Intact Rac Signaling Is Important for Leukemia Cell Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2885-2885
Author(s):  
Anja Troeger ◽  
Pascal-David Johann ◽  
Mumine Senturk ◽  
Michael D. Milsom ◽  
David A. Williams
Keyword(s):  
Western Blot ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Knock Down ◽  
Induced Apoptosis

Abstract Abstract 2885 Rho GTPases, Ras-related small G proteins, regulate multiple cell processes in hematopoietic cells. There is growing evidence that acute myeloid leukemia (AML) blasts and particularly MLL-rearranged AML blasts, rely on Rac activity (Mulloy JC et al, Blood, 2010). However, little is known about the role of these GTPases in acute lymphoblastic leukemia (ALL) and particularly precursor B cell ALL. To investigate the role of Rac and potential compensation by other GTPases in ALL, we first assessed the protein expression and activation of Rac in a number of B-ALL cell lines (SEM; RS4,11; REH; Nalm 6; Raji), compared with a T-ALL cell line (Jurkat) and several AML cell lines (ML2; MV4,11). Of these cell lines SEM; RS4,11; ML2 and MV4,11 are characterized by MLL-fusion genes. Jurkat and MLL-rearranged AML cell lines show higher expression of Rac proteins compared to B cell leukemia lines (Table 1). Overall, B-ALL cell lines exhibit highly variable levels of Rac expression and activity with no obvious correlation to the presence of MLL-fusion proteins. We then investigated proliferation and apoptosis in cell lines treated with the small molecule inhibitor NSC23766 (NSC), which blocks interaction of a subset of guanine exchange factors (GEFs) with Rac and thus inhibits its activation. Treatment with NSC led to ∼2-fold increase in cells arrested at G0/G1 and induced apoptosis in a dose-dependent fashion at NSC concentrations previously demonstrated to be non-toxic in normal hematopoietic cells (Muller LUW et al., Leukemia, 2008) (Table 2). The lymphoid cell lines Jurkat, Raji and SEM appeared less responsive to NSC with no increased apoptosis at 40μM NSC. There was no correlation between NSC response and baseline expression or activation status of Rac. However, cell lines resistant to NSC exhibited a paradoxical and transient early increase in Rac activation, suggesting the existence of compensatory activation mechanisms. To determine if the relative resistance observed in some cell lines was related to dependence on GEFs not targeted by NSC and to validate that the inhibitory effect of NSC was specifically due to Rac inhibition in sensitive cells, shRNAs were utilized to knock-down different members of the Rac subfamily. Effective shRNA-mediated knockdown was validated by western blot. Knockdown of Rac1 or Rac2 consistently induced apoptosis compared to non-targeting vector controls in NSC sensitive cell lines ML2 and Nalm6, with ML2 cells appearing slightly more sensitive to knock-down of Rac2 (Table 3). Knock-down of either Rac1 or Rac2 had little effect upon Jurkat cells which are resistant to NSC treatment. These data suggest that Jurkat cells are not dependent upon Rac signaling for survival; however we cannot discount the possibility that some compensation may occur between Rac1 and Rac2. These experiments demonstrate the importance of intact Rac signaling pathways for the survival of the majority of leukemia cell lines tested and demonstrate that dependence on Rac signaling is not restricted to leukemias characterized by MLL-rearrangements. Our observations also suggest that activation of different Rac isoforms may influence sensitivity towards pharmacological Rac inhibition. Table 1: Baseline Expression of Rac assessed by Western blot Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji Rac/b-actin expression* 1.6 2.5 1.7 0.5 0.7 0.8 1.0 1.0 (*arbitrary units, italics indicate cell lines carrying MLL-rearrangements) Table 2: % AnnexinV+ cells after treatment of the different cell lines with increasing doses the Rac-specific inhibitor NSC Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji control 6%+1.4 6%+1.3 9%+0.3 12%+3.6 9%+1.9 7%+1.5 9%+2 13%+2.3 20uM NSC 6%+1.4 9%+1.3 15%+0.3** 21%+8.5 8%+1.5 6%+1.9 25%+6.4 16%+3 40uM NSC 7%+1.8 24%+9.1 60%+4** 52%+11* 10%+1.3 10%+3.4 39%+11 16%+1.9 80uM NSC 15%+3.5* 73%+14.7** 97%+0.4** 80%+4** 17%+1.2* 46%+10.5** 62%+12.3* 22%+4 (Mean±SEM; n=5; * p<0.05; ** p<=0.01 versus control, bolded columns indicate increased NSC sensitivity) Table 3: % AnnexinV+ cells 7 days after lentiviral transduction of the different cell lines with Rac1 and Rac2-specific shRNA Cell line Jurkat ML-2 Nalm 6 non targeting control 4.3%+0.3 14.2%+8 11.4%+2.2 Rac1 shRNA* 8.0%+3.5 26.3%+7.9 36.8%+8.5 non targeting control 9.6%+4.2 8.1%+4.0 16.2%+3.1 Rac2 shRNA* 18.7%+4.5 35.5%+12.9 43.7%+7.1 (Mean±SEM; n=6; * second set of Rac1 and Rac2 shRNAs gave comparable results) Disclosures: No relevant conflicts of interest to declare.

