scholarly journals MiR-106b-25 Promotes Chemoresistance in Acute Myeloid Leukemia Via Abolishing Multiple Apoptotic Pathways

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4341-4341
Author(s):  
Mingying Zhang ◽  
Fangnan Xiao ◽  
Yunan Li ◽  
Zizhen Chen ◽  
Xiaoru Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Parental Cell ◽  
Drug Resistant ◽  
Caspase 7 ◽  
Direct Target ◽  
Underlying Mechanisms ◽  
Apoptotic Pathways

Abstract Introduction: Chemoresistance and disease relapse remain the main obstacles responsible for treatment failure in leukemia. MicroRNAs (miRNAs) play essential roles in various physiological and pathological processes, including cell proliferation, differentiation, metabolism, and cancer development. The miR-106b-25 cluster consists of three miRNAs: miR-106b, miR-93 and miR-25. We have previously reported that miR-106b-25 was associated with chemoresistance by negatively regulated EP300 in breast cancer, but its role in hematological malignancies has not yet been elucidated. Here, we aim to clarify the biological role and underlying mechanisms of miR-106b-25 on drug resistance in leukemia. Methods: To see whether the miR-106b-25 was associated with the poor prognosis of AML patients, enriched LSCs (CD34 + cells) were isolated from the bone marrow of 18 newly diagnosed AML patients, the expression of miR-106b, miR-93, and miR-25 were examined, respectively. The expression levels of miR-106b, miR-93 and miR-25 were further determined in the doxorubicin-resistant leukemia cell line K562/A02 and HL60/ADR, compared with their parental cell lines. In addition, K562 cells were transduced with lentiviral vectors carrying miR-106b-25, and cell proliferation, drug resistance, colony-forming assay, apoptosis assays were performed to explore the function of miR-106b-25 overexpression in leukemia cells in vitro. To investigate the role of miR-106b-25 on tumor growth and overall survival after drug treatment, we performed xenotransplantation in nude mice using miR-106b-25 overexpressed K562 cells. To further clarify the function of each microRNA function in this cluster, K562 cells were also transduced with lentiviral vectors carrying individual miR-25, miR-93, or miR-106b separately. Cell proliferation, colony forming assay and cell apoptosis assay were also carried out subsequently. Simultaneously, RNA-sequencing was performed to reveal the underlying mechanisms of miR-106b-25 in the chemoresistance of myeloid leukemia. To experimentally confirm the direct target of the miR-106b-25 cluster in AMLs, we further performed a dual-luciferase reporter assay. Results: Upregulated miR-106b, miR-93 and miR-25 expression in enriched LSCs were significantly associated with shortened overall survival of AML patients. We also found miR-106b, miR-93 and miR-25 were significantly upregulated in drug-resistant leukemia cell lines compared with its parental cell lines. Overexpression of miR-106b-25 cluster promoted cell proliferation, led to resistance of K562 cells to doxorubicin, imatinib and ABT-737 (BCL-2 inhibitor) in liquid culture and drug-resistant colony-forming assays. Overexpression of miR-93 or miR-106b accelerated cell growth, and all the three miRNAs can promote drug-resistant colony-forming and inhibit cell apoptosis. RNA-sequencing (RNA-Seq) data revealed that multiple critical genes related to apoptotic pathways were downregulated after overexpressing miR-25, miR-93, miR-106b as well as the whole cluster, such as TP73, BAX, BAK1, Caspase-7, CDKN1A and BTG2. RT-qPCR confirmed that these genes are reduced with or without ABT-737 treatment. Luciferase assay further identified TP73 was a direct target of miR-93 and miR106b, BAK1 was a direct target of miR-25, and CASPASE-7 was a direct target of all these three miRNAs. Conclusions: In summary, we made the novel observation that miR-106-25 is associated with AML drug-resisitance and disease prognosis and identified TP73, BAK1 caspase-7 as a novel direct target of this cluster. Further studies revealed that the biological effects of miR-106b-25 cluster on leukemic cell proliferation, chemoresistance and apoptosis were mediated through regulation of apoptotic pathway. These findings indicate a promising diagnostic biomarker and a potential target therapeutic strategy for AML patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sertad1 Antagonizes iASPP Function By Hindering Its Entrance into Nuclei to Interact with P53 in Leukemic Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5201-5201
Author(s):  
Shaowei Qiu ◽  
Jing Yu ◽  
Tengteng Yu ◽  
Haiyan Xing ◽  
Na An ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Expression Level ◽  
Leukemic Cells ◽  
Immunoblotting Analysis ◽  
Chemotherapy Drugs

