Background
T-cell acute lymphoblastic leukemia (T-ALL), which accounts for 25% of cases of adult ALL, is characterized by the malignant clonal expansion of immature T-cell progenitors [
1]. More than 50% of cases of T-ALL involve somatic activating mutations of Notch1 [
2], a potent regulator known to play an oncogenic role in many malignancies, affecting proliferation, invasion, chemoresistance, angiogenesis and cell fate determination [
3‐
5]. In the Notch1 signalling pathway, Notch1 transmembrane receptors become activated when ligands bind to their extracellular domains. This ligand binding results in two consecutive proteolytic cleavage events that liberate the intracellular Notch1 (ICN), which enters the nucleus and interacts with the DNA-binding protein CSL (CBF1/RBP-J, Su(H), Lag-1) to regulate expression of downstream genes [
6]. In murine models, constitutive activation of Notch1 signalling induced T-ALL, demonstrating the key role of Notch1 in the pathogenesis of T-ALL [
7]. Interference with aberrant Notch1 signalling in the context of T-ALL has the potential to inhibit proliferation and induce apoptosis, suggesting that Notch1 may be a pivotal oncogene [
8,
9].
In bone marrow, hematopoiesis occurs under relatively hypoxic conditions [
10]. Hypoxia induces cells to undergo a variety of biological responses, including up-regulation of a series of physiologically important genes such as erythropoietin, glucose transporter type 1 and vascular endothelial growth factor (VEGF) [
11,
12]. Activation of these hypoxia-induced genes enables cells to respond to oxygen deprivation by modifying cell growth, metabolism, erythropoiesis and angiogenesis. 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-1α is the unique, O
2-regulated subunit that determines HIF-1 activity [
13]. Tumor hypoxia and increased HIF-1α activity can act as major stimuli for tumor aggressiveness and progression [
14]. Hypoxia may also play a pivotal role in chemoresistance after leukemic chemotherapy. Severely hypoxic areas of bone marrow contain leukemic stem cells (LSCs) responsible for minimal residual disease [
15,
16], which results in hematologic relapse and may serve as a marker for chemoresistance [
17‐
19]. Moreover, it has been shown that Notch signalling is augmented under hypoxic conditions in human cervical, colon, ovarian and breast cancer cell lines. These reports seem to implicate activation of the hypoxia-mediated Notch pathway in tumor cell survival and invasiveness [
5,
20].
We down-regulated HIF-1α and Notch1 using small interference RNA (siRNA) in order to elucidate the influence of Notch1 signalling in hypoxia and also to assess the effect of hypoxia on T-ALL proliferation, invasion and chemoresistance. We provide evidence that hypoxia-induced proliferation, invasion and chemoresistance of T-ALL cells are dependent on HIF-1α-induced functional activation of Notch1 signalling.
Methods
Reagents
Primary antibodies for HIF-1α and Hes1 were purchased from Abcam (Cambridge, UK), and those for Notch1 ICN, Cyclin D1, cyclin-dependent kinase 2 (CDK2), p21, MMP2, MMP9, Bcl-2, Bcl-xL, cleaved caspase-3, cleaved caspase-9 and poly (ADP)-ribose polymerase (PARP) were purchased from CST (Beverly, MA). The primary antibody for β-Actin and all secondary antibodies were obtained from Zhongshan Golden Bridge Biotech (Shenzhen, China). Sources of other reagents are indicated in the text.
Cell culture and hypoxia
Jurkat and Sup-T1 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA) in an incubator maintained at 37°C in an atmosphere containing 5% CO2 and air.
Hypoxic conditions were achieved by culturing cells in a sealed, anaerobic work station (Concept 400, Ruskin Technologies, Pencoed, Wales, UK), in which the hypoxic environment (2% O2, 93% N2 and 5% CO2), temperature (37°C), and humidity (90%) was kept constant.
