Identification of Target Genes of an Erythroid Transcription Factor Complex Containing SCL (TAL1).

Abstract Haematopoiesis is the process whereby haematopoietic stem cells give rise to mature blood cell lineages. The SCL (TAL1) gene, originally identified by chromosomal translocations associated with T-cell acute lymphocytic leukaemia, encodes a key transcription factor (TF) which is expressed in various blood lineages and is essential for haematopoietic development. It has been shown that the SCL protein forms a multi-protein complex during erythroid development with other TFs (GATA1, E2A, LDB1, and LMO2) which binds to a sequence-specific motif to regulate the expression of glycophorin A and c-kit. We have used two complementary approaches to identify novel target genes regulated by this TF complex during erythroid development. In the first approach, we have transfected short interfering RNAs (siRNAs) into the K562 cell line to knockdown transiently the TFs of the erythroid complex. For all members of the complex, a knockdown efficiency of at least 70% was confirmed at the mRNA and protein level within 48 hours after transfection. The consequences of the knockdown at the level of gene expression were observed using Affymetrix GeneChips in order to identify downstream events associated with the erythroid complex in transcriptional programmes. In the second approach, chromatin immunoprecipitation (ChIP) was performed for each member of the complex in the K562 cell line and the ChIP material hybridised to a human transcription factor promoter microarray. A number of novel target genes for the SCL erythroid complex have been identified and verified independently using both approaches. Our data shows that members of the erythroid complex are involved in auto-regulation and regulate genes which control chromatin structure and function. These findings demonstrate that the expression of this TF complex is tightly controlled and point to an important role for it in orchestrating fundamental biological processes which have profound effects on gene expression in erythroid development.

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miR-101 sensitizes K562 cell line to imatinib through Jak2 downregulation and inhibition of NF-κB target genes

Tumor Biology ◽

10.1007/s13277-016-5205-9 ◽

2016 ◽

Vol 37 (10) ◽

pp. 14117-14128 ◽

Cited By ~ 15

Author(s):

Elham Farhadi ◽

Farhad Zaker ◽

Majid Safa ◽

Mohammad Reza Rezvani

Keyword(s):

Cell Line ◽

K562 Cell ◽

Target Genes ◽

K562 Cell Line

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7-Isopenthenyloxycoumarin, Arctigenin, and Hesperidin Modify Myeloid Cell Leukemia Type-1 (Mcl-1) Gene Expression by Hormesis in K562 Cell Line

Dose-Response ◽

10.1177/1559325818796014 ◽

2018 ◽

Vol 16 (3) ◽

pp. 155932581879601 ◽

Cited By ~ 7

Author(s):

Zahra Kafi ◽

Hamid Cheshomi ◽

Omid Gholami

Keyword(s):

Gene Expression ◽

Cell Line ◽

Dose Response ◽

K562 Cell ◽

Myeloid Cell ◽

Response Relationship ◽

Cell Leukemia ◽

Dose Response Relationship ◽

K562 Cell Line

Hormesis is a new concept in dose–response relationship. Despite of traditional dose–response curves, there is a low-dose stimulation and a high-dose inhibition in this case. Hormesis effect in apoptosis induction/inhibition by natural compounds is reported previously. Here, we searched this effect for myeloid cell leukemia type-1 (Mcl-1) gene expression by phytochemicals 7-isopenthenyloxycoumarin (7-IP), arctigenin (Arg), and hesperidin (Hsp). For this purpose, first we tested the cytotoxicity of various doses of these compounds against K562 leukemia cell lines for different times by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. After that we explored the effect of various doses of these phytochemicals on Mcl-1 gene expression for different times by real-time polymerase chain reaction method. We found that these phytochemicals have cytotoxicity against K562 cell line. Hesperidin is the most cytotoxic agent. We also found that these natural compounds have hormetic effect on Mcl-1 gene expression. The hormetic model in Mcl-1 gene expression is overcompensation stimulation. This phenomenon is reported for the first time. We conclude that 7-IP, Arg, and Hsp are cytotoxic against K562 cancerous cells and induce/inhibit Mcl-1 gene expression by hormesis dose–response relationship.