Abstract Introduction: As the important suprressor of P53, iASPP was found to be overexpressed in leukemia, and functioned as oncogene that inhibited apoptosis of leukemia cells. Sertad1 is identified as one of the proteins that can bind with iASPP in our previous study by two-hybrid screen. Sertad1 is highly expressed in carcinomas from pancreatic, lung and ovarian tissues, which considered Sertad1 as an oncoprotein. In this study, our findings revealed that Sertad1 could interact with iASPP in the cytoplasm near nuclear membrane, which could block iASPP to enter into nucleus to interact with P53, and inhibited the function of iASPP eventually. Methods: Co-immunoprecipitation and fluorescence confocal microscopic imaging were used to confirm the interaction between iASPP and Sertad1, the exact binding domains and the subcellular colocalization.The plasmids of iASPP and Sertad1 were transfected alone or co-transfected into K562 cells, the stable subclones that highly expressed iASPP, Sertad1 or both of them were then established by limiting dilution and named as K562-iASPPhi, K562-Sertad1hi, and K562-Douhi, respectively. The cell proliferation, cell cycle and apoptosis of above subclones were investigated by flow cytometry. Further, silence of the above two proteins was performed to confirm their functions. Immunoblotting analysis and immunofluorescence were performed to explore the possible mechanisms of difference between the biological functions of the above subclones. Results: Sertad1 expression level varied in leukemic cell lines and AML patients irrespectively of iASPP and P53. Interaction between iASPP and Sertad1 did exist in 293 cell and leukemic cells, both iASPP and Sertad1 scattered in the cytoplasm and nucleus, and their colocalizations were mainly in the cytoplasm, which encircled the nucleus. iASPP binds directly to Sertad1 through its PHD-bromo domain, C-terminal domain and Cyclin-A domain in a reduced order, and Serta domain failed to bind to iASPP. Overexpression of iASPP in K562 cells (iASPPhi) could result in the increased cell proliferation, cell cycle arrest in G2/M phase and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertad1 at the same time (Douhi) could slow down the cell proliferation, lead the cells more vulnerable to the chemotherapy drugs. As figure showed, in K562-Douhi cells, both iASPP and Sertad1 were obviously located in the cytoplasm, which encircled the nuclei, the subcellular colocalization was nearly outside the nuclei. The immunoblotting analysis further supported the conclusions. The resistance of iASPP to chemotherapeutic drug was accompanied by Puma protein expression in a p53-independent manner. By knocking down the expersssion of iASPP and Sertad separately, we found that iASPP is dispensable for maintenance of anti-apoptotic function and Sertad1 is indispensable for cell cycle in leukemic cells. Conclusions: In normal situation, the protein iASPP and Sertad1 scatter in the nucleus and cytoplasm, mainly in the cytoplasm. As convinced by our study, iASPP was overexpressed in the leukemia cell lines and primary AML patients, it could function as oncogene through its binding with P53 protein in the nucleus, inhibit the function of P53. When iASPPhi cells were exposed to apoptosis stimuli, Puma protein could play an important role in this process, irrespective of the expression level of P53. But when iASPP and Sertad1 were both overexpressed in the leukemic cells, Sertad1 could tether iASPP outside the nucleus mainly through its PHD-bromo domain, prevent it from inhibiting P53 function, suppress the leukemic cell growth and stimulate cell apoptosis by rescuing the P53 eventually. Our data provided a new insight to overcome iASPP protein, namely through its binding partners, when the similar proteins or drugs that can tether iASPP outside the nucleus such as Sertad1 are transfected into the leukemic cells, it may restore p53 function to eliminate the leukemic cells. Figure 1 Figure 1. Disclosures Wang: Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

Aberrant Stabilization of c-Myc Protein in Lymphoblastic and Myelogenous Leukemia Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1532-1532
Author(s):  
Suman Malempati ◽  
Rosalie C. Sears
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Half Life ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Proteasome Inhibition ◽  
Protein Stabilization ◽  
Hematopoietic Malignancies ◽  
Protein Levels ◽  
Leukemia Cell Lines

