestingly, no significant cytotoxicity was observed up to concentrations of 5μM. However, in all cell lines we observed a significant reduced number of colonies formed in methylcellulose, associated with morphological differentiation as observed in Giemsa stained cytospots. Similar to the murine leukemic cell lines we found that I-CBP112 did not cause immediate cytotoxic effects but impaired colony formation and induced cellular differentiation of a series of 18 human leukemic cell lines. Reduced colony formation upon I-CBP112 treatment was also observed of human primary AML blasts but not of CD34+ hematopoietic stem cells from two healthy donors. I-CBP112 effects were studied in more detail in three human leukemia cell lines: SEM (MLL-AF4+), MOLM13 (MLL-AF9+) and Kasumi-1 (AML1-ETO+). Long-term exposure of these cells to I-CBP112 in liquid medium, resulted in a dose-dependent G1 cell cycle arrest, with Kasumi-1 being the most sensitive to the inhibitor, demonstrating further morphological signs of differentiation and apoptotic cell death. Importantly, combination of I-CBP112 with the BET-BRD inhibitor JQ1 or Doxorubicin revealed a clear synergistic effect on cell survival of the AML cell lines except for the combination of I-CBP112 with Doxorubicin on MOLM13. Surprisingly only modest effects of I-CBP112 exposure on the transcriptional programs of SEM, MOLM13 and Kasumi-1 cells were found by microarray expression profiling. Genes found affected were mainly immune response regulators or NFkappaB targets suggesting that attenuation of NFkappaB downstream signals might impair the leukemia initiation capacity reflected by reduced colony formation. Extreme limited dilution assays (ELDA) in methylcellulose, as well as bone marrow transplantations in limiting dilutions using MLL-AF9-transformed murine leukemic blasts revealed that I-CBP112 significantly impaired self-renewal of the leukemic stem cell compartment in vitro and reduced the leukemia-initiating potential in vivo. Taken together, these data demonstrate that selective interference with the CBP/p300 BRD by I-CBP112 has the potential to selectively target leukemic stem cells and opens the way for novel combinatory “BRD inhibitor” therapies for AML and other human cancers. Disclosures No relevant conflicts of interest to declare.

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