The Roles Of Hypoxia And Notchl In T-cell Acute Lymphoblastic Leukemia And Their Mechanisms

Background:T-cell acute lymphoblastic leukemia (T-ALL) ischaracterized by a clonal expansion of immature T-cell progenitors that do not retain the capacity to differentiate normally to mature blood cells. More than50%of cases of T-ALL involve somatic activating mutations of Notch1, a potent regulator known to play an oncogenic role in many malignancies including hematologic diseases, affecting proliferation, invasion, chemoresistance, angiogenesis and cell fate determination.Hypoxia enables cancer cells to respond to oxygen deprivation by promoting cell growth, inducing chemoresistance and leading to poor prognosis. It has been shown that bone marrow is a regional hypoxic microenvironment. Moreover, bone marrow in patients with leukemia is generally very hypoxic because of the clonal expansion of immature leukemic cells and secondary anemia. Hypoxia induces cells to undergo a variety of biological responses, including up-regulation of a series of physiologically important genes. Hypoxia-inducible factor-1(HIF-1), a heterodimeric transcription factor consisting of HIF-1α and HIF-1β subunits, plays a critical role in cellular response to hypoxic conditions. It is well established that HIF-1a is the unique, O2-regulated subunit that determines HIF-1activity. Notch signalling is augmented under hypoxic conditions in many human solid cancers, which seem to implicate activation of the hypoxia-mediated Notch pathway intumor cell survival, invasiveness and chemoresistance. Section IThe study of HIF-la and Notchl signalling pathways in hypoxia and their mechanismsObjective:To investigate the activation statusof HIF-la and Notchl signalling pathways in hypoxia; to study the interaction between HIF-la and Notch1signalling pathways;and to cast light on their mechanisms.Materials and methods:1. T-ALL cell culture in hypoxia:Hypoxic conditions were achieved by culturing Jurkat and Sup-T1cells in a sealed, anaerobic work station in which the hypoxic environment (2%O2,93%N2and5%CO2) was kept constant.2. Quantitative real-time RT-PCR:Total RNA was extracted from cells using Trizol reagent and reverse transcription was performed with M-MLV reverse transcriptase. The expressionof HIF-la, VEGF, Notch1and Hesl was detected.3. Western blot:Cells were solubilized in RIPA lysis buffer and total proteins were fractionated using SDS-PAGE. The expression of HIF-la, Notchl ICN and Hesl was detected.4. Dual luciferase reporter assay:Cells were transfected with a RBP-Jk responsive firefly luciferase reporter together with an expression vector of Renilla luciferase. After24h, cells were washed and then cultured under hypoxic conditions for48h. Dual luciferase reporter assay was performed to determine the activity of Notch1signalling pathway.5. RNA interference:cells were transfected with specific siRNA targeting HIF-1α or Notchl using Lipofectamine2000for72h. Blocking efficiency was determined byreal-time RT-PCR and Western blot.6. Quantitative real-time RT-PCR:Transfected cells were cultured in hypoxia for48h and the expression of HIF-1α, Notch1and Hes1was determined.7. Western blot:SiRNA transfected cells were cultured in hypoxia for48h and the expression of HIF-la, Notch1ICN and Hesl was determined.Results:1. Hypoxia induced accumulation of HIF-la and potentiated Notchl signalling: Real-time RT-PCR showed that expression of Notchl, Hesl and VEGF was elevated by hypoxia. Western blot showed that under hypoxic conditions, expression of HIF-1α, Notchl ICN and Hesl was also up-regulated. Analysis of Notchl transcriptional activity using luciferase reporter assay revealed a significant increase in Notchl activity when T-ALL cells were exposed to hypoxic conditions2. HIF-la/Notchl siRNA were transfected into T-ALL cells:Real-time RT-PCR showed that HIF-1α/Notchl expression was significantly down-regulated in both Jurkat and Sup-T1cell lines, which was consistent with the results of Western blot.3. Notchl activation