RNA interference
The following double-strand RNA oligos specific for HIF-1α (5′-GUUGCCACUUCCACAUAAUTT-3′) and Notch1 (5′-UACAGUACUGACCUGUCCACUCUGG-3′) were synthesized by Shanghai GenePharma (Shanghai, China). Commercially available siRNA to random noncoding sequences were used for control transductions (Shanghai GenePharma). To obtain HIF-1α or Notch1 knock-down cells with transient transfection, cells were transfected with siRNA duplexes at the final concentration of 100 nM using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA).
The luciferase assay was performed with a Notch dual-luciferase assay kit (Qiagen, Valencia, USA) following the manufacturer’s instructions. Briefly, cells (3 × 104 cells/well in a 96-well plate) were transfected with a RBP-Jκ responsive firefly luciferase reporter together with an expression vector of Renilla luciferase using Lipofectamine LTX and Plus™ Reagent (Invitrogen). After 12 h, cells were washed and then cultured under hypoxic conditions for 48 h. The luciferase assay was performed with the Dual Luciferase Assay by Promega using Renilla luciferase as an internal control.
Proliferation assay
The effect of hypoxia on the viability of T-ALL cells was evaluated by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, a monosodium salt (WST-8) assay (Dojindo Molecular Technologies, Tokyo, Japan). Briefly, cells were seeded at a density of 5 × 103 cells per well in 96-well microplates and placed in the hypoxic incubator for 24 h, 48 h or 72 h, 10 ul WST-8 solution was applied to each well and they were incubated for 4 h at 37°C. Absorbance was measured at 450 nm using a microplate reader (Benchmark Microplate Reader, BIO-RAD) with a reference wavelength of 655 nm.
Cell cycle distribution analysis
For cell cycle analysis, T-ALL cells were plated on 6-well plates and cultured under hypoxic conditions for 48 h. Cells were fixed in 70% alcohol for 1 h at 4°C, resuspended with propidium iodide (PI) solution (0.04 mg/ml) containing 0.2 mg/ml RNase A (both from Sigma-Aldrich, St. Louis, USA), and incubated at room temperature in the dark for 30 min. DNA content was then analyzed using FACS Calibur (Becton Dickinson, CA, USA).
Drug treatment under hypoxic conditions
Jurkat and Sup-T1 cells were incubated for 24 h under hypoxic conditions, with a pulse of 1 μM dexamethasone (Sigma-Aldrich, St. Louis, USA) added for 48 h under continuous hypoxic conditions. Cells were collected for the apoptosis assay and Western analysis of Bcl-2, Bcl-xL, cleaved caspase-3, cleaved caspase-9 and cleaved PARP.
Apoptosis assay
The apoptosis assay was performed using an Annexin V/PI Apoptosis Detection Kit (Jingmei Biotech, Shenzhen, China). Cells were harvested, resuspended in 100 μl of binding buffer, labeled with 5 μl Annexin V-biotin followed by 10 μl PI and incubated for 15 min in the dark. Then 400 μl binding buffer was added and Annexin V-PI was measured by FACS Calibur (Becton Dickinson, CA, USA).
