The Role Of Notch Signaling In T-ALL Cells And Involved Mechanism

Section I The effects of down-regulation of Notch1 mediated by siRNA on the proliferation and apoptosis of T-ALL cellsBackground:T-cell acute lymphoblastic leukemia (T-ALL) ranks a serious type of hematological malignancies in patients, constituting a substantial fraction of ALL, both in children and in adults. Current treatment is primarily based on combination chemotherapy with low long-term survival rate particularly in adult patients, emphasizing the need for improved therapy. A large body of evidence has been accumulated showing that oncogene activation and antioncogene inactivation can lead uncontrolled proliferation, defective differentiation and abnormal apoptosis, and then leukemias develop. The molecular mechanisms underpinning T-ALL are likely to be complex.The involvement of Notch1 was first observed in a rare t(7;9)(q34;q34.3) translocation, which brings an activated form of the Notch-1 receptor gene under the control element of the T-cell receptor gene. More recently, it was shown that more than 50% of all T-ALL patients carried Notch1 gain-of-function mutations that generate an activated form of Notch. Mutant Notch1 could represent an important new target for therapy of T-ALL patients. Since the generation of activated Notch1 can be inhibited by gamma-secretase inhibitors (GSIs), GSIs are valuable tools for delineating the cell biological function of the Notch cascade, but the efficacy of this strategy has been questioned, as GSIs seem to be active in only a small fraction of human and mouse T-ALL cell lines with constitutive Notch1 activity. GSI are not specific for Notch1 and turn off all four Notch receptors. Furthermore, gamma-secretase might affect other proteins involved in proliferation. Therefore, it is important to explore the role of Notch1 in T-ALL using RNA interference technology.Objective:To investigate Notch1 on the effects of biological behavior of T-ALL cell proliferation, apoptosis and cell cycle, so as to reveal the pathogenesis of T-ALL and provide thebasis for molecular targeted therapy.Materials and Methods:1. Cell culture. Human T-ALL cell lines SupT1, Jurkat were cultured in RPMI medium 1640. Total RNA was extracted by Trizol according to the manufacturer's instructions. Synthesis of first-strand cDNA was carried out with Revert Aid? First Strand cDNA Synthesis Kit. RT-PCR was used to detect the expression of Notchl,HES-1 mRNA.2. Immunocytochemical stain. SupT1 and Jurkat cells on cover slides were fixed in 95% ethanol, and then the expression of Notch1 protein was detected using Notch1 antibody according to the manufacturer's instructions.3. SiRNA transfection. 8-16×10⁵/ml of SupT1 and Jurkat cells were seeded in 24-well plates. And then cells were treated with 100nM Notch1 siRNA/control siRNA. Seventy-two hours after siRNA transfection, cells were used for MTT, real-time PCR, western blot, and other experiments.4. Cell growth inhibition by MTT assay. SupT1 and Jurkat cells were incubated at a density of 5,000 cells/well in 96-well plates, and subsequently transfected with Notch1 siRNA or control siRNA. 24, 48, 72, 96 hours after tansfection, 20μl of MTT was added each well. 4 additional hours later, color development was measured on a microplate reader at 570 nm.5. Flow cytometry and cell cycle analysis. Notch1 siRNA, or control siRNA transfected SupT1, Jurkat cells were collected and stained with PI. DNA content was detected on a FACS Calibur.6. Apoptosis Assay. 72 hours after transfection, Notch1 siRNA/control siRNA transfected SupT1 and Jurkat cells were collected and were labeled with annexin V-biotin followed by PI. Annexin V/PI were measured by FACS Calibur and analyzed with the Modfit Software.7. For the chemotherapeutic drug assays, 24 hours after transfection, cells were exposed to different concentration of adriamycin respectively for 48 hours, and then cells were collected and analyzed as above.8. Western Blot Analysis. Total protein was extracted from transfected cells with Notch1 siRNA/control siRNA cells. Total proteins were fractionated using SDS-PAGE and transferred onto nitrocellulose membrane. After incubation with blocking buffer, the membrane was incubated with primary and secondary antibodies. The protein bands were detected using the enhanced chemiluminesence detection system.Results:1. Notch1 and HES-1 mRNAs were all expressed in human T-ALL cell lines SupT1 and Jurkat.2. Immunocytochemical stain results show that Notch1 protein is expressed and located at different places in human T-ALL cell lines