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.

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.

Inhibition of WT1 and bcl2 in leukemic cell lines by small interfering RNA (siRNA)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 16504-16504
Author(s):  
W. Glienke ◽  
E. Milz ◽  
N. Bauer ◽  
L. Bergmann
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Transfected Cells ◽  
Leukemic Cell Lines ◽  
Blast Cells ◽  
Induced Apoptosis ◽  
Interfering Rna

16504 The expression of Wilm‘s tumor gene-1 (wt1) and bcl-2 is considered to have a proliferating and survival supporting effect in leukemia blast cells. The downregulation of wt1 by means of antisense-oligonucleotides and ribozymes revealed an inhibition of cell proliferation and induction of cell death. Here we describe the effect of siRNA against wt1 and bcl-2 in leukemic cell lines. RT-PCR and western blot analyses were performed to examine wt1 and bcl-2 gene expression in transfected leukemia cell lines. Apoptosis was detected with FACS analysis. K562 and HL-60 cell lines transfected with wt1 siRNA showed decreasing levels of wt1 mRNA and protein expression after 24 and 48 hours. The cell proliferation was reduced between 45% and 76% 48 hours after transfection, and apoptosis increased from 6.6 % in control cells to 12.2 % 24 hours after transfection in transfected cells. 48 hours after transfection the amount of apoptotic cells increased up to 45 % in transfected cells. Bcl-2 siRNA only induced apoptosis in about 15% of the cells. The combination of wt1 and bcl-2 siRNA had no additive effect on the induction of apoptosis. The expression of wt1 seems to be more important for cell survival than expression of the anti-apoptotic gene bcl-2. We therefore consider siRNA targeting human wt1 as possible tool against leukemic cells overexpressing wt1. No significant financial relationships to disclose.

scholarly journals Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 100-106 ◽  
Author(s):  
HN Steinberg ◽  
AS Tsiftsoglou ◽  
SH Robinson
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Cell Phenotype ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Leukemic Cell Lines

Abstract The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.

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.

Priming Effect with G-CSF Enhances In Vitro Apoptosis by Treatment with Ara-C and VP-16 in Leukemic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4333-4333
Author(s):  
Jun-ichi Kitagawa ◽  
Takeshi Hara ◽  
Hisashi Tsurumi ◽  
Nobuhiro Kanemura ◽  
Masahito Shimizu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Priming Effect ◽  
Low Dose ◽  
Combined Treatment ◽  
Annexin V ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Significant Difference

Abstract Introduction: We have recently reported that the effectiveness of low dose Ara-C, VP-16 and G-CSF (AVG therapy) for elderly AML patients who were ineligible for intensive chemotherapy (Hematol Oncol, in press). G-CSF has been reported to potentiate in vitro anti-leukemic effect of Ara-C. The mechanism of the potentiation is assumed to recruit quiescent G0 leukemic cells into cell cycle. We hypothesized that the enhanced cytotoxicity was due to the apoptosis by the effect of the priming of G-CSF, and the effect was depended on the cell cycle. In order to afford proof of this hypothesis, we assayed proliferation, apoptosis, and cell cycle in leukemic cell lines. Materials: Ara-C, VP-16, G-CSF was provided by Nippon Shinyaku, Nihonkayaku, Chugai pharmacy, respectively, Tokyo, Japan. 32D and HL-60 were obtained from RIKEN Bioresource Center Cell Bank (Ibaragi, Japan), Ba/F3 was generous gifts from Dr. Kume, Jichi medical school, Tochigi, Japan. Methods: 5 x 105/ml HL60, 32D and Ba/F3 were cultured with various concentrations of Ara-C and/or VP-16 in the presence or absence of G-CSF 50ng/ml for 3 days. At the end of the culture, cell proliferation and viability were determined by using the trypan blue. The Annexin V-binding capacity of treated cells was examined by flow cytometry using ANNEXIN V-FITC APOPTOSIS DETECTION KIT I purchased from BD Pharmingen™. Cell cycle analysis was done with BrdU Flow KIT purchased from BD Pharmingen™. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies, and the levels of cell-associated BrdU are then measured by flow cytometory. Result: Ara-C and VP-16 inhibited proliferation and decreased viability of leukemic cell lines dose-dependently. Half killing concentration (IC50) was redused in combination of Ara-C and VP-16 than Ara-C or VP-16 alone. In G-CSF dependent cell line (32D), IC50 was redeced in the presence of G-CSF than absence of G-CSF at G-CSF, and there was no significant difference between with and without G-CSF in G-CSF independent cell lines (HL-60, Ba/F3) (p<0.05). In combined treatment of low dose Ara-C (10−7M) and VP-16 (10−7M), the percentage of apoptotic cells were increased to 20.67% from 13.04% by addition of G-CSF in 32D, and there was no significant differencebetween with and without G-CSF in HL-60 and Ba/F3 (p<0.05). At combined treatment of low dose Ara-C and VP-16, the percentage of G0/G1 phase cells were decreased to 43.94% from 35.63% and S phase cells were increased to 29.50% from 24.05% in 32D by addition of G-CSF, and there was no significant difference between with and without G-CSF in HL-60 and Ba/F3 (p<0.05). Discussion: We first showed a combination effect of Ara-C and VP-16. Next we demonstrated that the potentiation of the cytotoxicity was mediated through the mechanism of apoptosis, and apoptosis played an important role for eradicating leukemic cells by low dose Ara-C and VP-16. And G-CSF recruited cells G0/G1 phase into S phase in G-CSF dependent cells by addition of G-CSF. These results suggest that priming effect of G-CSF significantly potentiate the cytotoxicity mediated by AVG chemotherapy. Conclusion: The priming effect of G-CSF might be admitted at least of a part in AML cells.