Abstract The c-Myc oncoprotein is a key regulator of cell fate decisions including proliferation, differentiation, and apoptosis. Orderly control of c-Myc protein levels is important in maintaining regulated cell proliferation in normal cells. While c-Myc overexpression is seen in many hematopoietic malignancies, the reason for high protein levels in most cases is unknown and, in general, is not the result of translocations or gene amplification. C-Myc levels vary with cell cycle and are kept very low in quiescent cells. Protein half-life is controlled by phosphorylation at two specific N-terminal sites, Serine 62 and Threonine 58, which regulate c-Myc degradation by the ubiquitin proteasome pathway. Two Ras-dependent signaling pathways (Raf/MEK/ERK and PI(3)K/Akt) modulate phosphorylation at Serine 62, which stabilizes the protein, and Threonine 58, which targets Myc for ubiquitination and subsequent degradation. We recently reported that a stabilized form of c-Myc (c-Myc T58A) contributes to oncogenic transformation of human cells in culture (Yeh et al, Nat. Cell Bio.6:308–318, 2004). Here we describe the role of c-Myc protein stabilization in 2 pediatric ALL cell lines (REH and Sup-B15), 1 AML cell line (HL-60), and 1 CML cell line (K562). Markedly higher expression of c-Myc protein was seen in all 4 cell lines as compared to normal peripheral blood mononuclear cells (PBMCs). FISH analysis demonstrated amplification of the c-myc gene in HL-60 cells as has been previously reported, but not in REH, Sup-B15, or K562 cells. Using [35S]methionine pulse-chase analysis we demonstrate that the half-life of c-Myc in REH (55 minutes), Sup-B15 (47 minutes), and K562 (40 minutes) cells is longer than in normal PBMCs (9 to 15 minutes), but is not significantly prolonged in HL60 cells. We provide additional functional evidence for aberrant protein stabilization based on greater elevation of c-Myc protein after proteasome inhibition in PBMCs and HL-60 cells than in REH, Sup-B15, or K562 cells. These results suggest that that abnormalities in c-Myc degradation exist upstream of ubiquitination in the ALL and CML cell lines. Consistent with this hypothesis, experimental inhibition of the PI(3)K pathway knocked down c-Myc levels in REH and Sup-B15 cells, an effect that was abrogated by concomitant proteasome inhibition. This result suggests that abnormal activation of the PI(3)K pathway could participate in c-Myc stabilization in these cells. In addition, destabilization of c-Myc by PI(3)K inhibition correlated with a significant decrease in cell proliferation. In conclusion, we demonstrate that aberrant stabilization of c-Myc protein occurs in human leukemia cell lines. Affecting the c-Myc degradation pathway in hematopoietic malignancies that have stabilized c-Myc may constitute a novel therapeutic target. Additional experiments are ongoing to assess c-Myc stability in primary cells from leukemic bone marrow samples.

scholarly journals miR-153-3p Suppresses Inhibitor of Growth Protein 2 Expression to Function as Tumor Suppressor in Acute Lymphoblastic Leukemia

2019 ◽  
Vol 18 ◽  
pp. 153303381985299 ◽  
Author(s):  
Jian Jiang ◽  
Yan Liu ◽  
Yanxia Zhao ◽  
Fei Tian ◽  
Gaoyan Wang
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Western Blot Assay ◽  
Leukemia Cell Lines ◽  
Direct Target

Alterations in microRNAs expression can accelerate the development of human cancers. However, the role of miR-153-3p in acute lymphoblastic leukemia remains unknown. The expression of miR-153-3p in acute lymphoblastic leukemia cell lines was measured by quantitative real-time polymerase chain reaction. Effects of miR-153-3p expression on acute lymphoblastic leukemia cell proliferation, migration, and invasion were examined by Cell Counting Kit-8 assay, wound healing assay, and Transwell invasion assay, respectively. We then validated inhibitor of growth protein 2 as a direct target of miR-153-3p through bioinformatics analysis, luciferase activity reporter assay, and Western blot assay. The miR-153-3p expression was decreased in acute lymphoblastic leukemia cell lines. Cell proliferation, migration, and invasion of acute lymphoblastic leukemia were obviously decreased by miR-153-3p overexpression. Moreover, inhibitor of growth protein 2 was validated as a direct target of miR-153-3p and the overexpression of inhibitor of growth protein 2 reversed the suppressive effects of miR-153-3p on acute lymphoblastic leukemia cell behaviors. Based on these results, we provided evidence that miR-153-3p might be a target for the treatment of acute lymphoblastic leukemia.