CD45RO regulates the HIV-1 gp120-mediated apoptosis of T cells by activating Lck

2018 ◽  
Vol 399 (6) ◽  
pp. 583-591
Author(s):  
Kelei Li ◽  
Zhe Cong ◽  
Zhuoying Peng ◽  
Ting Chen ◽  
Jing Xue ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Tyrosine Phosphatase ◽  
Extracellular Domain ◽  
Jurkat Cells ◽  
Transfected Cells ◽  
Induced Apoptosis ◽  
Hiv 1

Abstract CD45 has been reported to regulate the HIV-1 gp120-induced apoptosis of Jurkat cells. Here, we demonstrate that the extracellular domain of CD45 plays an important role in this function. We observed that CD45RO-transfected cells, but not cells transfected with other CD45 isoforms, underwent significant apoptosis induced by gp120. However, a CD45RA-transfected cell line treated with an O-glycan inhibitor was able to undergo apoptosis. The role of the extracellular domain of CD45 was further confirmed using CD45 isoform-transfected cell lines by analyzing the phosphorylation of Lck, which is a direct substrate of CD45 tyrosine phosphatase, and by using an Lck inhibitor. These results suggest that CD45RO modulates HIV-1 gp120-induced apoptosis by regulating the activity of Lck.

scholarly journals Sirtuin 2 in Endometrial Cancer: A Potential Regulator for Cell Proliferation, Apoptosis and RAS/ERK Pathway

2020 ◽  
Vol 19 ◽  
pp. 153303382098078
Author(s):  
Yanjuan Guo ◽  
Nannan Zhao ◽  
Jianli Zhou ◽  
Jianxin Dong ◽  
Xing Wang
Keyword(s):  
Cell Proliferation ◽  
Endometrial Cancer ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Erk Pathway ◽  
Uterine Epithelial Cell ◽  
Knock Down ◽  
Ishikawa Cells ◽  
And Control

Objective: The present study aimed to explore the function of sirtuin 2 (SIRT2) on cell proliferation, apoptosis, rat sarcoma virus (RAS)/ extracellular signal-regulated kinase (ERK) pathway in endometrial cancer (EC). Methods: SIRT2 expression in human EC cell lines and human endometrial (uterine) epithelial cell (HEEC) line was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. SIRT2 knock-down and control knock-down plasmids were transfected into HEC1A cells, respectively; SIRT2 overexpression and control overexpression plasmids were transfected into Ishikawa cells, respectively. After transfection, SIRT2, HRas proto-oncogene, GTPase (HRAS) expressions were evaluated by RT-qPCR and western blot. ERK and phosphorylated ERK (pERK) expressions were evaluated by western blot. Meanwhile, cell proliferation and cell apoptosis were measured. Results: Compared to normal HEEC cell line, SIRT2 mRNA and protein expressions were increased in most human EC cell lines (including HEC1A, RL952 and AN3CA), while were similar in Ishikawa cell line. In HEC1A cells, SIRT2 knock-down decreased cell proliferation but increased apoptosis. In Ishikawa cells, SIRT2 overexpression induced cell proliferation but inhibited apoptosis. For RAS/ERK pathway, SIRT2 knock-down reduced HRAS and inactivated pERK in HEC1A cells, whereas SIRT2 overexpression increased HRAS and activated pERK in Ishikawa cells, suggesting that SIRT2 was implicated in the regulation of RAS/ERK pathway in EC cells. Conclusion: SIRT2 contributes to the EC tumorigenesis, which appears as a potential therapeutic target.

Breakpoint junctions of chromosome 9p deletions in two human glioma cell lines

1994 ◽  
Vol 14 (11) ◽  
pp. 7604-7610
Author(s):  
H M Pomykala ◽  
S K Bohlander ◽  
P L Broeker ◽  
O I Olopade ◽  
M O Díaz
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Repetitive Sequences ◽  
Leukemia Cell ◽  
Chromosome 9 ◽  
Breakpoint Junction ◽  
Leukemia Cell Lines ◽  
Long Interspersed Nuclear Elements

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.

