Upregulation of HLA-G Gene Expression in Malignant Hematopoietic Cells Treated with 5-Aza-2′- Ddeoxycytidine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4567-4567
Author(s):  
Firas Alsabty ◽  
Martin Mistrik ◽  
Katarina Polakova
Keyword(s):  
Gene Expression ◽  
G Protein ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Dna Demethylation ◽  
Lymphocytic Leukemia ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Demethylating Agent

Abstract Abstract 4567 Introduction Human leukocyte antigen G (HLA-G) is a nonclassic HLA class I antigen with restricted distribution in normal tissues. It exerts multiple immunregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Ectopic HLA-G expression observed in some pathological conditions such as malignant transformation may be triggered by epigenetic modifications such as DNA demethylation or histone acetylation. Materials and Methods Mononuclear cells were isolated from peripheral blood of newly diagnosed previously untreated patients with acute myeloblastic leukemia (AML) (n=9) and chronic lymphocytic leukemia (CLL) (n=5) by standard Ficoll-Hypaque density gradient centrifugation. Isolated cells were resuspended in RPMI 1640 medium supplemented with 2mM L-glutamine, 200 μg/ml gentamicin, 0.125 μg/ml amphotericin B and 10% heat-inactivated fetal bovine serum. Demethylating treatment of cells was carried out with100 μM 5-aza-2x- deoxycytidine (AdC) (Sigma) for 3 days. Human choriocarcinoma cell lines JEG3 and JAR (ATCC, Rockville, MD) were used as HLA-G positive and negative controls, respectively. Real time polymerase chain reaction (RT-PCR) and semiquantitative RT-PCR were performed using the ABI Prism 7000 Sequence Detection System and AmpliTaq Gold DNA polymerase to detect HLA-G mRNAs transcriptions. The HLA-G protein expression was examined by western blot analysis using mAb 4H84. Results HLA-G transcripts in AdC untreated leukemia samples were demonstrated in 3 out of 5 patients (60%) with B-CLL and in 5 out of 9 patients (56%) with AML. Treatment with demethylating agent AdC resulted in up-regulation of HLA-G transcription and expression of HLA-G protein in 5 out of 8 (63%) examined leukemia cell lines (Table 1). Conclusions we conclude that DNA methylation is an important control mechanism of HLA-G gene expression, and treatment of human leukemia with demethylating agent AdC may up-regulate HLA-G gene expression and induce HLA-G protein synthesis in some patients that may allow leukemic cells to escape recognition and destruction by cytotoxic T-cells or NK cells. Therefore patients should be monitored for HLA-G expression in order to follow risk of AdC therapy. 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.

Effect of sodium dichloroacetate on apoptotic gene expression in human leukemia cell lines

2019 ◽  
Vol 71 (2) ◽  
pp. 248-256 ◽  
Author(s):  
Jagoda Abramek ◽  
Jacek Bogucki ◽  
Marta Ziaja-Sołtys ◽  
Andrzej Stępniewski ◽  
Anna Bogucka-Kocka
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Human Leukemia Cell ◽  
Apoptotic Gene ◽  
Leukemia Cell Lines

scholarly journals Expression of L-myc and N-myc proto-oncogenes in human leukemias and leukemia cell lines

Blood ◽  
1991 ◽  
Vol 78 (11) ◽  
pp. 3012-3020 ◽  
Author(s):  
H Hirvonen ◽  
V Hukkanen ◽  
TT Salmi ◽  
TP Makela ◽  
TT Pelliniemi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Mrna Processing ◽  
Lymphocytic Leukemia ◽  
Human Leukemia ◽  
Hematopoietic Malignancies ◽  
Cell Proliferation And Differentiation ◽  
Leukemia Cell Lines ◽  
Proliferation And Differentiation ◽  
Nuclear Phosphoproteins