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Imatinib-induced apoptosis could be enhanced by Triptolide in Imatinib-resistant chronic myeloid leukemia cell line through down-regulation of HIF-1αand Nrf2

Blood ◽

10.1182/blood.v122.21.4928.4928 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4928-4928

Author(s):

Bing Xu ◽

Feili Chen ◽

Yuejian Liu ◽

Shiyun Wang ◽

Rongwei Li ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Cell Line ◽

K562 Cell ◽

Myeloid Leukemia ◽

Target Genes ◽

Leukemia Cell Line ◽

K562 Cell Line ◽

Down Regulation ◽

G Cells ◽

Induced Apoptosis

Abstract Background The emergence of Imatinib has brought a new era for the treatment of chronic myeloid leukemia. However, resistant to Imatinib might lead to the treatment failure and death of patients. Thus, finding a drug which could enhance the Imatinib-induced apoptosis in vitro could provide the experimental base for the treatment of chronic myeloid leukemia. K562 cell line is a common cell line used in the study of chronic myeloid leukemia while K562/G cell line which is resistant to Imatinib is derived from K562 cell line. Aim This study aims to explore whether low-dose Triptolide(TPL) could enhance the Imatinib-induced apoptosis in K562/G cells and related mechanism. Methods K562/G cells were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes. Results Triptolide is highly cytotoxic to K562/G cells in a concentration-dependent manner. To determine the combination effect of TPL and anticancer agents, K562/G cells were exposed to Imatinib(50μM) with or without TPL (25nM) for 24h. The apoptotic cells were determined by PI/Annexin V staining and flow cytometric analysis. Apoptotic ratio of cells treated by Imatinib together with TPL is significantly increased compared to cells treated by Imatinib alone (24.78±1.12 vs. 77.52±7.75, P<0.01). Next, the underlying mechanism was investigated. High expression of Hypoxia-inducible factor-1α (HIF-1α) has been shown to be related with bad clinical outcome in patients while Nrf2 is responsible for chemo-resistanct in cancer cells. Imatinib in combination with TPL could decrease Nrf2 and HIF-1α expression at protein and mRNA levels. Down-stream genes of Nrf2 e.g NQO1, GSR and HO-1 as well as target genes of HIF-1α e.g BNIP3, VEGF and CAIX are also down-regulated at mRNA level. Conclusion TPL could significantly increase the Imatinib-induced apoptotic ratio in K562/G cells through down-regulation of HIF-1α and Nrf2. Disclosures: No relevant conflicts of interest to declare.

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All-trans retinoic acid enhances the effect of 5-aza-2′-deoxycytidine on p16INK4a demethylation, and the two drugs synergistically activate retinoic acid receptor β gene expression in the human erythroleukemia K562 cell line

Oncology Letters ◽

10.3892/ol.2014.2133 ◽

2014 ◽

Vol 8 (1) ◽

pp. 117-122 ◽

Cited By ~ 8

Author(s):

LILI XIANG ◽

WEIMIN DONG ◽

RONG WANG ◽

JIANG WEI ◽

GUOQIANG QIU ◽

...

Keyword(s):

Gene Expression ◽

Retinoic Acid ◽

Cell Line ◽

K562 Cell ◽

Retinoic Acid Receptor ◽

K562 Cell Line ◽

Acid Receptor ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid

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Gene Expression Profile Regulated by CREB in K562 Cell Line

Transplantation Proceedings ◽

10.1016/j.transproceed.2016.04.023 ◽

2016 ◽

Vol 48 (6) ◽

pp. 2221-2234 ◽

Cited By ~ 2

Author(s):

H. Xia ◽

Z. Gong ◽

Y. Lian ◽

J. Zhou ◽

X. Wang

Keyword(s):

Gene Expression ◽

Cell Line ◽

Gene Expression Profile ◽

Expression Profile ◽

K562 Cell ◽

K562 Cell Line

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The Transcription Factor SCL/TAL-1 Plays a Positive Role in the Erythropoietic Differentiation Via MEK/ERK Pathway in EPO-Induced K562 Cell Line

Blood ◽

10.1182/blood.v118.21.4799.4799 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4799-4799

Author(s):

Yuping Gong ◽

Ruiqing Zhou ◽

Ting Niu

Keyword(s):