SupT1 and Jurkat.3. Notch1 siRNA effectively down-regulated the expression level of Notch1 in human T-ALL cells. Notch1 and HES1 mRNAs were decreased by more than 90% in both SupT1 and Jurkat cells. Protein levels were also greatly reduced in Notch1 siRNA transfected cells compared with control siRNA transfected cells.4. Down-regulation of Notch1 expression by siRNA inhibited the proliferation of human T-ALL cells. Cell viability was determined by the MTT assay after transfection with Notch1 siRNA or control siRNA. Down-regulation of Notch1 expression by siRNA markedly inhibited cell proliferation in SupT1 cells at 48, 72 and 96 h (P<0.05), whereas no apparent changes in Jurkat cells occurred (P> 0.05).5. Down-regulation of Notch1 expression by siRNA induced G₀/G₁ phase cell cycle arrest in SupT1 cells. The Notch1 siRNA transfected SupT1 cells demonstrated a phase arrest pattern (62.85% vs. 46.17%) at 72 hours after transfection as compared with control cells. No alteration in cell cycle distribution was observed in Jurkat cells.6. Down-regulation of Notch1 expression by siRNA led to apoptosis in human T-ALL cells. 72 hours after transfection, cells were stained with Annexin V/PI, and analyzed by flow cytometry. The early and late apoptosis rates were 9.01% and 12.41% in Notch1 siRNA-transfected SupT1 cells, compared with 4.13% and 4.84% in control siRNA-transfected group respectively. There was no apparent change in early and late apoptosis of Jurkat cells.7. Down-regulation of Notch1 expression by siRNA increased chemosensitivity. We have used siRNA to specifically knock down the expression of Notch1 in SupT1 and Jurkat cells, and then examined cell viability and drug-induced apoptosis. Results showed that the IC50 value of adriamycin in SupT1 cells transfected with control siRNA was 0.21 (μg/ml) and SupT1 cells transfected with Notch1 siRNA was more sensitive, with an IC₅₀ value of 0.05 (μg/ml). The combination of Notch1 siRNA and adriamycin group increased the apoptosis rate 2.4 fold in comparison with the combination of control siRNA and adriamycin group in SupT1 cells. However, there was no significant difference in Jurkat cells between the combination of Notch1 siRNA and adriamycin group and the combination of control siRNA and adriamycin group.8. Down-regulation of Notch1 expression affected the levels of cyclin proteins and p-Akt protein. Results showed that p-Akt, cyclin D1 and CDK2 protein expressions were decreased while p21 protein expression was increased in Notch1 siRNA-transfected SupT1 cells. However, there was no significant change of the expressions of p-Akt, cyclin D1 and CDK2 proteins, and p21 protein expression was slightly increased in Notch1 siRNA-transfected Jurkat cells.Conclusion:1. Notch1 siRNA effectively down-regulated the expression level of Notch1.2. Down-regulation of Notch1 mediated by siRNA can inhibit the proliferation of T-ALL cells by increasing apoptosis and inducing cell cycle arrest. 3. Down-regulation of Notch1 mediated by siRNA can increase T-ALL cells chemosensitivity to adriamycin.4. These effects may be through the repressing of Akt pathway.5. Notch pathway interacts with Akt pathway and Notch signaling can regulate Akt signaling.Section II The role of Notch1 in BMSCs mediated drug resistance of T-ALL cellsBackground:Resistance to chemotherapy is one of the most serious problems in treating patients with ALL and other hematologic malignancies. Recently, increasing evidence suggests that the interaction between tumor cells and elements of their microenvironment results in resistance to chemotherapy in leukemia and myeloma. However, the molecular mechanisms of BMSCs-mediated drug resistance of tumor cells are poorly understood.To address this question, we have focused on the Notch family of receptors and transcriptional regulators that are critically important for interaction between BMSCs and hematopoietic cells. Notch signaling is a pathway highly conserved through evolution which regulates various physiological processes, including stem cell maintenance, differentiation, proliferation and apoptosis. In mammals, key components of the Notch pathway include four transmembrane receptors (Notch 1-4) and five ligands (Delta-like 1, Delta-like 3, Delta-like 4, Jagged 1, Jagged 2). Direct binding of a ligand from a signaling cell to a Notch receptor on the membrane of the receiving cell initiates two successive proteolytic cleavages by TACE (TNF-α-converting enzyme) and theγ-secretase/presenilin complex, which ultimately results in the release of the intracellular domain (Notch-IC). Notch-IC