in hypoxia is HIF-la-dependent:Real-time RT-PCR and Western blot showed that under hypoxic conditions, silencing HIF-la inhibited Notchl signalling, as shown by decreased expression of Notchl, Notchl ICN and Hes1, whereas silencing Notchl did not affect HIF-1α expression.Conclusion:1. Hypoxia induced accumulation of HIF-1α protein in T-ALL cells.2. Expression of HIF-la transcription factor is regulated at the level of protein by hypoxia.3. Hypoxia induced activation of Notchl signalling at the level of transcription.4. Hypoxia potentiated Notchl signalling, which is dependent on accumulation of HIF-1α. Section IIThe study of hypoxia on the biological behavior and chemoresistance in T-ALL cells and their mechanismsObjective:To investigate the effect of hypoxia on the biological behavior and chemoresistance in T-ALL cells; to study the role of HIF-1α and Notchl signalling pathways in the effect of hypoxia and their mechanisms, so as to provide the basis for molecular targeted therapy.Materials and methods:1. The effects of hypoxia and HIF-la on proliferation of T-ALL cells and their mechanisms.1.1CCK8assay:Cell proliferation assays were performed at baseline and after24h,48h and72h exposure to hypoxia.1.2Cell cycle analysis by flow cytometry:Jurkat and Sup-T1cells were exposed in hypoxia for48h and cell cycle distribution was analyzed by flow cytometry using propidium iodide (PI) staining.1.3Western blot:T-ALL cells were solubilized in RIPA lysis buffer after48h exposure to hypoxia. Expression of p21, Cyclin D1and cyclin-dependent kinase2(CDK2) was determined.1.4The effect of HIF-la/Notch1siRNA on T-ALL cells proliferation:Cells were exposed in hypoxia after transfection with HIF-la/Notchl siRNA. CCK8assays wereperformed to detect cell growth. Flow cytometry was used to determine cell cycle. Western blot was performed to detect expression of p21, Cyclin D1and CDK2.2. The effects of hypoxia and HIF-1a on T-ALL cells invasion and their mechanisms.2.1Transwell invasion assay:The invasive potential of T-ALL cells was examined using transwell inserts fitted with polycarbonate filters coated with matrigel. T-ALL cells were seeded in the upper compartment and exposed in hypoxia for48h. Cells that had passed through the filter were counted.2.2Real-time RT-PCR:Jurkat and Sup-T1cells were cultured in hypoxia for48h and collected for total RNA extraction. Real time RT-PCR was used to detect the expression of MMP2and MMP9.2.3Western blot:Jurkat and Sup-T1cells were exposed in hypoxia for48h and solubilized in RIPA lysis buffer. Expression of MMP2and MMP9was determinedusingWestern blot.2.4The effect of HIF-la/Notchl siRNA on T-ALL cells invasion:Cells transfected with HIF-la/Notchl siRNA were exposed in hypoxia for another48h. Transwell assay was used to examine invasiveness of T-ALL cells. Real-time PCR and Western blot were used to determine MMP2and MMP9expression.3. The effects of hypoxia and HIF-la on chemoresistance of T-ALL cells and their mechanisms.3.1Jurkat and Sup-T1cells were incubated for24h under hypoxic conditions, with a pulse of1μM dexamethasone added for48h under continuous hypoxic conditions. Cells were collected for the apoptosis assay and Western analysis.3.2Apoptosis assay by flow cytometry:The apoptosis assay was performed using Annexin V/PI staining.3.3Western blot:Cells were solubilized in RIPA lysis buffer and expression of proteins related with apoptosis was determined.3.4The effect of HIF-la/Notchl siRNA on chemoresistance of T-ALL cells: SiRNA transfected cells were treated with1μM dexamethasone. After48h exposure in hypoxia,apoptosis assay was used to determine the proportion of apoptotic cells.Expression of proteins related with apoptosis was determined by Western blot.Results:1. Hypoxia and HIF-1α may potentially promote proliferation of T-ALL cells via activation of the Notch1signalling pathway.1.1Hypoxia promoted T-ALL cells proliferation:CCK8assay showed hypoxia for72h led to consistently accelerated cell growth compared with normoxic