Lymphoblastic Leukemia Cell Line Jurkat Research Articles

Indigofera suffruticosa aerial parts extract induce G2/M arrest and ATR/CHK1 pathway in Jurkat cells

BackgroundIndigofera suffruticosa Mill. is used as a folk medicine for treating patients with leukemia, however very little is known regarding the molecular mechanism of its anti-leukemic activity and the chemical profile of the active extract. The present study aimed to reveal the molecular effect of I. suffruticosa aerial parts extract (ISAE) on leukemia cells and its chemical constituents.MethodsCytotoxicity of ISAE were determined by resazurin viability assay, multitox – Glo multiplex cytotoxicity assay, and Annexin V staining assay. Cell cycle profiles were revealed by propidium iodide staining assay. The effects of ISAE on G2/M arrest signaling and DNA damage were evaluated by Western blot assay and phospho-H2A.X staining assay. The chemical profile of ISAE were determined by tandem mass spectroscopy and molecular networking approach.ResultsWe showed that the acute lymphoblastic leukemia cell line Jurkat cell was more responsive to ISAE treatment than other leukemia cell lines. In contrast, ISAE did not induce cytotoxic effects in normal fibroblast cells. Cell cycle analysis revealed that ISAE triggered G2/M arrest in Jurkat cells in dose- and time-dependent manners. Elevation of annexin V-stained cells and caspase 3/7 activity suggested ISAE-induced apoptosis. Furthermore, ISAE alone could increase the phosphorylation of CDK1 at Y15 and activate the ATR/CHK1/Wee1/CDC25C signaling pathway. However, the addition of caffeine, a widely used ATR inhibitor to ISAE, reduced the phosphorylation of ATR, CHK1, and CDK1, as well as G2/M arrest in Jurkat cells. Moreover, increased phospho-H2A.X stained cells indicated the involvement of DNA damage in the anti-leukemic effect of ISAE. Finally, qualitative analysis using UPLC-tandem mass spectroscopy and molecular networking revealed that tryptanthrin was the most abundant organoheterocyclic metabolite in ISAE. At equivalent concentrations to ISAE, tryptanthrin induced G2/M arrest of Jurkat cells, which can be prevented by caffeine.ConclusionsISAE causes G2/M arrest via activating ATR/CHK1/CDK1 pathway and tryptanthrin is one of the active components of ISAE. Our findings provide subtle support to the traditional use of I. suffruitcosa in leukemia management in folk medicine.

Vincristine induces procoagulant activity of the human lymphoblastic leukemia cell line Jurkat through the release of extracellular vesicles.

Thromboembolic events are frequent and serious complications of acute lymphoblastic leukaemia treatment. The importance of chemotherapy in the pathogenesis of this increased risk is enhanced by the fact that thrombosis rarely occurs at diagnosis. Our study aims at investigating the effect of chemotherapy on pro-coagulant activity (PCA), phosphatidylserine (PS) exposure, tissue factor (TF) activity and derived extracellular vesicles (EV) of Jurkat cells. Jurkat cells were treated with two commonly used chemotherapeutics: Vincristine (VCR) or Daunorubicin (DNR), at relevant concentrations. PCA of cells and derived EV were evaluated using Thrombin generation Assay (TGA). Cells or EV were incubated with annexin V or anti TF antibodies to assess the respective contribution of TF and PS. PS exposure on cells was analysed by flow cytometry. Derived EV were evaluated in fluorescence microscopy and flow cytometry. Untreated Jurkat cells and EV support thrombin generation. Thrombin generation was abolished when PS activity was inhibited by annexin V. VCR treatment resulted in a time dependent increase of thrombin generation. After VCR exposure, TF activity increased as well as PS exposure increased on the cell surface. The increase in TF activity was abolished by annexin V indicating that PS was required. A spontaneous release of EV from Jurkat cells was observed and VCR treatment increased the number of generated EV. Our results indicate that VCR increased the PCA of Jurkat cells predominantly through PS exposure and increased EV generation. Lymphoid blasts derived EV could be biomarkers to determine high thrombotic risk ALL patients.