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.

A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2267-2267
Author(s):  
Eva Sahakian ◽  
John Powers ◽  
Jie Chen ◽  
Allison Distler ◽  
Jennifer Rock-Klotz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cytokine Production ◽  
Hematopoietic Cells ◽  
Negative Regulator ◽  
Over Expression

Abstract Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production. Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts. We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures: No relevant conflicts of interest to declare.

Interactions Between BAFF and BAFF Receptors Are Involved in Macrophage-Induced Bortezomib Resistance in Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4429-4429
Author(s):  
Jing Chen ◽  
Donghua He ◽  
Xing Guo ◽  
Qingxiao Chen ◽  
Xuanru Lin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Western Blot ◽  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Counting ◽  
Therapeutic Modality ◽  
Second Primary ◽  
Induced Apoptosis

Abstract Background:B-cell-activating factor (BAFF) is a member of the TNF family that critical for maintenance of B-cell development and homeostasis. BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) are three BAFF receptors. It has been reported that BAFF is expressed by neutrophils, monocytes, dentritic cells and macrophages and modulates the proliferation, survival and drug resistance of multiple myeloma (MM) cells. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by direct interaction with MM cells. We hypothesized that BAFF/BAFF receptors play a role in macrophage-induced bortezomib resistance in myeloma. Methods: First, the expression levels of BAFF and its three receptors in primary MM cells, MM cell lines and peripheral blood monocyte(PBMC)-induced macrophages were detected by semiquantitative real time-polymerase chain reaction (qPCR),Western blot and flow-cytometry. Also the concentration of BAFF in the supernatants of MM patients' bone marrow, MM cell lines and macrophages were determined by ELISA. Second, Primary MM cells and MM cell lines were cocultured with macrophages for the indicated time (usually 4-6h and 24h), for some experiments, we added bortezomib to the coculture system. Cell viability and apoptosis of MM cells were verified by Cell Counting Kit-8(CCK8) after treated with recombinant human (rh) BAFF, BAFF neutralizing antibody and BAFF siRNA. The interactions between BAFF and its receptors are unveiled by flow-cytometry. Then, cell survival signaling activations that may confer MM drug resistance were examined by Western blot. Results: Two receptors of BAFF, TACI and BCMA were highly expressed in various MM cell lines. The expressions of BAFF in PBMC-induced macrophages were heterogeneous. Functional studies showed that rhBAFF promoted RPMI8226 and ARP1 myeloma cells growth (P<0.05) and protected them from bortezomib-induced apoptosis (P<0.05). Then we verified macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, RPMI8226) with PBMC-derived macrophages from healthy donors. The macrophage-induced bortezomib resistance was attenuated by neutralizing antibodies(P<0.05) and siRNA of BAFF(P<0.01) . Next we found that in MM cells cocultured with macrophages, bortezomib-induced PARP and caspase-3 cleavages were highly repressed and phosphorylated Src ,AKT and Erk1/2 were upregulated which indicated that BAFF-mediated MM drug resistance may be through ERK1/2 and Src pathway .In addition, BAFF induced activation of NF-κB2,a pathway critical for the growth and survival of these cells. Conclusions: Our data show that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors, leading to a reduction in caspase proteins and subsequent activation of Src and Erk1/2 kinases and NF-κB2 pathways .These studies reveal a promising unknown role for BAFF/BAFF receptors, suggesting that targeting macrophage-MM interactions may represent a promising therapeutic modality. Disclosures No relevant conflicts of interest to declare.

TET2 Downregulation Promotes AML Cell Proliferation Via Pim-1 Expression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5085-5085
Author(s):  
Qingxiao Chen ◽  
Jingsong He ◽  
Xing Guo ◽  
Jing Chen ◽  
Xuanru Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Lines ◽  
Target Genes ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Rna Seq ◽  
Knock Down

