Low Expression of hOCT1 May Be a Key Point Mediating Low Intracellular Imatinib Accumulation in Imatinib Mesylate Resistance K562 Cell Line.

Abstract Objective To establish an imatinib resistance cell line and to study its resistant principia. Methods K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. Clone imatinib resistance cell lines by limited dilution culture. MTT assay, real time PCR and Semi-quantity PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance. Results Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5μmol/L. K562R cells were maintained in the media containing 5μmol/L imatinib. Proliferation data showed that cell growth of K562R was not inhibited in 5 μmol/L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2μM imatinib. The IC50 of K562R was about 7.5μmol/L which was ten times higher than that of the parental cell. HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27.8-fold). MDR1 were not detected in mRNA (by RT-PCR)and protein(by flow cytometry) levels on K562R cell, whereas hOCT1 level measured by semi-quantity PCR showed lower expression in K562R cell lines than that of parental sensitive cell, indicating that low intracellular imatinib concentration may be due to lower affluence of imatinib by low level of hOCT1. (5) Real time PCR analysis showed no BCR-ABL/G6PD gene amplification and sequence analysis of the 374bp ABL kinase domain showed no mutation in K562R cell lines. Conclusion An imatinib resistance cell line K562R has been successfully established. Low expression of hOCT1 may be a key point mediating low intracellular imaitnib accumulation in K562R cell lines.

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Long-Term Increase in Fetal Hemoglobin Expression in Nonhuman Primates Following Transplantation of Autologous Bcl11a Nuclease-Edited HSCs

Blood ◽

10.1182/blood.v126.23.2035.2035 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2035-2035 ◽

Cited By ~ 2

Author(s):

Olivier Humbert ◽

Hans-Peter Kiem

Keyword(s):

Flow Cytometry ◽

Real Time ◽

Real Time Pcr ◽

Peripheral Blood ◽

Nonhuman Primates ◽

Pcr Analysis ◽

Beta Globin ◽

Gamma Globin ◽

Cd34 Cells

Abstract Elevated levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms of beta-thalassemia and sickle cell anemia. The transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is required for silencing of gamma-globin expression in adult erythroid cells and functions as a switch from fetal to adult hemoglobin production in humans. BCL11A therefore constitutes a therapeutic target for the treatment of hemoglobinopathies. We inactivated BCL11A function by double-strand DNA break-induced mutagenesis using Transcription Activator-Like Effector Nucleases (TALENs). 20 to 30% gene editing could be achieved in vitro in human and nonhuman primate CD34+ cells by TALEN mRNAs electroporation targeting exon 2 of Bcl11a. Colony-forming efficiency was slightly lower in Bcl11a-edited CD34+ cells but lineage differentiation potential was unchanged. Erythroid differentiation of CD34+ cells in culture showed increased Fetal to Beta hemoglobin ratio in both human and primate Bcl11a-modified cells as compared to control cells, thus validating our editing approach to increase HbF production. To determine if Bcl11a-edited hematopoietic stem cells (HSCs) could be engrafted and give rise to HbF-producing erythrocytes, we transplanted a pigtail macaque with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. We detected about 1 % gene disruption in vivo early post-transplant and disruption frequency gradually declined to reach a set point of about 0.3% starting at day 28 post-transplantation. In this analysis, which we have so far taken out to 42 days, single clones could be tracked based on their mutation signature, and we found that several clones persisted over time, confirming engraftment of Bcl11a-modified cells. Since the transplantation procedure and chemo-radiotherapy conditioning can raise HbF production, three control animals that were transplanted using similar conditions as with the Bcl11a-edited HSCs and one untransplanted animal were also included in our analysis. Flow cytometry measurement of HbF in peripheral blood showed a rapid increase in F-cell production in all animals, reaching levels that ranged from 10% to 40% by 30 days, while the untransplanted control showed basal HbF expression of about 0.5% (Fig. 1A). The peak for HbF expression lasted for about 140 days and eventually returned to basal levels that averaged 0.5% for all control animals. In comparison, the animal transplanted with Bcl11a-edited cells showed significantly higher HbF levels starting at day 140 post-treatment (1-1.5%), and HbF production has remained constant for at least 150 days. This result was confirmed by hemoglobin mRNA analysis in peripheral blood using real-time PCR. We found a rapid increase in gamma globin expression following transplantation, before returning to near basal levels. As compared to controls, the animal transplanted with Bcl11a-edited cells showed a 5 to 10-fold increase in gamma to beta globin ratio at day 140 and this ratio has remained constant ever since (Fig. 1B). We are currently working on ways to enhance Bcl11a-editing and to select for Bcl11a-modified HSCs using targeted integration of the chemoselection cassette P140K MGMT to ultimately achieve curative HbF production. Potential TALEN off-target sites will also be examined as well as any side effect associated with the inactivation of BCL11A. Overall, our data demonstrate that transplantation of Bcl11a-edited HSCs results in elevated HbF production in nonhuman primates. Furthermore, we show that nonhuman primates can serve as a useful model for novel gene editing strategies toward the treatment of hemoglobinopathies. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Disclosures No relevant conflicts of interest to declare.