Abstract The myc proto-oncogenes encode nuclear phosphoproteins, which are believed to participate in the control of cell proliferation and differentiation. Deregulated expression of c-myc has been implicated in several human hematopoietic malignancies. We have studied the expression and mRNA processing of human L-myc, N-myc, and c-myc genes in a panel of human leukemias, leukemia cell lines, and normal hematopoietic cells. L-myc mRNA was expressed in three acute myeloid leukemias (AML) studied and in several myeloid leukemia cell lines. Only low expression levels were observed in adult bone marrow and in fetal spleen and thymus. The K562 and Dami leukemia cell lines showed a unique pattern of L-myc mRNA processing, with approximately 40% of L- myc mRNA lacking exon III and intron I. N-myc was expressed in five of six AML cases studied, in one of nine acute lymphocytic leukemia (ALL) cases, and in several leukemia cell lines, while c-myc mRNA was detected in all leukemias and leukemia cell lines studied. Coexpression of all three myc genes was observed in Dami and MOLT-4 cell lines and in two AMLs, and either L-myc or N-myc was coexpressed with c-myc in several other cases. These results show that in addition to c-myc, the L-myc and N-myc genes are expressed in some human leukemias and leukemia cell lines, and suggest a lack of mutually exclusive cross- regulation of the myc genes in human leukemia cells.

Alternation of Cellular Morphology and Proliferation Induced by microRNA in Human Leukemia Cell Line, UT-7/EPO.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4203-4203
Author(s):  
Nobuyoshi Kosaka ◽  
Yusuke Yamamoto ◽  
Nami Nogawa ◽  
Keiichi Sugiura ◽  
Hiroshi Miyazaki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Macrocytic Anemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Morphological Studies

Abstract Mature microRNA (miRNA) originated from primary miRNA (pri-miRNA) is a new group of potential regulator for cell differentiation, apoptosis, proliferation and oncogenesis. Some miRNAs were recently identified in hematopoietic cells, while the roles of miRNAs in erythrocytic and megakaryocytic cells had not been well examined. As a first step to explore for miRNAs specific for hematopoietic lineage, the expressions of several known primary microRNAs in erythrocytic and megakaryocytic cell lines, such as TF-1, HL-60, HEK293 and UT-7 leukemia cells, were examined by RT-PCR. We consequently focused on the pri-miR-10a, a primary transcript of miR-10a located within Hox gene clusters, and found the significant expression in TF-1 cells and UT-7/EPO cells. The UT-7/EPO cells were a subline established from the original UT-7 cells, as well as UT-7/GM and UT-7/TPO cells; therefore it was suitable for the further comparative analysis. Interestingly, in UT-7/EPO cells, the expression of pri-miR-10a increased under stimulation of erythropoietin (EPO; 1U/mL and 10U/mL). Based on these observations, it was postulated that pri-miR-10a might involve in modulating erythrocyte differentiation or proliferation. To clarify the role of pri-miR-10a in UT-7/EPO, we have established clonal cell lines by transfecting UT-7/EPO cells with either the control vector or the pri-miR-10a expression vector pCMV-pri-miR10a. Overexpression of pri-miR-10a in the UT-7/EPO cell line (miR10a-UT-7/EPO) was confirmed by RT-PCR. MiR10a-UT-7/EPO showed higher proliferation rate even at low concentration of EPO (0.1 mU/mL). Overexpression of pri-miR-10a did not appear to affect HOXB4 and HOXA1 expression, as similar mRNA levels were seen in both cell lines. It was notable that the cellular size of miR10a-UT-7/EPO became larger than its parental cells. Morphological studies of miR10a-UT-7/EPO were performed in detail. It is possible that miR-10a was capable to modulate morphological features particularly in cellular size relating to cell cycle regulation. For instance, loss of the E2F family members result in marked macrocytic anemia with megaloblastic features in adult mice (Mol Cell. 2000 Aug;6(2):281–91., Mol Cell Biol. 2003 May;23(10):3607–22., Blood. 2006 Aug 1;108(3):886–95.). Data presented here hypothesized that the roles of miR-10a in erythroid cells are tightly associated with cell cycle.

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.