Abstract Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults which is still incurable although novel drugs and new combination of chemotherapies are used . With the development of genetic and molecular biology technologies, more and more genes are found to be related to leukemogenesis and drug resistance of AML. TET2, a member of the ten-eleven-translocation gene family which can modify DNA by catalyzing the conversion of 5-mehtyl-cytosine to 5-hydroxymethyl-cytosine , is often inactivated through mutation or deletion in myeloid malignancies. Recent research reported that TET2 knock-down can promote proliferation of hematopoietic stem cells and leukemic cells. Also, several clinical studies showed that patients with TET2 mutation or low levels of TET2 expression have more aggressive disease courses than those with normal levels of TET2. However, the mechanism of the phenomenon is unknown. Our aim is to uncover how TET2 protein level is negatively correlated with AML cell proliferation and to provide a better view of target therapy in AML. Methods: We determined the expression levels of TET2 and other target genes in acute leukemia cell lines, bone marrow AML specimens, and peripheral blood mononuclear cells from healthy donors by qRT-PCR and Western blot. We also determined the mutation status of TET2 in AML cell lines. CCK8 and flow cytometry were used to determine cell proliferation, cell apoptosis, and cell cycle profile. Methylation-specific PCR were used to examine the methylation status in gene promoter regions. Also, we developed TET2 knock-down lentivirus to transfect AML cell lines to examine the effect of TET2 depletion. Last, RNA-seq was used to compare gene expression level changes between TET2 knock-down cell lines and the control cell lines. Results: AML cells from AML cell lines (KG-1,U937, Kasumi, HL-60, THP-1, and MV4-11) and AML patients' specimens expressed lower levels of TET2 than those of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expressed TET2, while KG-1 expressed TET2 at a relatively higher level than those of other AML cell lines. We constructed a TET2 shRNA to transfect KG-1,THP-1,MV-4-11,Kasumi,and HL-60, and used qRT-PCR and western blot to verify the knock-down efficiency. CCK8 confirmed that knocking down TET2 could increase leukemia cell proliferation (P<0.05). Flow cytometry showed that cell cycle profile was altered in TET2 knock-down cells compared to the negative control cells. In order to identify target genes, we performed RNA-seq on wildtype and TET2 knockdown KG-1 cells and found that the expression of cell cycle related genes, DNA replication related genes, and some oncogenes were changed. We focused on Pim-1, an oncogene related to leukemogenesis, which was significantly up-regulated in the RNA-seq profile. Western blot and qPCR verified the RNA-seq results of Pim-1 expression in the transfected cells . Also, AML patients' bone marrow samples (n=35) were tested by qPCR and 28 of them were found to express low TET2 but high Pim-1 with the other 7 being opposite. For detailed exploration in expression regulation of Pim-1 via TET2, we screened genes affecting Pim-1 expression and found SHP-1, a tumor suppress gene which is often silenced by promoter methylation in AML. Western blot band of SHP-1 was attenuated in TET2 knockdown KG-1 cells. Moreover, methylation-specific PCR showed that after knocking down TET2 in KG-1 cell line, the promoter regions were methylated much more than the control cells. These results indicated that the function of TET2 in epigenetic modulation plays an important role in regulating Pim-1 expression. Finally, using flow cytometry and CCK8 we surprisingly found that knocking down TET2 expression could lead leukemic cells (KG-1, THP-1 and MV-4-11) more sensitive to Pim-1 inhibitor (SGI-1776 free base) and decitabine (a demethylation agent treating MDS and AML) (P<0.05). Conclusion: Our study showed that knocking down TET2 promoted leukemic cell proliferation. This phenomenon may correlate to Pim-1 up-regulation. Our clinical data also showed that the expression of TET2 and Pim-1 have an inverse relationship. The mechanism of TET2 regulating Pim-1 expression may be related to the epigenetic modulation function of TET2. Finally, we found TET2 downregulation could increase leukemia vulnerability to Pim-1 inhibitor and decitbine, and provide a novel view of target therapy in AML. Disclosures No relevant conflicts of interest to declare.

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

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 HOX GENE EXPRESSION IN HUMAN B-CELL PROGENITOR LEUKEMIA CELL LINES EXPRESSING E2A-PBX1 ONCOGENE

2020 ◽  
Vol 19 (1) ◽  
pp. 89-95
Author(s):  
E. A. Shestakova
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Chain Reaction ◽  
Hoxa Cluster ◽  
Oncogenic Potential ◽  
Leukemia Cell Lines ◽  
Hoxa Genes ◽  
Polymerase Chain