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Hypericin Exerts Detrimental Effect on Huh-7 As a Delegacy of Hepatocellular Carcinoma: A P53 Dependent Pathway

Galen Medical Journal ◽

10.31661/gmj.v9i0.1896 ◽

2020 ◽

Vol 9 ◽

pp. 1896

Author(s):

Maedeh Olya ◽

Hamid Zaferani Arani ◽

Amirhossein Shekarriz ◽

Amirhossein Zabolian ◽

Hadi Zare Marzouni ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

Liver Cancer ◽

Cell Line ◽

Real Time ◽

Real Time Pcr ◽

Huh7 Cell ◽

P53 Expression ◽

Huh7 Cells ◽

Huh7 Cell Line

Background: Hepatocellular carcinoma is the most common type of liver cancer which arises from the main cells in the liver. We address many studies investigating anti-cancer role of hypericin, however the proposing corresponding molecular pathway seems to be still a debate. Therefore, the present study aimed to evaluate the apoptotic effect of hypericin on the Huh7 as the liver cancer cell line and its relation with the gate keeper gene P53. Materials and Methods: In this study, the Huh7 cell line and fibroblast cells (as control group) were treated with different concentrations of hypericin for 24 and 48 hours. Detection of cell death was performed by MTT assay and flow cytometry. The expression of bax, bcl2 and p53 mRNAs was evaluated by Real-time PCR. Also, Immunocytochemistry (ICC) analysis was used for further evaluation of P53expression. Results: The results showed that hypericin has a dose-dependent cytotoxic effect on the Huh7 cell line, with no or marginal effect on fibroblastic cells. According to flow cytometry results, about 53%cells underwent apoptosis after exposure to LD50 of hypericin for 24 hours. Real-time PCR data demonstrated that the pro-apoptotic genes Bax and P53 expression level increased. Expectedly ICC results confirmed the up-regulation of P53 proteins in treated samples. Conclusion: Our results indicate the cytotoxicity of hypericin on Huh7 cells by affecting the expression of the gate keeper gene P53; furthermore it is suggested that this herb can be utilized simultaneously with modalities targeting P53 up-regulation or related molecular pathways. [GMJ.2020;9:e1896]

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Profiling of 95 MicroRNAs in Pancreatic Cancer Cell Lines and Surgical Specimens by Real-Time PCR Analysis

World Journal of Surgery ◽

10.1007/s00268-008-9833-0 ◽

2008 ◽

Vol 33 (4) ◽

pp. 698-709 ◽

Cited By ~ 201

Author(s):

Yuqing Zhang ◽

Min Li ◽

Hao Wang ◽

William E. Fisher ◽

Peter H. Lin ◽

...

Keyword(s):

Pancreatic Cancer ◽

Real Time ◽

Cell Lines ◽

Cancer Cell ◽

Real Time Pcr ◽

Pancreatic Cancer Cell ◽

Cancer Cell Lines ◽

Pcr Analysis

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Aldehyde Dehydrogenase (ALDH) Activity Resolves Neuroblastoma (NB) Cells and Hematopoietic Stem and Progenitor Cells (HPC) in Graft Material: Potential Method for Clinical Purging.