Krüppel-like Factors KLF4 and KLF2 Regulate microRNA-150 Expression in Myeloid Leukemias

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 874-874
Author(s):  
Valerie A. Morris ◽  
Carrie Cummings ◽  
Brendan Korb ◽  
Sean M. Boaglio ◽  
Vivian Oehler
Keyword(s):  
Cell Proliferation ◽  
Transcription Factors ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Promoter Activity ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Myeloid Differentiation ◽  
Klf4 Expression ◽  
Proliferation And Differentiation

Abstract Background: Acute myeloid leukemia (AML) is characterized by increased self-renewal of leukemia stem/progenitor cells and failure of differentiation to mature myeloid cells. MicroRNAs (miRNAs) are small single stranded non-coding RNAs 19 to 24 nucleotides in length that regulate expression of tens to hundreds of genes via mRNA degradation or translational repression. MiRNA contributions to normal hematopoiesis have been described and deletion of key miRNA processing enzymes in murine and human cells suggests that miRNA loss contributes to the cancer phenotype and aberrant differentiation in leukemia. By combining observations of miRNA expression in normal hematopoietic progenitor cells and patient AML cells and high-throughput lentiviral expression library screening approaches in AML cell lines we have identified candidate miRNAs that contribute to altered proliferation and differentiation in AML cells. We have previously established 1) that miR-150 expression is decreased in a large subset of primary patient AML samples, in particular poor risk cytogenetic groups, 2) and that miR-150 re-expression induces myeloid differentiation and decreases cell proliferation of normal hematopoietic progenitor cells and AML cell lines and primary patient cells in part through downregulation of MYB expression. MiR-150 loss is relevant in other hematopoietic and solid tumor malignancies where re-expression inhibits cell proliferation, promotes apoptosis and induces reversal of endothelial to mesenchymal transition. Transcription factors are important regulators of myeloid differentiation and cell proliferation. Moreover, as highlighted by recent sequencing of the AML genome, alterations in myeloid transcription factors through mutation, gene rearrangement, and altered expression play a significant role in leukemogenesis. Consequently, we have focused on how myeloid transcription factors regulate miRNA expression, specifically for miR-150. Results: Using 5’RACE from healthy bone marrow RNA, we identified a major transcription start site at 214 basepairs upstream of the pre-miR-150 hairpin. We identified the minimal miR-150 promoter region as -266 to +259 basepairs from the major transcription start site using miR-150 promoter truncation luciferase constructs assayed in myeloid leukemia cell lines (THP-1, K562, and KG1a) and a lymphoid leukemia cell line (Jurkat). We identified DNA binding sites for the Krüppel-like factor (KLF) family of transcription factors that are necessary for miR-150 promoter activity using site-directed DNA mutagenesis of the luciferase reporters. KLFs regulate proliferation, differentiation, pluripotency, migration and inflammation. Depending on cell type and context, KLFs can function as tumor suppressors or oncogenes. To identify which KLF isoforms regulate miR-150 expression, we assayed the ability of KLFs 2, 3, 4, 5, 6, 7, 9, and 10 to induce miR-150 promoter activity using the luciferase reporters and endogenous miR-150 expression by quantitative PCR. KLF2 and KLF4 overexpression increased miR-150 promoter activity in luciferase assays 50-fold and 450-fold respectively in K562 cells. Furthermore, KLF2 and KLF4 induced endogenous miR-150 expression 20-fold and 100-fold respectively as detected by quantitative PCR in both THP-1 and K562 cells. Prior work has established that KLF2 and KLF4 regulate the differentiation of monocytes. We then confirmed that KLF2 and KLF4 overexpression promotes myeloid differentiation of THP-1 cells by flow cytometry and gene expression that was partially reversed by inhibition of miR-150 expression. Conclusions: Previous studies have determined that KLF2 and KLF4 expression are decreased or absent in a significant subset of AML cases. Our observations suggest that loss of KLF2 and KLF4 expression contributes to decreased miR-150 expression which in turn alters cell proliferation and differentiation. Other studies have implicated the cell cycle inhibitor p21WAF1/CIP1 and altered PPAR gamma signaling downstream of KLF4. Nonetheless, our mechanistic understanding is limited. Our work suggests that the loss of miR-150 and other miRNAs downstream of these transcription factors also contributes. Understanding the interactions between KLFs, miR-150 and other miRNAs has broader significance as KLF2 and KLF4 expression is altered in other hematopoietic and solid tumors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dye-mediated photolysis is capable of eliminating drug-resistant (MDR+) tumor cells