Effect of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells on the Proliferation and Apoptosis of Leukemic Cells and Its Mechanism

To investigate the effects of human umbilical cord blood-derived mesenchymal stem cells(HUCMSC) on the leukemic cell line HL-60 and acute lymphoblastic leukemia cell line Jurkat as well as the role of CXCL12/CXCR4. HL-60 cells and Jurkat cells were co-cultured with human umbilical cord blood mesenchymal stem cell (HUCMSC), and the model was treated with G-CSF, AMD3100 and their combination. The cell viability and cell cycle were measured by Cell Counting Kit-8 (CCK-8), the apoptosis and the cell-cycle analysis were assessed by flow cytometry with the Annexin V/PI double staining. The expression of surface CXCR4 protein and total CXCR4 protein of leukemic cells were detected by flow cytometry and Western blot respectively. HUCMSC could decrease the viability of HL-60 cells and Jurkat cells, as well as the percentage of apoptotic cells, they could also increase the number of G0/G1 cells, while G-CSF and AMD3100 could reduce the proliferation of HL-60 cells and Jurkat cells in HUCMSC co-culture model, destructed the anti-apoptotic effect of HUCMSC on HL-60 cells and Jurkat cells, and the combination of 2 drugs resulted in a synergistic effect. The G-CSF could reduce the expression of surface CXCR4 protein and total CXCR4 protein in leukemic cells, while AMD3100 could only decrease the expression of surface CXCR4 protein of leukemia cell membrane, having no effect on the expression of CXCR4 protein in cytoplasm. Human umbilical cord blood mesenchymal stem cells can inhibit the proliferation and apoptosis of acute leukemia cells and increase the number of G0/G1 phase cells in leukemic cells. The AMD3100 can decrease the expression of surface CXCR4 protein in leukemia cells, G-CSF can decrease expression of total CXCR4 protein as well as membrane CXCR4 protein. Both of them can block the CXCL12/CXCR4 signal axis, weakening the relationship between leukemia cells and microenvironment. And on the basic of HUCMSC influenced leukemia cells' growth and proliferation, the cell viability will be weakened, its apoptosis will be promoted, and the percentage of G0/G1 phase cells in leukemia cells will be decreased.

Influence of Biological Character of Acute Lymphoblastic Leukemia Cell Linet after Overexpression of C/EBPa and PU.1

Objective: To observe the influence of biological character of acute lymphoblastic leukemia cell line Jurkat in vitro and in vivo after overexpression of C/EBPa and PU.1.Methods: To establish transducing plasmids, using 293T cells to package lentiviral vector and to transfect Jurkat cell line. Flow cytometry is used to analyze the expression of membrane cluster differentiation antigen, apoptosis rate, proliferation and cell cycle of cells. Cell morphology and Q-PCR was used to analyze the changes of cell morphology and molecule gene. Six to eight weeks old immunodeficiency (NSI) mice were transplanted by tail vein injected with 2x106 cells after transduction. To observe the biological behavior and survival time, using Flow cytometric, cell morphology, pathological HE staining, immunohistochemistry to analyze the invasion of leukemia cells.Results: The expression of CD3 (mark antigen of lymphocyte) fall as low as 20% after overexpression of C/EBPa and PU.1, CD11b (mark antigen of myeloid cell) increase as high as 30%, which has significant difference with Jurkat-gfp group. The apoptosis rate has significantly increase, proliferation rate has significantly decrease after overexpression of C/EBPa, which blocked in S period. Adding myeloid cytokine (GM-CSF, M-CSF, IL-3, FLT-3L,100ng/mL) on the supernatant of transduction cells, Jurkat cells can be reprogrammed to attached cells from suspension after overexpression of C/EBPa and PU.1 in the 5th day. Attached cells can chemotaxis and consuming yeast. Cell multicore and small amount of purple particles in cytoplasm has observed in Jurkat after overexpression of C/EBPa and PU.1, high expression of myeloid gene (CD14, CD64) and low expression of lymphocyte gene (BCL-11B, GATA-3) has observed in cells after transduction. All the mice have leukemia between 1 to 2 months after injection with transfected cells. The survival time is longest in overexpression of C/EBPa group, which shows significant difference (P<0.001). Cells of peripheral blood, liver, spleen and bone marrow can partly transduce to myeloid cells after overexpression of C/EBPa and PU.1, additionally, the expression of CD11b is observed in liver and spleen cells by immunohistochemistry.Conclusion: Jurkat cells can be partly reprogrammed to monocyte- macrophages, while with the help of myeloid cytokines, it can be reprogrammed to macrophages with chemotactic and phagocytic function. Jurkat cells can be partly reprogrammed to monocyte- macrophages in vivo and prolong the survival time after overexpression of C/EBPa and PU.1. DisclosuresNo relevant conflicts of interest to declare.

Salinomycin Enhances the Apoptosis of T-cell Acute Lymphoblastic Leukemia Cell Line Jurkat Cells Induced by Vincristine

This study was aimed to investigate the effect of salinomycin combined with vincristine on the proliferation and apoptosis of Jurkat cells and its possible mechanisms. The proliferation of Jurkat cells was examined by CKK-8 assay. Flow cytometry was used to assess cellular apoptosis. Levels of BCL-2, caspase-3, and caspase- 8 were measured by Western blot. The salinomycin or vincristine, either alone or in combination, inhibited the proliferation of Jurkat cells in a dose-dependent manner. Salinomycin combined with vincristine produced more obveous inhibition of cell proliferation than either compound used alone (P<0.05). Western blot analysis showed that the combined use of Sal and VCR reduced the expression of BCL-2 protein, and increased expression of caspase 3 and caspase 8 protein, more significantly. Furthermore, combination of Sal and VCR synergistally promoted apoptosis of the Jurkat cells (P<0.05). The combination of salinomycin and vincristine synergistically inhibits proliferation and promotes apoptosis of T-cell acute lymphoblastic leukemia Jurkat cells.