Introduction. Acute lymphoblastic leukemia (ALL) is diagnosed mainly in children (2/3 of diseases) making this type of leukemia one of the most common oncological diseases among children. Oncogenes are involved in the development of ALL, in particular the product of chromosomes 1 and 19 translocation, the oncogene E2A-PBX1 that codes for E2A-PBX1 chimeric oncoprotein with strong transcription activation properties as well as oncogenes of HOX family, mainly HOXA and HOXB cluster genes. E2A-PBX1 chimeric oncoprotein and НОХА proteins are associated in vivo with factors participating in epigenetic regulation of gene expression such as chromatin modifying and remodeling enzymes that partially determines their oncogenic properties. In previous studies we obtained data indicating genetic interactions of E2A-PBX1 and НОХ genes participating in leukemia development.The aim of this research was to confirm the role of Е2А – РВХ1 oncogene in the activation of the expression of НОХА cluster genes coding for the proteins with high oncogenic potential.Materials and methods. The objects of the study were four B cell progenitor (pre-B) leukemia cell lines: RCH-ACV, KASUMI-2, 697 and NALM-6. Standard polymerase chain reaction (PCR) was used for the identification of chromosome 1 and 19 translocation product, E2A-PBX1 oncogene and its expression. Method of reverse transcription coupled with quantitative polymerase chain reaction (Q-RT-PCR) was used for the analysis of 11 HOXA cluster genes expression.Results. It is demonstrated that E2A-PBX1 oncogene is present and expressed in three studied human pre-B leukemia cell lines, RCH-ACV, KASUMI-2 and 697, while its expression is absent in NALM-6 cell line. High expression of 7 from 11 HOXА cluster genes is revealed in RCH-ACV, KASUMI-2 and 697 cell lines expressing E2A-PBX1 oncogene, whereas NALM-6 cell line, that does not express E2A-PBX1 oncogene, also does not express HOXA genes except low expression of two genes from this cluster.Conclusions. The data obtained in this study demonstrate that RCH-ACV, KASUMI-2 and 697 human leukemia pre-B cell lines, containing and expressing Е2А-РВХ1 oncogene, also express most of HOXA genes (7 genes of 11 genes) at high level in contrast to control NALM-6 cell line that does not comprise Е2А-РВХ1 oncogene and almost does not express НОХА genes. Therefore, the results of this study suggest the participation of strong transcriptional activator, chimeric oncoprotein Е2А-РВХ1, associated with chromatin modifying and remodeling enzymes, in the expression activation of HOXA cluster genes that also possess high oncogenic potential.

The Novel, Orally Active Proteasome Inhibitor, NPI-0052, Induces Apoptosis in Leukemia Lymphoma Cell Lines and Patient Specimens.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 241-241
Author(s):  
Claudia P. Miller ◽  
Kechen Ban ◽  
Stacey L. Ruiz ◽  
Saskia Neuteboom ◽  
Michael Palladino ◽  
...  
Keyword(s):  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Proteolytic Activities ◽  
Induced Apoptosis ◽  
Orally Active

Abstract NPI-0052 is the second proteasome inhibitor with potential for clinical use since the FDA approval of bortezomib. NPI-0052 is a novel, orally active, non-peptide small molecule inhibitor discovered by Nereus Pharmaceuticals during the fermentation of a new marine Gram-positive actinomycete, Salinospora sp. In human erythrocyte derived 20S proteasomes, with EC50 values in the picomolar and nanomolar range, NPI-0052 inhibits all three proteolytic activities: the chymotrypsin-like, the trypsin-like and caspase-like activities. In the present study, exposure of 1uM NPI-0052 for 1 h inhibited the chymotryptic and caspase-like activities by greater than 90% in Jurkat and ML-1 cells. The trypsin-like activity was also inhibited to a lesser extent. NPI-0052 demonstrated varying degrees of apoptosis in cell lines representative of AML (ML-1), ALL (Jurkat), and Burkitt lymphoma (BL-41) and in mononuclear cells isolated from CLL and ALL patients, as measured by propidium iodide staining and subsequent FACS analysis. In addition, treatment of Jurkat cells with NPI-0052 resulted in activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP). Further experiments revealed that caspase activity might be initiated differently in myeloid versus lymphoid leukemia cell lines. NPI-0052 caused cleavage of caspase-8 as demonstrated by SDS-PAGE analysis and when combined with an inhibitor specific for caspase-8 (IETD-fmk), Jurkat cells (of lymphoid origin) were protected against NPI-0052 induced apoptosis whereas ML-1 (of myeloid origin) were not. The cleaved product of Bid was detected by immunoblotting in NPI-0052 treated Jurkat cells, suggesting amplification of caspase-8 activity through mitochondria. NPI-0052 induced loss of mitochondrial membrane potential and release of cytochrome c in Jurkat cells. Cell lines of lymphocytic origin exposed to 4h of NPI-0052 resulted in increased levels of peroxide and superoxide prior to cell death. Furthermore, the antioxidant, N-acetyl cysteine (NAC), conferred protection in Jurkat cells against NPI-0052 induced apoptosis. CLL and Ph+ ALL patient material confirmed that lymphocytes from these patients are protected from NPI-0052 induced apoptosis by antioxidants. In summary, NPI-0052 inhibits all three major proteolytic activities of 20S proteasome in leukemia cells and induces apoptosis in leukemic cells and patient samples. The cytotoxic effects of NPI-0052 in leukemia and lymphoma cells warrant further testing to determine if this compound is clinically effective.

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