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.

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.

Dasatinib Is An Effective Inhibitor of Proliferation and Inducer of Apoptosis in the KASUMI Cell Line Bearing the T(8;21)(q22;q22) and the N822K KIT Mutation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5054-5054
Author(s):  
Vassiliki Pappa ◽  
F. Kontsioti ◽  
E. Liakata ◽  
S Papageorgiou ◽  
A. Spathis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cell Lines ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Kit Mutation ◽  
Free Survival ◽  
Leukemic Cell Lines ◽  
Imatinib Resistant ◽  
Proliferation And Apoptosis

Abstract Introduction. Within the group of core binding factor (CBF) AML, the presence of the t(8;21)(q22;q22) confers a favorable prognosis based on high complete remission rates and high survival probabilities. However within this subgroup the presence of KIT mutations and in some studies specifically mutations at codon 816 in exon 17 have been associated with inferior event free survival, relapse free survival, cumulative incidence of relapse and overall survival. Dasatinib a dual SRC/ABL kinase inhibitor is an active agent already approved for the treatment of imatinib resistant or intolerant chronic myelogenous leukemia which has shown in vitro activity against KIT exon 17 mutations including the D816 imatinib resistant mutation. The aim of the present study was the investigation of the activity of dasatinib on cell proliferation and apoptosis of leukemic cell lines with or without KIT mutations. Materials and methods. The leukemic cell lines ME-1, NB4 and KASUMI were cultured in RPMI. Following RNA extraction RT-PCR was performed for the amplification of the extracellular (exon 8,9), transmembrane/juxtamembrane (exon 10,11) and tyrosine kinase 2 domains (exon 17,18) of c-Kit.Following sequencing only the KASUMI cell line derived from a t(8;21)(q22;q22) AML was found to bear the N822K KIT mutation at exon 17, also described in patients’samples. The KASUMI, the K562 cell line bearing the t(9;22) used as a positive control and the NB4 cell line without KIT mutations used as a negative control, were subsequently cultured under the presence of dasatinib at the concentrations of 1nM, 10nM, 100nM, 500 nM. Cell proliferation, was determined at 24, 48, 72 h using the Cell Proliferation Elisa, BrDU protocol and apoptosis was determined by the method of annexin using flow cytometry at the same time points. Results The BrDU value of K562 cells at 48h without the drug was 1.046 significantly higher compared to those of cells cultured under the presence of Dasatinib at 1nM, 10nM, 100nM, 500 nM (0.6485, 0,5647, 0,4770, 0.4755 respectively) (p&lt;0.001). Similarly the BrDU value of K562 cells without the drug at 72h was 1.320 significantly higher to those under the presence of the drug at 10, 100, 500 nM (0.8137, 0.7292, 0.6637 respectively) (p&lt;0.001). The level of apoptosis was significantly induced by the drug at all concentrations at 24h(p&lt;0.001) and at the concentrations of 10nM, 100nM, 500 nM at 48h (p&lt;0.001) but not at 72h.Ôhere was no effect of the drug on the proliferation and apoptosis of the NB4 cell line. In the KASUMI cells there was a significant reduction of the BrDU values by the presence of dasatinib at the concentrations of 10nM, 100nM, 500nM at 48h (0.9517 vs 0.6462, 0.5653, 0.3467, p=0.038, 0.011, 0.002 respectively). The same was true at the concentrations of 100nM and 500nM at 72h (0.9538 vs 0.2412, 0.1907, p=0.002, 0.004 respectively). Dasatinib significantly increased the level of apoptosis of the KASUMI cells at 24h at 1nM, 10nM, 100nM (2.45 vs 1.41, 1.71, 2.18, p&lt;0.001, &lt;0.001, 0.026 respectively) At 48h dasatinib significantly increased the level of apoptosis at the concentrations of 1nM, 10nM, 100nM (0.84vs 1.03, 1.49, 2.81, p=0.02, p&lt;0.001, p&lt;0.001 respectively). At 72h there was a significant induction of apoptosis by the drug at all concentrations (0.16 vs 1.11, 1.94, 2.93, 1.88 p&lt;0.001) Conclusion. Dasatinib is an effective suppressor of proliferation and inducer of apoptosis of the KASUMI cell line with the t(8;21)(q22;q22) and the N822K KIT mutation. These encouraging results need to be confirmed on patients’ cells with the view to integrate the drug in conventional chemotherapy regimens in future clinical trials.

Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Induces Apoptosis In Leukemic Cells through Mitochondria-Dependent Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2889-2889
Author(s):  
Mohammad Reza Mirlashari ◽  
Ingrid Randen ◽  
Jens Kjeldsen-Kragh
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Cell Types ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
Leukemic Cell Lines ◽  
Cell Death Pathway ◽  
Gsk 3Β

Abstract Abstract 2889 GSK-3β is a multifunctional kinase that plays a role in several signaling pathways. Due to the contradictory roles of GSK-3β as a mediator of both cell survival and apoptosis, we have examined the role of GSK-3β for proliferation and apoptosis in leukemic cell lines KG1a, K562 and CMK. GSK-3β was selectively inhibited by the small-molecule SB-415286. Treatment of leukemia cells with SB-415286 (40 μM) for 72 hr approximately halved cell growth in all three cell lines. SB-415286 also showed a concentration-dependent stabilization of intracellular β-catenin: In KG1a cells the mean fluorescence intensity (MFI) [± 95% CI] was 3.1 [± 1.7] in untreated cells vs. 423 [± 24] in treated cell. The figures for the K562 and CMK cell lines were: 2.8 [± 1.6] vs. 353.2 [± 11.1], and 6.8 [± 4.0] vs. 320.2 [± 23.7], respectively. Cell cycle analysis was carried out to examine if the growth inhibition was caused by arrest in cell cycle and/or induction of apoptosis. We found that SB-415286 caused cell cycle arrest in the G2/M phase and accumulation of events corresponding to the subG1 phase, indicative of DNA fragmentation. The subG1 population was 45%, 34% and 17% in KG1a, K562 and CMK cells, respectively. To confirm that the increase of the subG1 fraction represented an apoptotic effect of the GSK-3β inhibition, we analyzed phosphatidylserine (PS) externalization and plasma membrane integrity. We found that SB-415286 caused a considerable increase of the proportion of early apoptotic cells, i.e. cells that were annexin V-positive and 7-AAD-negative: Mean [± 95% CI] in KG1a cells increased from 6.2% [± 1.2%] in untreated cells to 38% [± 3.1%] in treated cells. The figures for the K562 and CMK cell lines were: 3.0% [± 1.2%] vs. 29% [± 3.3%], and 3.9% [± 1.0%] vs. 16.0% [± 1.1%], respectively. Apoptosis signaling can be initiated by extracellular (death receptor) and/or intracellular (mitochondrial) signals. Flow cytometric analysis of cells stained by a dual-fluorescent mitochondrial dye JC-1 showed that 5–11% of untreated leukemic cells had low mitochondrial membrane potential. After 72 hr exposure to SB-415286 the mean [±95% CI] loss of the mitochondrial potential was found in 23% [± 2.0%], 33% [± 3.5%] and 42% [± 3.8%], in CMK, K562 and KG1a cells, respectively. Since drug treatment in some cell types may result in activation of both the intrinsic or extrinsic cell-death pathway in a parallel manner, we investigated if the external pathway is involved in SB-415286-induced apoptosis. For this purpose we assessed caspase-8 activation by flow cytometry. After 72 hr of treatment of CMK, K562 and KG1a cells the caspase-8 activities compared, to untreated cells, had increased 3.7-fold, 3.9-fold, and 4.4-fold, respectively. In some cell types, the extrinsic cell-death pathway leads to the cleavage of Bid (pro-apoptotic member of the Bcl-2 family) by caspase-8, generating a truncated version of the protein (tBid) which in turn activates the mitochondrial apoptotic pathway. Therefore, we determined whether depolarization of the mitochondrial membrane in the leukemic cell lines was an effect of activated caspase-8 or a direct effect of SB-415286. For this purpose Z-IETD-FMK (25 μM), a specific inhibitor of caspase-8, was applied to the cells for 2 hr. We found that inhibition of caspase-8 did not prevent SB-415286-induced apoptosis assessed by PS externalization. This indicates that activation of caspase-8 is part of the intrinsic apoptotic pathway and occurs downstream of mitochondria membrane potential depolarization mediated by other caspases. Taken together, our observations suggest that inhibition of GSK-3β induces apoptosis of leukemic cells by depolarizing the mitochondria membrane. Thus, inhibition of GSK-3β could be an attractive target for treatment of leukemia. Disclosures: No relevant conflicts of interest to declare.