RNA extraction and quantitative real-time PCR
Total RNA was prepared from cell lines using TRIzol reagent (Takara, Dalian, China) according to the manufacturer’s protocol. For reverse transcription, double-stranded cDNA was synthesized from about 1 μg of total RNA using M-MLV RTase cDNA Synthesis Kit (Takara). Quantitative real-time PCR (qRT-PCR) was performed using a LightCycler 2.0 Instrument (Roche, Penzberg, Germany) with SYBR Green PCR Master Mix (Toyobo, Osaka, Japan). Samples were run in triplicate and amplified in a 20 μl reaction according to the manufacturer’s experimental protocol. The housekeeping gene β-Actin, which has relatively constant expression in T-ALL cell lines, was used as an internal control. Primer sequences were as follows (5′-3′): Notch1 forward: GGG TCC ACC AGT TTG AAT GG; Notch1 reverse: GTT TGC TGG CTG CAG GTT CT; Hes1 forward: TGA TTT GGA TGC TCT GAA GAA AGA TA; Hes1 reverse: GCT GCA GGT TCC GGA GGT; HIF-1α forward: TTT GCT GAC ACA GAA GCA AAG A; HIF-1α reverse: TTG AGG ACT TGC GCT TTC AGG; VEGF forward: GAG CCT TGC CTT GCT GCT CTA C; VEGF reverse: CAC CAG GGT CTC GAT TGG ATG; MMP2 forward: CAG GGA ATG AAT ACT GGA TCT ACT; MMP2 reverse: GCT CCA GTT AAA GGC GGC ATC CAC; MMP9 forward: GCC TGC AAC GTG AAC ATC T; MMP9 reverse: TCA AAG ACC GAG TCC AGC TT; β-Actin forward: CGG GAC CTG ACT GAC TAC CT; β-Actin reverse: AAG CAT TTG CGG TGG A.
Western blot
Cells were harvested in ice-cold RIPA lysis buffer in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (both from Beyotime, Haimen, China) for 30 min, and lysates were cleared by centrifugation. Protein concentrations in the supernatant were quantified with the bicinchoninic acid assay protein reagent kit (Sangon, Shanghai, China) according to a standardized curve. Proteins were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Non-specific sites were blocked with 5% nonfat milk in PBS/0.1% Triton X-100 and incubated with appropriate primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG at room temperature for 1 h. After washing, immunoreactive bands were detected by enhanced chemiluminescence (Millipore, Billerica, MA).
Transwell invasion assay
The invasive potential of T-ALL cells was examined using transwell inserts fitted with polycarbonate filters (5 um pore size, Costar, Cambridge, MA) coated with matrigel (BD Biosciences, Bedford, MA). Matrix solutions within transwells were polymerized at 37°C for 1 h and dried onto the transwells overnight at room temperature. Cells in FBS-free medium were seeded in the upper compartment while lower wells contained 10% FBS medium. After 48 h of hypoxic incubation, the cells in the upper chamber were removed while other cells, which had passed through the filter on the underside of the membrane, were fixed with 3.7% paraformaldehyde, stained with 0.2% crystal violet, counted and captured at 100× and 400× magnification using the camera on inverted microscope. Contents of the lower compartments were collected and migrated cells were also counted. The rate of migration was expressed as the fraction of migrated cells out of the total number of cells placed in the upper compartment.
Statistical analysis
Experiments were performed three times and data are expressed as mean ± SD. Statistical analysis was conducted using Student’s t-test. Unless otherwise specified, P < 0.05 was considered significant.
Discussion
There is increasing evidence that tumor cells often reside in a low oxygen tension environment that promotes accumulation of HIF-1α and is involved in tumor progression and aggressiveness. However, the effect of hypoxia on propagation of T-ALL cells and the mechanisms by which hypoxia exerts its effects are still not clear. Notch1 signalling has been shown to play an oncogenic role in the majority of hematological malignancies including T-ALL. Further, Notch1 is related to resistance to chemotherapy, a major cause of treatment failure and poor prognosis in T-ALL. Hypoxia-induced expression of Notch receptors and ligands has been demonstrated in stem cells and malignant tumors [
22,
23]. In the present study, we showed that Notch1 signalling was activated by hypoxia and its transducer HIF-1α. Furthermore, we provide the first evidence that hypoxia/HIF-1α promoted the progression of T-ALL through activation of the Notch1 pathway, resulting in altered expression of downsteam genes regulating cellular proliferation, invasion and chemoresistance. HIF-1α-dependent overexpression of Notch1 in T-ALL cells is one of the major mechanisms underlying T-ALL aggressiveness and resistance to chemotherapy.