Effects of Sam68 gene silence on proliferation of acute T lymphoblastic leukemia cell line Jurkat

This study was purpose to investigate the effect of Sam68 gene silence on proliferation of human acute T lymphoblastic leukemia cell line Jurkat. The sequence of shRNA targeting the site 531-552 of Sam68 mRNA was designed and chemically synthesized, then a single-vector lentiviral, Tet-inducible shRNA-Sam68 system (pLKO-Tet-On) was constructed; next the Jurkat cells were infected with lentivirus to create stable cell clones with regulatable Sam68 gene expression. The inhibitory efficiency of Sam68 gene was assayed by Real-time PCR and Western blot; the cell activity of Jurkat cells was detected with MTT assay; the change of colony forming potential of Jurkat cells was analyzed by colony forming test; the cell cycle distribution was tested by flow cytometry. The results indicated that the expression of Sam68 in experimental cells was statistically decreased as compared with that of the control cells; the cells activity and colony forming capacity of the Jurkat cells with Sam68 gene silence were significantly inhibited; with Sam68 gene silencing, the percentage of S phase cells was significantly increased, while the percentage of G2 phase cells was significantly decreased. It is concluded that the silencing Sam68 gene using shRNA interference can effectively inhibit the proliferation of human acute T lymphoblastic leukemia cell line Jurkat.

The apoptotic and anti-proliferative activity of Origanum majorana extracts on human leukemic cell line

Scientists are constantly searching for phytochemicals and compounds with anti-cancer and antioxidant activity. In this study, the anti-proliferative activity of plant extracts from Origanum majorana (marjoram) was tested on human lymphoblastic leukemia cell line Jurkat. Cytotoxicity was examined using non-radioactive cytotoxicity assay and the IC 50 was calculated. At non-cytotoxic concentrations, the viability of cells decreased with increase of concentration of plant extract. The anti-proliferative effect was also found to be dose-dependent. Analysis via flow cytometry shows that marjoram extracts stimulated apoptosis. Induction of apoptosis was caused by an up-regulation of p53 protein levels and down-regulation of Bcl-2α. Marjoram exhibited a strong scavenging activity (SC 50 = 0.03 mg dry weight). The conclusions from this study suggest that marjoram extracts exhibit anti-proliferative effect and high antioxidant activity. For that it merits further investigation as a potential therapeutic agent.

Cyp33-MLL Interaction Modulates Human HOX Gene Expression and Chromatin Structure.

The MLL gene is involved in translocations and fusion genes associated with human leukemia. We have identified a protein, Cyp33, which interacts with the PHD fingers of MLL, and modulates the expression of HOX genes regulated by MLL. In order to define possible mechanisms for this modulation, we set up an experimental system in which the Cyp33 protein is conditionally expressed from a transgene under the control of a tetracycline inducible promoter. Two different cell lines with such inducible gene were established, based on the human kidney epithelial cells HEK293, and the human lymphoblastic leukemia cell line Jurkat, respectively. In these systems we have shown that induction of Cyp33 expression over endogenous levels, leads to downregulation of the HOXC8 gene, but this downregulation is reversible, the HOXC8 being re-expressed when Cyp33 is de-induced and its levels go back to normal. This indicates that Cyp33 overexpression does not erase an epigenetic mark for HOXC8 gene expression. Concordantly, no change in methylation of histone H3 at lysines 4 or 9 is observed upon Cyp33 overexpression. We also showed that both MLL and Cyp33 bind to the promoter of HOXC8, and that histones H3 and H4 at this promoter are de-acetylated upon Cyp33 overexpression. This deacetylation, as well as the downregulation of HOXC8 expression, are prevented by cyclosporin A and by trichostatin A, suggesting that the prolyl-isomerase activity of Cyp33 is necessary for these effects, and that histone deacetylation mediates these effects. We have previously shown that overexpression of Cyp33 enhances the recruitment of HDAC1 to the repression domain of MLL. We now show that this enhanced recruitment is inhibited by cyclosporin A. These results suggest a model in which Cyp33 is recruited to MLL and targets the MLL repression domain to promote the recruitment of histone deacetylases, thus allowing or promoting the silencing of the MLL regulated genes. In this model Cyp33 functions downstream of the gene activation or silencing epigenetic marks.