EphrinB1 Activation As a Potential New Treatment Option in AML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5235-5235
Author(s):  
Arja ter Elst ◽  
Kim R Kampen ◽  
Sander H Diks ◽  
Steven M. Kornblau ◽  
Guillermo Garcia-Manero ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Hl60 Cells ◽  
Ephrin Receptors ◽  
Apoptotic Protein ◽  
Ephb Receptors ◽  
New Treatment

Abstract Abstract 5235 Aberrant Ephrin signaling has been shown to be an important pathway that contributes to the pathogenesis of many solid tumors (Surawska et al. Cytokine & Growth factor reviews 2004). Deregulated ephrin receptor (Eph) and ligand (Efn) expression is often associated with poor prognosis in solid tumors. Ephrin receptor and ligand overexpression can result in tumorigenesis through induced tumor growth, tumor cell survival, angiogenesis and metastasis (Surawska et al. Cytokine & Growth Factor Reviews 2004; Campbell et al. Curr. Isues Mol. Biol. 2008; Chen et al. Cancer Research 2008). In normal cells Eph receptors and ligands play key roles in vascular patterning, where they function in endothelial cell migration, and proliferation (Adams et al, Genes Dev. 1999; Zhang et al., Blood 2001). Thus far particularly EphB4 receptor and ephrin-B2 ligand have been implicated in the process of normal angiogenesis. In acute myeloid leukemia (AML) patients it was found that bone marrow biopsies at diagnosis exhibited enhanced microvessel density (MVD) (de Bont ES et al., BJH 2001; Byrd JC et al., Blood 2002; Padro et al., Blood 2000). Normal hematopoietic stem cells (HSCs) express the following mRNA transcripts ephrin receptors EphA1, EphA2, EphB2, and EphB4 and ephrin ligands EfnA3, EfnA4, and EfnB2. Moreover, overexpression of EphB4 receptor in HSCs (from cord blood) resulted in enhanced differentiation towards megakaryocytes (Wang et al. Blood 2002). In AML cell lines there is a common co-expression on protein level observed between EphB4 receptor and ephrin-B2 ligand. Recently, an aberrant DNA methylation of ephrin receptors and ligands was described in acute lymphocytic and myelocytic leukemia cell lines (Kuang et al. Blood 2010). In addition, restoration of EphB4 expression in an acute lymphoid leukemia cell line resulted in reduced proliferation and apoptotic cell death. These data suggests that the ephrin signaling pathway might play an important role in leukemia. In a previous study we have found high kinase activity of EphB receptors and high phosphorylation levels of EphB receptors in AML samples, as measured using kinase arrays and proteome profiler arrays. In this study, we have found extensive membrane expression of EphB1 on AML cell lines and primary AML blasts. To identify the role of Ephrin signaling in AML, two AML cell lines THP-1 and HL60 with an EphB1 membrane expressing cell percentage of 70% and 20% respectively were chosen for stimulation with Ephrin-B1 ligand. Treatment of these cell lines with Ephrin-B1 ligand resulted in a decreased proliferation 30% in THP-1 cells versus 22% in HL60 cells and increased apoptosis 23% in THP-1 cells and 4% in HL60 cells. Of note, the most prominent effect of Ephrin-B1 stimulation was found in THP-1 cells, this cell line contained a higher percentage of EphB1 membrane expressing cells. We further investigated the mechanism through which EphB1 reduces leukemic cell growth and induces leukemic cell death in THP-1 cells. Westernblot analysis of cell cycle regulators showed that expression of the anti-apoptotic protein BCL2 is reduced upon Ephrin-B1 ligand stimulation and the expression of the pro-apoptotic protein BAX is induced. In addition, mRNA expression of the cell cycle inhibitor of cell cycle progression p21 was found to be 2,5 fold upregulated in ephrin-B1 ligand treated cells compared to untreated control cells. MGG stainings of Ephrin-B1 treated cells revealed multiple cells with two nuclei in both THP-1 and HL60 cells. These results indicate that a high percentage of AML cells express EphB1 receptor on the membrane and that stimulation of these cells with Ephrin-B1 ligand results in reduced leukemic growth and increased cell death. EphrinB1 activation in AML deserves further investigation considering EphB1 as a putative new treatment option for AML patients. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document