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 793-800 ◽  
Author(s):  
RM Lemoli ◽  
T Igarashi ◽  
M Knizewski ◽  
L Acaba ◽  
A Richter ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Light Exposure ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Drug Resistant ◽  
Colony Forming Unit

Abstract We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte- macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst- forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long- term marrow culture at levels equal to untreated controls. The T- lymphoblastic leukemia cell line CEM and its vinblastine (VBL)- resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.

Effects of rmhTRAIL Combined with Daunorubicin in Inducing Apoptosis of Leukemia Cell Lines and Its Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1986-1986
Author(s):  
Xuejun Zhang ◽  
Li Wen ◽  
Fuxu Wang ◽  
Ling Pan ◽  
Jianmin Luo ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Mrna Level ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Expression Level ◽  
U937 Cells ◽  
Leukemia Cell Lines ◽  
Before And After

Abstract Tumor Necrosis factor (TNF)-related apoptosis- inducing ligand (TRAIL) is a new member of TNF superfamily discovered recently. Several studies showed that TRAIL can preferentially induce apoptosis in a variety of tumor cells, while most normal cells tested do not appear to be sensitive to TRAIL. In the present study, we treated K562 and U937 leukemia cell lines with recombinant mutant human TRAIL (rmhTRAIL) alone or together with daunorubicin (DNR) to investigate the apoptosis of the treated cells and the synergistic reaction of rmhTRAIL and DNR. The normal cell line MRC-5 was used as control. The expression of four TRAIL receptors mRNA (death receptor DR4 and DR5, decoy receptor DcR1 and DcR2) in the cells lines were detected before and after the treatment by DNR. (1) AO-EB double staining and TUNEL staining were used to evaluate the morphological change of leukemia cell lines before and after the treatment. The results showed that rmhTRAIL could induce the apoptosis of leukemia cell lines and a dose-dependent manner was found in leukemia cell lines but not in MRC-5 cell lines. (2) The growth inhibition rate of leukemia cell lines induced by rmhTRAIL alone or combined with DNR was examined with MTT assays. Different concentrations of rmhTRAIL(8, 40, 200, 1000ng/mL)alone or combined with DNR(8, 40, 200, 1000ng/mL) was used. The result showed a dose-dependent growth inhibition by rmhTRAIL alone for K562- and U937-cell line (P<0.05) also, but not for MRC-5 cell line (P>0.05). The IC50 for K562 cells and for U937 cells had no statistic difference (538.80 vs 301.56ng/mL, P>0.05). In leukemia cell lines, the growth inhibition rates in combination groups were much higher than in rmhTRAIL or DNR alone groups (P<0.05), and no synergistic killing effects was found in MRC-5 cells (P<0.05). It was concluded that rmhTRAIL had synergistic effects with DNR in the growth inhibition of K562 and U937 cells. (3). To explore the antitumor mechanisms of rmhTRAIL combined with DNR, the expression level of the DR4, DR5 and DcR1, DcR2 mRNA in these three cell lines was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) before and after the treatment with DNR. The high expression of DR4,DR5 mRNA in the tested cells were observed before the treatment of DNR, while very low or even undetectable expression level of DcR1 and DcR2 mRNA were observed in U937 and K562 cells, and a high expression level of DcR1 and DcR2 mRNA in MRC-5 cells were observed. After 24 hours treatment of three cell lines with DNR (200ng/ml), the expression level of DR5 mRNA increased in K562 and U937 cells (P<0.05). DR4 mRNA also increased in K562 cells but not in U937 cells. There was no change in DcR1 and DcR2 mRNA level in three cell lines. The four receptors’ mRNA level in MRC-5 cells was not influenced by DNR. Our results indicated that rmhTRAIL could induce the apoptosis of leukemia cell lines, and DNR could enhance significantly the sensitivity of K562 and U937 cells to apoptosis induced by rmhTRAIL through up-regulation of death receptors. Therefore, we presumed TRAIL might be act as a new agent for biological therapy in leukemia.