then translocates into the nucleus and directly interacts with the DNA binding protein CBFl/Su(H)/Lagl(CSF) that activates the transcription of target genes including the HES (hairy/enhancer-of-split) and hey.Notch1 has been shown to inhibit T-cell receptor-induced apoptosis in mature cells and dexamethasone-mediated apoptosis in thymocytes and to be antiapoptotic in T cells. More and more evidence suggests cancers with activated Notch1 signal are chemo-resistant. We suggest that Notch1 signaling may play an important role in the drug resistance mediated by stromal cells.Objective:To investigate BMSCs on the effect of drug-induced apoptosis of T-ALL cells and explore the role of Notch1 signaling in the drug resistance mediated by stromal cells, so as to reveal the pathogenesis of T-ALL and provide the basis for molecular targeted therapy for drug resistance.Materials and Methods:BMSCs were isolated and culture-expanded from human bone marrow; Jurkat cells were transfected by siRNA using the Lipofectamine 2000 reagent, and then co-cultured with BMSCs; co-cultured Jurkat cells were selected using CD3+ magnetic micro beads for real-time reverse transcription-PCR, apoptosis analysis and Western blot; real time quantitative polymerase chain reaction (RQ-PCR) was used to detect the mRNA; Flow cytometry was used to detect apoptosis; Western blot were applied to detect the protein expression.Results:1. BMSCs protect drug-induced apoptosis in Jurkat cells. Jurkat cells were co-cultured with BMSCs followed by cytotoxic drugs. Jurkat cells cocultured with BMSCs were resistant to dexamethasone and adriamycin treatment. Treatment of Jurkat cells with 1μM dexamethasone induced 67.91% apoptosis of cells maintained in suspension, compared with a value of 17.30% of cells co-cultured with BMSCs. Similarly, Jurkat cells contact with BMSCs followed by adriamycin also underwent less apoptosis than those in suspension (9.46% vs. 22.19%).2. Notch1 ligands are detected in BMSCs and Notch-1 signaling is further activated in Jurkat cells following contact with BMSCs. The protein expression of Notch-1 and its target gene HES-1 were examined. Jurkat cells were incubated for 48 hours in suspension or on the monolayer of BMSCs. Notch1-ICD (the activated form of Notch1) and HES-1 protein were increased in Jurkat cells with BMSCs compared with those in suspension.3. P-Akt is up-regulated in Jurkat cells following contact with BMSCs. Western blot analysis with antibodies specific for phosphorylated Akt was performed. Level of phosphorylated Akt was elevated in co-cultured Jurkat cells compared with that in suspension.4. Notch1 siRNA effectively down-regulates the expression level of Notch1 in Jurkat. The efficiency of Notch1 siRNA for knockdown of Notch1 mRNA and protein was confirmed by real time RT-PCR and Western blot. Notch1 protein level was significantly reduced and almost completely knocked down by Notch1 siRNA compared with control siRNA-transfected cells in Jurkat cells. And Knock-down of Notch1 in Jurkat cells resulted in decreased protein levels of Notch target gene HES-1. Knock-down of Notch1 and HES-1 in Jurkat cells with siRNA paralleled the reduced amount of specific mRNA, as demonstrated by real time RT-PCR amplification5. Down-regulation of Notch1 enhances the effect of chemotherapy on co-cultured Jurkat cells. Notch1 was down-regulated by siRNA in Jurkat cells, and then drug-induced apoptosis of co-cultured Jurkat cells was examined. Control siRNA-or Notch1 siRNA-transfected Jurkat cells following contact with BMSCs were exposed to dexamethasone or adriamycin as described, drug-induced apoptotic rates was detected. Notch1 siRNA-transfected Jurkat cells following contact with BMSCs were significantly susceptible to apoptosis induced by dexamethasone or adriamycin compared with control siRNA-transfected Jurkat cells following contact with BMSCs6. Down-regulation of Notch1 affects the level of p-Akt protein. p-Akt protein was examined by Western blot analysis. When Notch1 was down-regulated in Jurkat cells following contact with BMSCs, the expression of p-Akt was decreased. Conclusion:1. BMSCs protect drug-induced apoptosis in Jurkat cells2. Interactions of leukemic and BMSCs result in activation of Notch1-HES and Akt signaling in leukemic cells.3. Down-regulation of Notch1 restores of sensitivity to chemotherapy of co-cultured Jurkat cells. The possible mechanisms of restoration of sensitivity to chemotherapy of Jurkat cells following contact with BMSCs by down-regulation of Notch1 could be associated with repressed the activity of Akt signaling.