Transcription Factor ◽

Cell Line ◽

K562 Cell ◽

K562 Cells ◽

Erythroid Differentiation ◽

Mrna Levels ◽

Erk Pathway ◽

Rt Pcr ◽

K562 Cell Line ◽

Positive Role

Abstract Abstract 4799 Objective In the present study, EPO-induced K562 cell line was used to be the cell model of erythroid differentiation, the role and mechanism of transcript factor SCL/TAL-1 in the erythropoiesis was investigated. Methods Three plasmids, which included pTRIPdU3-RNAiTALh -EF1a-GFP (SCL/TAL1 shRNA to reduce the expression of SCL/TAL-1), pTRIP-EF1a-TAL1 (SCL/TAL-1 cDNA to enhance the expression of SCL/TAL-1) and control plasmid pTRIP-dU3-RNAiluc-EF1-GFP expressing EGFP gene, were transfected into K562 cell line via lentiviral vector system, and K562 SCL/TAL-1low, K562 SCL/TAL-1high and K562 LUC (control) were established and the effect of reducing or enhancing the expression of SCL/TAL-1 on the erythropoiesis of these three cell lines was investigated. After incubated with EPO-RPMI1640 medium in which EPO induced K562 cell line into erythropoiesis for 5 day, the mRNA levels of SCL/TAL-1 and erythroid related RhD, GPA, CD47 were detected by RT-PCR assay and erythroid antigen CD71, CD235a were examined by flow cytometry in the three cell lines. Effect of SCL/TAL-1 on key phosphorylated proteins, including p-PTEN, p-Akt, p-mTOR, p-P70 and p-4EBP-1 from PI3K/Akt/mTOR pathway and p-c-Raf, p-MEK and p-ERK1/2 from Raf/MEK/ERK pathway, in the downstream of EGFR signaling pathway were checked by Western Blot assay. Effect of MEK-ERK 1/2 inhibitor U0126 on the expression of SCL/TAL1 also examined. Results 1. After 48h of transfect, more than 95% of K562 cells were GFP positive under the fluorescence microscope, indicating that infection rate of the plasmids in the K562 cells was more than 95%. 2. The results of RT-PCR showed SCL/TAL-1 mRNA expression in the K562 SCL/TAL-1low was significantly lower than that in the K562 LUC control (P <0.05). The mRNA levels of CD47 and RhD was also significantly lower and however, GPA just decreased slightly in comparison with the control. The mRNA levels of above erythroid antigens increased a little in K562-SCL/TAL-1high. 3. The FCM results showed the expression of CD71, CD235a obviously reduced in the K562 SCL/TAL-1low and positive rates were 10.4% and 76.5%, while the positive rates in the LUC control were 94.3% and 83.6%. The expression of CD71 and CD235a in K562-SCL/TAL-1high was similar to the control. 4. The level of p-MEK and p-ERK1/2 increased with transfect of SCL/TAL-1 cDNA and decreased after SCL/TAL-1 RNA interference. However, there were no obvious changes to be observed in PI3K-Akt-mTOR pathway, another important signal pathway. 5. There was no obvious alteration in SCL/TAL-1 level after treatment of MEK-ERK1/2 inhibitor, although MEK-ERK1/2 level reduced. Conclusion Our findings suggest that transcription factor SCL/TAL-1 plays a positive role in erythroid differentiation in EPO-induced K562 cell line. SCL/TAL-1 is located in the upstream of MEK-ERK1/2 and may regulate erythroid differentiation by affecting the phosphorylation levels of MEK-ERK1/2 pathway. Grant support: National Natural Science Foundation of China (No.30770912), Foundation of the Science & Technology Department of Sichuan Province (No.2008SZ0017). Disclosures: No relevant conflicts of interest to declare.

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Zinc-sensitive genes as potential new target genes of the metal transcription factor-1 (MTF-1)

Biochemistry and Cell Biology ◽

10.1139/o04-133 ◽

2005 ◽

Vol 83 (2) ◽

pp. 221-229 ◽

Cited By ~ 12

Author(s):