A Novel Microarray Gene Expression Analysis of Murine AML Cell Lines Provides Clues to the Development of Drug Resistance to Ara-C

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5017-5017
Author(s):  
Susan K Rathe ◽  
David Largaespada
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Ribosomal Subunit ◽  
Nuclear Factor Κb ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Drug Resistant ◽  
Microarray Gene Expression ◽  
Expression Microarrays

Abstract Acute myeloid leukemia (AML) has the ability to evade cell death in the presence of chemotherapeutic cocktails containing cytosine arabinoside (Ara-C). This lab previously developed two highly resistant murine AML cell lines, B117H and B140H, by introducing increasing concentrations of Ara-C to their parental cell lines, B117P and B140P, respectively. B117H and B140H can tolerate Ara-C concentrations ~1000X that of their drug sensitive parental cell lines. mRNA from all four cell lines were used in gene expression microarrays for the purpose of comparing Ara-C drug resistant murine AML cell lines with their Ara-C drug sensitive parental lines. A novel algorithm was developed to evaluate the changes in gene expression between the drug resistant and drug sensitive cells. The algorithm differed from more conventional algorithms in two key ways. First, the detection data was normalized by using ribosomal subunit 9 (Rsp9) as the normalization gene, and secondly it calculated fold change by comparing the minimum value of one population to the maximum value of the other population. The output of this algorithm was a list of genes with significant gene expression changes. These genes were next submitted to the Ingenuity Pathway Analysis (IPA) process. IPA implicated nuclear factor-κB (NFκB) in the Ara-C resistance process. Cell growth assays confirmed that the Ara-C drug resistant B117H cell line was significantly more sensitive to NFκB inhibition than its Ara-C sensitive parental cell line. This leads us to believe that the selection of Ara-C resistance may also concomitantly make some AML cells highly sensitive to killing by NFκB inhibition. This theory is being tested further through the use of drug combination assays, to determine if a synergistic or antagonistic relationship exists between Ara-C and various drugs that affect the NFκB pathway.

Myeloid Leukemias Directly Suppress T Cell Proliferation Through STAT3 and Arginase Pathways

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3885-3885 ◽  
Author(s):  
Samantha Miner ◽  
Sawa Ito ◽  
Kazushi Tanimoto ◽  
Nancy F. Hensel ◽  
Fariba Chinian ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Immune Editing

Abstract The immune-editing effect of myeloid leukemia has recently been reported in several studies. We previously demonstrated that the K562 leukemia-derived cell line suppresses T cell proliferation, which suggests that myeloid leukemia may function in a similar way to myeloid derived suppressor cells (MDSC). While the mechanism of suppression in leukemia is not fully understood, recent murine and human studies suggest that the STAT3 and arginase pathways play a key role in the immunosuppressive function of MDSC. We hypothesized that myeloid leukemia utilizes the MDSC STAT3 and arginase pathway to evade immune control, and block anti-leukemic immune responses. To evaluate the suppressive capacity of myeloid leukemia on T cell proliferation, we isolated CD34+ blasts and myeloid derived suppressor cells (MDSC: CD11b+CD14+) from blood of primary leukemia samples by FACS sorting (n=5). These cells were co-cultured with CFSE-labeled CD4+ T cells (n=9), previously isolated from healthy donor PBMCs using an automated cell separator (RoboSep). After stimulating with CD3/CD28 Dynabeads (Invitrogen, New York, USA) for 72 hours, proliferation was measured by CFSE dilution of the viable cell population. In three myeloid leukemias studied, CD4+ T cell proliferation was significantly suppressed in the presence of primary CD34 blasts and MDSC cells (p<0.001). Interestingly, CD34 blasts demonstrated a greater suppressive effect on T cells compared to MDSC cells for these samples (not statistically significant p=0.61). Next we repeated the proliferation assay using five leukemia cell lines: THP-1 and AML1 (derived from AML), K562 and CML1 (derived from CML), and the Daudi lymphoid-derived leukemia cell line. After staining with cell tracer dye and irradiating 100Gy, the cells were co-incubated with CFSE-labeled CD4+ T cells from healthy volunteers (n=6). We found that CD4+ T cell proliferation in the presence of the myeloid leukemia cell lines was significantly suppressed (mean proliferation 5.7±0.9% to 26.1±10.7%: p<0.0001 to 0.05) compared to lymphoid cell lines (mean proliferation 76.3±8.2%: p>0.05), consistent with the results obtained with the primary leukemia samples. To evaluate the impact of STAT3 and arginase on the immunosuppressive function of myeloid leukemia, the five cell lines were primed overnight with either arginase inhibitor (N(ω)-Hydroxy-nor-L-arginine; EMD Biosciences, Inc., California, USA) or two STAT3 inhibitors (STAT3 Inhibitor VI or Cucurbitacin I; EMD Millipore, Massachusetts, USA). Then, CD4+ T cells from healthy donors (n=3) were cultured with either (1) leukemia without any inhibitor (2) leukemia in the presence of inhibitor (3) leukemia primed with inhibitor. Priming leukemia with arginase inhibitor and STAT3 inhibitors almost completely abrogated their suppressive effect of T cell proliferation (p<0.001). We conclude that myeloid leukemia, like MDSC, directly immunosuppresses T cells, through STAT-3 and arginase. This finding may underlie the immune-editing of T cells by myeloid leukemia. Our results suggest that STAT3 inhibitors could be used to augment leukemia-targeted immunotherapy. Further investigation of T cell biology within the leukemia microenvironment is needed to further define immune editing mechanisms in myeloid leukemia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures: No relevant conflicts of interest to declare.