Blood ◽

10.1182/blood.v104.11.2869.2869 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2869-2869

Author(s):

Sandra J. Foster ◽

Lisa Winstead ◽

Timothy A. Driscoll ◽

Andrew E. Balber

Keyword(s):

Bone Marrow ◽

Real Time ◽

Tumor Cells ◽

Cell Lines ◽

Real Time Pcr ◽

Pcr Analysis ◽

Graft Material ◽

Gene Products ◽

Aldh Activity ◽

Flow Sorting

Abstract High dose, myeloablative chemotherapy followed by autologous HPC transplantation is used clinically to treat NB in children. The presence of NB cells in transplant material adversely affects outcome. Grafts have been purged by using monoclonal antibodies to select CD34+ HPC for transplantation or to remove tumor cells expressing NB surface antigens from graft material. We are exploring the feasibility of purging NB autografts by flow sorting HPC and NB cells based on differential expression of ALDH. Cell populations with high levels of ALDH activity and low side scatter (ALDHbrSSClo cells) can be identified in mobilized peripheral blood (PBSC), bone marrow (BM), and umbilical cord blood using the ALDEFLUOR™ [StemCo Biomedical, Inc.] method for flow cytometric measurement of cellular ALDH activity. ALDHbrSSClo cells isolated by flow sorting are highly enriched for HPC activity. We report here that none of three established NB cell lines (SK-N-SH, SK-N-BE(2), and IMR-32) express detectable ALDH activity in the ALDEFLUOR™ assay. Furthermore, when any of these NB lines was labeled with the stable, red fluorescent intracellular marker SNARF™ [Molecular Probes, Inc.] and then mixed with BM or PBSC, NB were readily resolved from the green fluorescent ALDHbrSSClo cells. In mixtures containing equal numbers of tumor cells and PBSC or BM cells 0–6 events were detected in the ALDHbrSSClo region out of a total of 3-10 x 105 events in the SSC vs. FSC gate. Even when mixtures containing 9 tumor cells to 1 leukocyte were used, fewer than 21 SNARF-labeled NB were detected in the ALDHbrSSClo region out of a total of 2-9 x 105events in the SSC vs. FSC gate. Additional studies were done by sorting mixtures of SNARF-labeled NB cells and PBSC or BM and measuring tumor burden post-sort. In a representative study, pre-sort NB content of 30% was reduced to 0.04% when care was taken to eliminate NB-HPC doublets by using pulse width discrimination; without this precaution, NB contamination in the sorted cells was 1.3%. RNA was prepared from pre- and post-sorted cells, and real time PCR analysis was performed using probes to NB gene products tyrosine hydroxylase, GD2 synthase and PRAME. PCR signals were readily detected in the starting sample and were reduced by >1000-fold in the sorted ALDHbrSSClo cells. To extend these findings with NB cell lines to clinical material, a bone marrow sample was obtained from a NB patient following appropriate institutional consent procedures and was analyzed by multiparameter flow analysis using ALDEFLUOR™ and a monoclonal antibody to GD2 synthase, an antigen strongly expressed by NB cells. Approximately 60% of the cells in this BM sample expressed GD2 synthase, but only 0.03% of the NB cells were detected among ALDHbrSSClo events. RNA was prepared from 100,000 flow-sorted ALDHbrSSClo cells, and real-time PCR analysis was performed. NB gene products were readily detected in the donor BMs, but no signal was detected in the sorted cell preparations. The NB burden in the flow-sorted ALDHbrSSClo population was reduced >100-fold compared to the unpurged, heavily contaminated BM. We are extending this analysis to maximize purging obtainable with clinical material. As ALDEFLUOR™ does not impair any tested functional characteristics of HPC, flow sorting based ALDH activity may provide a new method to purge autografts of NB cells for clinical transplantation.