Deacetylase Inhibitor Cocktails Provide Striking Synergistic Interactions in Human Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4404-4404
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Floriana Fruscione ◽  
Santina Bruzzone ◽  
Gabriele Zoppoli ◽  
...  
Keyword(s):  
Hydroxamic Acid ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia

Abstract Abstract 4404 Cancer cells almost invariably exhibit aberrant histone deacetylase (HDAC) activity leading to changes in chromatine structure, altered gene expression, poor differentiation, impaired apoptosis and increased proliferation. Accordingly, virtually all the HDAC inhibitors currently available show some degree of antitumor activity in preclinical cancer models and several of these compounds are currently under investigation or already approved for the treatment of human malignancies. Such is the case of the hydroxamic acid derivative suberoylanilide hydroxamic acid (Vorinostat, Zolinza), approved for the treatment of cutaneous T cell lymphomas. Sirtuins are a large family of deacetylases characterized by a unique, NAD+-dependent enzymatic mechanism. In addition to their established role in metabolism and longevity, recent evidence points to an emerging role for sirtuins in carcinogenesis. In the attempt to identify drug combinations that would increase the activity of traditional HDAC inhibitors we have explored the combination of valproic acid (VA) and butyrate (BU) with the sirtuin inhibitors cambinol and sirtinol in primary B-cell chronic lymphocytic leukemia (B-CLL) cells (n=35), acute myelogenous leukemia (AML) cells (n=10) and leukemia cell lines. Cell viability was assessed by propidium iodide staining and flow cytometry. Combination indices were determined using the median-effect method. In leukemia cells, exposure to sirtuin inhibitors synergistically increased VA and BU mediated cytotoxicity. Conversely, these drugs were poorly active and failed to show any cooperation in healthy cells, including peripheral blood mononuclear cells and fibroblasts, suggesting a cancer-specific mode of action. Similar results were obtained by combining VA or BU with the Nampt inhibitor APO866, which reduces intracellular NAD+ levels and thereby prevents sirtuin activity. Remarkably, SIRT1 and SIRT6 inhibition per se did not seem to account for cell demise upon HDAC inhibition since expression of a dominant negative SIRT1 isoform or RNA interference-mediated SIRT6 silencing failed to increase VA and BU activity. Our data indicate a specific requirement by leukemia cells for sustained sirtuin activity when classical HDACs are inhibited. This feature is suitable to be therapeutically exploited by combining sirtuin inhibitors or APO866 with classical HDAC inhibitors especially for the treatment of hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

Comprehensive Mapping of Transposon Insertions in Human Hematopoietic Neoplasias.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1103-1103
Author(s):  
Kathleen H. Burns
Keyword(s):  
Human Genome ◽  
Cell Lines ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Clonal Evolution ◽  
Leukemia Cell ◽  
Human Tumor ◽  
Lymphocytic Leukemia

Abstract 1103 Poster Board I-125 Our genomes are replete with mobile DNAs, many of which are retrotransposons that have accumulated over time by “copy-and-paste” mechanisms involving reverse transcription of RNA intermediates. Subsets of human transposable elements have been recently active or remain active today, resulting in many insertional polymorphisms in modern populations. In vitro studies in human tumor cell lines have unequivocally shown that expressed retrotransposons can generate new insertions and potentiate large scale genomic rearrangements. Though normally transposon sequences are highly methylated and thus stably suppressed in somatic cells, loss of methylation has been described in some malignant states, including chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and ‘blast crisis’ phase chronic myeloid leukemia (CML). This has led to speculation that derepressed transposons contribute to clonal evolution of these pathologies, though experimental evidence for this has been lacking due to an inability to detect new genomic insertions. In collaboration with Jef Boeke's laboratory, I have developed an array-based transposon insertion profiling method (TIP-chip) for mapping mobile retrotransposons in the human genome. Early application of this technology in leukemia patients and leukemia cell lines shows numerous novel insertions of L1 LINEs, AluYb SINEs, and HERV-K transposons, including several insertions in genes known to be involved in leukemogenesis. We expect the technology will add a new dimension to our understanding of the human genome, including genetic predispositions to cancer development, and will enable tests of the hypothesis that insertional mutagenesis by endogenous transposons is a driving force in hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.

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