STUDY ON ANTIANGIOGENENIC EFFECT OF ACANTHOPANAX SENTICOSUS IN HL60 CELL LINES

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4857-4857
Author(s):  
Chen Fangyuan ◽  
Zhang Minyue ◽  
Cai Jiayi ◽  
Shen Lijing
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Concentration Dependence ◽  
Cell Lines ◽  
Western Blotting ◽  
Leukemia Cell ◽  
Hl60 Cell ◽  
Vegf Receptor ◽  
Acanthopanax Senticosus ◽  
Hl60 Cells

Abstract Introduction Many studies have been confirmed that neovascularization, the formation of new blood vessels from existing vasculature, plays an essential role in growth, development and metastasis of acute leukemia. At present, antiangiogenic therapy of leukemia become the new hot spot. Acanthopanax senticosus(Chianese name Ci Wu Jia ,CWJ) is a kind of Chinese herb, which contain natural flavonoid compounds and have been proven to inhibit leukemia cell proliferation. But there is no detailed report about the relationship with the inhibition of leukemia cells and the inhibition of angiogenesis effect. In this study, we should demonstrate the inhibition of leukemia cell growth and antiangiogenic mechanism through HL60 cell lines, further confirm the inhibitory effect on leukemia and antiangiogenic effect of Acanthopanax senticosus, Methods HL60 cells were treated with different concentrations of Acanthopanax senticosus (25°¢50°¢75°¢100°¢200µg/ml). Cell proliferation were detected using Cell Counting Kit-8. Kinds of transcription factors in Dll4/Notch (Delta-like 4 is the only ligand of Notch expressing in endothelium) and VEGF(R) signaling pathway were evaluated using quantitative real-time PCR (qRT-PCR) and Western blotting. Results Acanthopanax senticosus inhibited the growth of HL60 cells, and the time and concentration dependence(Fig.1). We extracted RNA and protein from these cells at 12hr, 24hr and 48hr respectively, found that Acanthopanax senticosus remarkably results in VEGF, VEGFR2(VEGF Receptor 2), DLL4 down-regulation based on the time and the concentration dependence, and mild inhibit VEGFR1(VEGF Receptor 1) and Notch1 factors gene expression(Fig. 2). Western blotting also showed a significant inhibition protein of VEGFR2, DLL4 and Notch1, mild inhibited the expression of VEGF and VEGFR1 protein, and with time and concentration dependence (Fig. 3). Summary Acanthopanax senticosus can inhibited proliferation of HL60 cells in vitro and anti-angiogenesis effect mainly via inhibition of VEGFR2-mediated signaling. It has an instantaneous effect on Dll4/ Notch signaling pathway. The data have elucidated the potential roles of several key signaling pathways in angiogenesis. Disclosures: No relevant conflicts of interest to declare.

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