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The Role and Mechanism Study of Lncrnas Involved in Glucocorticoid Resistance in Paediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.5136.5136 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5136-5136

Author(s):

Zhai Xiaowen ◽

Cuiqing Fan ◽

Hongsheng Wang

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Signaling Pathways ◽

Cell Line ◽

Real Time ◽

Pathway Analysis ◽

Real Time Pcr ◽

Lymphoblastic Leukemia ◽

Resistant Cell ◽

Sensitive Cell ◽

Mechanism Study

Abstract Objective: Glucocorticoids (GCs) are key components in the treatment of childhood acute lymphoblastic leukemia (ALL) and most ALL therapeutic failures can be explained by cellular resistance to GCs. However, the mechanisms of GC resistance are poorly understood. LncRNAs are involved in normal hematopoiesis and leukemia development, whereas the roles of lncRNAs in GC resistance are still unknown. Our goal was to investigate the role of lncRNAs involved in glucocorticoid resistance in paediatric acute lymphoblastic leukemia, and also elucidate the mechanism preliminarily. Methods: In this study, lncRNA microarray was performed on GC-resistant cell line CEM-C1 and GC-sensitive cell line CEM-C7 to screen the differential expression of lncRNAs. Five up-regulated and five down-regulated lncRNAs were randomly chosen for validation by Real-time PCR. GO-Pathway analysis was done to investigate potential signaling pathways regulated by the lncRNAs. Results: The microarray revealed that 4286 lncRNAs differed (p<0.05 and fold change>2.0) in GC-sensitive cell from those in GC-resistant cell. 826 lncRNAs changed more than 5-fold, while 356 lncRNAs changed more than 10-fold. Among them, 203 lncRNAs were up-regulated and 153 lncRNAs were down-regulated. Expression of ten selected lncRNAs was validated by Real-time PCR. GO analysis indicated that differentially expressed lncRNAs were involved in apoptotic process related GO terms. Pathway analysis revealed that these lncRNAs were involved in apoptosis, cell cycle, mTOR and some other signaling pathways. Conclusions: Our study showed that lncRNA expression profile was altered in GC-sensitive and GC-resistant cells, indicating that differentially expressed lncRNAs may play important functional roles in GC resistance of ALL. And these lncRNAs may be involved in GC resistance by regulating signaling pathways associated with cell proliferation, differentiation and apoptosis. Our study provides new biological foundations for further mechanism study in GC resistance and also provides a new strategy for therapeutic development of ALL. Disclosures No relevant conflicts of interest to declare.

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Autophagy Overactivity Regulate Terf1 and Terf2 in Positive and Negative-Telomerase Cancer Cell Lines

10.21203/rs.3.rs-1102455/v1 ◽

2021 ◽

Author(s):

mohammad panahi ◽

Saeed Samani ◽

Nasrin Mohajeri ◽

Akram Sadat Tabatabee Bafroee ◽

Kazem Baesi ◽

...

Keyword(s):

Flow Cytometry ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Huh7 Cell ◽

Gfp Expression ◽

Transfected Cells ◽

Autophagy Induction ◽

Huh7 Cells ◽

The Relationship

Abstract A recent suggestion for cancer therapy is targeting intracellular homeostatic signaling pathways like autophagy providing the balance between metabolism and cell cycling. Our study focused on investigating the relationship between autophagy activation by Beclin1 transfection and assessing Terf1 and Terf2 expression as shelterin proteins. The beclin1-containing plasmid was introduced to the U-2OS and Huh7 cell lines using Lipofectamine. The LC3-II as an intracellular autophagosomal marker was detected in transfected cells by flow cytometry. Also, the cells were treated with 3-methyladenine and metformin as autophagy inhibitors and inducers, respectively. Finally, the expression levels of Terf1 and Terf2 were analyzed by real-time PCR. Fluorescent images and flow cytometry results proved excellent GFP expression in the transfected cells. The results of real-time PCR demonstrated that autophagy induction by Beclin1 was increased Terf1 expression level in U-2OS cells up to 451%, while Huh7 cells suffered from the decreased expression of Terf1. Altogether, Terf2 expression was enhanced significantly in both cell lines after 48h treatment in comparison with 24h treatment. The obtained data provided that Beclin1-based activation of autophagy leads to overexpression of some protective shelterin proteins.

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