Birgit Kindermann ◽

Frank Döring ◽

Jan Budczies ◽

Hannelore Daniel

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Steady State ◽

Cell Line ◽

Target Genes ◽

Complement Factor ◽

Mrna Levels ◽

Control Of Gene Expression ◽

Conditional Expression ◽

Ht 29

Zinc is an essential trace element that serves as a structural constituent of a large number of transcription factors, which explains its pivotal role in the control of gene expression. Previous studies investigating the effect of zinc deficiency and zinc supplementation on gene expression in the human adenocarcinoma cell line HT-29 led to the identification of a considerable number of genes responding to alterations in cellular zinc status with changes in steady state mRNA levels. For 9 of 20 genes from these previous screenings that were studied in more detail, mRNA steady state levels responded to both high and low media zinc concentrations. As they are primarily zinc-dependent, we assessed whether these genes are controlled by the zinc-finger metal transcription factor MTF-1. To test this hypothesis we generated a doxycyline-inducible Tet-On HT-29 cell line overexpressing MTF-1. Using this conditional expression system, we present evidence that Kruppel-like factor 4 (klf4), hepatitis A virus cellular receptor 1 (hhav), and complement factor B (cfbp) are 3 potential new target genes of MTF-1. To support this, we used in silico analysis to screen for metal-responsive elements (MREs) within promotors of zinc-sensitive genes. We conclude that zinc responsiveness of klf4, hhav, and cfbp in HT-29 cells is mediated at least in part by MTF-1.Key words: zinc-sensitive genes, target genes, MTF-1, HT-29 cells, metal-response element.

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A New Series of Antileukemic Agents: Design, Synthesis, In Vitro and In Silico Evaluation of Thiazole-Based ABL1 Kinase Inhibitors

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200824100408 ◽

2020 ◽

Vol 20 ◽

Author(s):

Ebru Zeytün ◽

Mehlika D. Altıntop ◽

Belgin Sever ◽

Ahmet Özdemir ◽

Doha E. Ellakwa ◽

...

Keyword(s):

Molecular Docking ◽

Anticancer Activity ◽

Cell Line ◽

Antiproliferative Activity ◽

In Silico ◽

K562 Cell ◽

Kinase Inhibitors ◽

K562 Cell Line ◽

Cytotoxic Effects ◽

Atp Binding Site

Background: After the milestone approval of imatinib, more than 25 antitumor agents targeting kinases have been approved, and several promising candidates are in various stages of clinical evaluation. Objectives : Due to the importance of thiazole scaffold in targeted anticancer drug discovery, the goal of this work is the design of new thiazolyl hydrazones as potent ABL1 kinase inhibitors for the management of chronic myeloid leukemia (CML). Methods: New thiazolyl hydrazones (2a-p) were synthesized and investigated for their cytotoxic effects on K562 CML cell line. Compounds 2h, 2j and 2l showed potent anticancer activity against K562 cell line. The cytotoxic effects of these compounds on other leukemia (HL-60, MT-2 and Jurkat) and HeLa human cervical carcinoma cell lines were also investigated. Furthermore, their cytotoxic effects on mitogen-activated peripheral blood mononuclear cells (MA-PBMCs) were evaluated to determine their selectivity. Due to its selective and potent anticancer activity, compound 2j was benchmarked for its apoptosis-inducing potential on K562 cell line and inhibitory effects on eight different tyrosine kinases (TKs) including ABL1 kinase. In order to investigate the binding mode of compound 2j into the ATP binding site of ABL1 kinase (PDB: 1IEP), molecular docking study was conducted using MOE 2018.01 program. The QikProp module of Schrödinger’s Molecular modelling package was used to predict the pharmacokinetic properties of compounds 2a-p. Results: 4-(4-(Methylsulfonyl)phenyl)-2-[2-((1,3-benzodioxol-4-yl)methylene)hydrazinyl]thiazole (2j) showed antiproliferative activity against K562 cell line with an IC50 value of 8.87±1.93 µM similar to imatinib (IC50= 6.84±1.11 µM). Compound 2j was found to be more effective than imatinib on HL-60, Jurkat and MT-2 cells. Compound 2j also showed cytotoxic activity against HeLa cell line similar to imatinib. The higher selectivity index value of compound 2j than imatinib indicated that its antiproliferative activity was selective. Compound 2j also induced apoptosis in K562 cell line more than imatinib. Among eight TKs, compound 2j showed the strongest inhibitory activity against ABL1 kinase enzyme (IC50= 5.37±1.17 µM). According to molecular docking studies, compound 2j exhibited high affinity to the ATP binding site of ABL1 kinase forming significant intermolecular interactions. On the basis of in silico studies, this compound did not violate Lipinski's rule of five and Jorgensen's rule of three. Conclusion: Compound 2j stands out as a potential orally bioavailable ABL1 kinase inhibitor for the treatment of CML.

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