conditions.1.2Hypoxia reduced the number of cells in the GO/G1phase of the cell cycle: Flow cytometry for cell cycle revealed that48h exposure to hypoxia substantially reduced the number of cells in the GO/G1phase of the cell cycle while concomitantly increasing the number of cells in the S and G2/M phases.1.3Hypoxia reduced expression of p21protein and increased expression of Cyclin D1and CDK2in T-ALL cells:Western blot revealed that hypoxia reduced expression of p21protein, the key regulator of the G1-S phase transition, while increasing protein expression of Cyclin D1and CDK2in T-ALL cells.1.4Knockdown HIF-1α expression under hypoxic conditions reduced cell growth: CCK8assay showed that down-regulation of HIF-1α under hypoxic conditions reduced cell growth significantly. Cell cycle analysis revealed that silencing HIF-la increased the proportion of cells in the GO/G1phase compared with control. Western blot showed that knockdown HIF-la reduced protein expression of CDK2and Cyclin D1while elevating protein expression of p21.1.5Knockdown Notchl expression reduced cell proliferation rate in hypoxia: CCK8assay showed that silencing Notchl under hypoxic conditions reduced cell proliferation rate.An increased proportion of cells in GO/G1phase was detected by flow cytometry in Notchl siRNA transfected cells. Western blot revealed that CDK2and Cyclin D1were inhibited while p21was increased in Notchl down-regulation cells.2. Hypoxia/HIF-la-induced T-ALL cell invasion requires Notch1signalling.2.1Hypoxia induced a higher invasiveness in T-ALL cells:Transwell assay showed that hypoxia increased penetration through the matrigel-coated membrane.2.2Expression of MMP2and MMP9were elevated by hypoxia:Real-time RT-PCR and Western blot showed that hypoxia transcriptionally up-regulated MMP2and MMP9.2.3Knockdown HIF-la abrogated hypoxia-induced cell invasion:Transwell assay showed that down-regulation of HIF-la decreased cell penetration through the matrigel-coated membrane in hypoxia. Real-time RT-PCR and Western blot revealed that silencing HIF-la attenuated the up-regulation of MMP2and MMP9in hypoxia.2.4Knockdown Notch1abrogated hypoxia/HIF-la-induced cell invasion: Transwell assay showed higher invasiveness was abrogated in Notchl knockdown cells. Real-time PCR and Western blot revealed that the increased expression of MMP2and MMP9induced by hypoxia was attenuated.3. Notchl signalling is required for hypoxia/HIF-la-induced T-ALL chemoresistance.3.1Hypoxia induces chemoresistance of T-ALL cells:Jurkat and Sup-T1cells were treated with1μM dexamethasone in hypoxic conditions for48h. Apoptosis analysis showed the proportion of apoptotic cells was significantly lower in hypoxia than in normoxia.3.2Hypoxia increased expression of anti-apoptotic proteins Bcl-2and Bcl-xL:Western blot of cells subjected to dexamethasone treatment in hypoxia revealed increased expression of anti-apoptotic proteins Bcl-2and Bcl-xL. Moreover, a significantly decreased levels of the active products of caspase3, caspase9and PARP was detected.3.3Knockdown HIF-la abrogated hypoxia-induced chemoresistance:Apoptosis assay showed a significantly higher rate of cell apoptosis after dexamethasone treatment in HIF-la knockdown cells. Western blot revealed that silencing HIF-la expression decreased protein levels of Bcl-2and Bcl-xL and increased caspase3and caspase9activity.3.4Knockdown Notchl abrogated hypoxia/HIF-la-induced chemoresistance to dexamethasone:Apoptosis assay showed that in hypoxic conditions, down-regulation of Notchl increased the proportion of apoptotic cellsinduced by dexamethasone. Western blot of Notchl knockdown cells revealed a decrease in Bcl-2and Bcl-xL and activity of caspase3and caspase9wasconcomitantlyincreased.Conclusion:1. Proliferation, invasion and resistance to chemotherapy were stimulated when T-ALL cells were exposed to hypoxic conditions, due in part to activation of Notchl signalling.2. Pharmacological inhibition of Notchl or HIF-la signalling might have potential for improving T-ALL therapy.