The role of hypoxia and HIF-1α in cellular proliferation has been investigated in various cell types. It has been revealed that hypoxia and HIF-1α convey stimulating or inhibiting effects on cellular proliferation and viability, depending on cell type. Studies on epithelial ovarian tumors and esophageal cancer showed that HIF-1α overexpression correlated with tumor apoptosis and patient survival [
24,
25]. However, in an animal model of chronic myeloid leukemia (CML), inhibition of HIF-1α impaired propagation of CML by impairing cell cycle progression and inducing apoptosis of LSCs, suggesting that HIF-1α plays a crucial role in survival maintenance of LSCs [
26]. In the current study, we showed that hypoxia and HIF-1α, by reducing the proportion of cells in the G0/G1 phase of the cell cycle, promote the proliferation of T-ALL cells. Molecular investigation revealed that, under hypoxic conditions, expression of Cyclin D1 and CDK2 was increased whereas p21 expression was decreased. We also found that Notch1 ICN was augmented by hypoxia and this effect was dependent on HIF-1α accumulation, consistent with previous results [
23]. Potentiation of Notch1 signalling by hypoxia was further corroborated by elevated expression of the Notch1 downstream gene Hes1 in hypoxic conditions. Inhibition of Notch1 activity using Notch1-targeted siRNA reversed hypoxia-induced changes in expression of cell cycle regulatory proteins (CDK2, CyclinD1 and p21), thus repressed the hypoxia-induced proliferation of T-ALL cells. On the basis of these results, we concluded that hypoxia potentiated Notch1 signalling in T-ALL, leading to altered expression of cell cycle regulatory proteins and increased cell proliferation.
T-ALL is characterized as leukemic cell infiltration of various organs such as lymph nodes, liver, spleen and lungs. MMPs, in particular the gelatinases MMP2 and MMP9, are an important group of zinc- and calcium-dependent proteolytic enzymes responsible for degradation of the vascular basement membrane and the extracellular matrix of lymphoid tissues [
27]. A previous study suggested that hypoxia and HIF-1α overexpression contribute to tumor cell invasion and dissemination, probably through activation of MMPs [
28]. Considerable evidence has accumulated that MMP9 and MMP2 play an important role in the invasiveness and propagation of several hematological malignancies [
29‐
31]. Our studies indicate that hypoxia may be an initiating event that results in enhanced Notch1 signalling, increased expression of MMP2 and MMP9, and ultimately increased invasiveness of T-ALL. Furthermore, inhibition of Notch1 signalling abrogated hypoxia/HIF-1α-induced cell invasion, probably through down-regulation of MMP2 and MMP9. This indicates that Notch1 may serve as a critical intermediary that transforms the hypoxic response into invasion.
In addition to cellular invasion, as discussed above, chemoresistance has been recognized as another major cause of treatment failure among patients who suffer from T-ALL. Previous studies have shown that hypoxia is closely related to tumor resistance to anticancer therapy in a wide spectrum of neoplastic cells [
32,
33]. We hypothesized that chemoresistance to dexamethasone, an anti-leukemia drug commonly used in T-ALL treatment, was acquired by T-ALL cells in response to hypoxia. Under our hypoxic culture conditions (2% oxygen tension), dexamethasone exhibited less effectiveness. Further investigation revealed that hypoxia and HIF-1α increased expression of Bcl-2 and Bcl-xL, leading to a marked decrease in caspase activity.
Several studies have demonstrated the contribution of the Notch pathway to chemoresistance in human malignancies. Previous studies on solid tumors suggested that Notch is involved in the formation of cancer stem cells and the acquisition of the epithelial-mesenchymal transition phenotype, which are both critically associated with chemoresistance [
34,
35]. In myeloma and other malignant lymphoid cell lines, studies have demonstrated that Notch1 is closely related to bone marrow stroma–mediated drug resistance, and that inhibition of Notch signalling sensitizes cells to chemotherapy and prevents bone marrow-mediated chemoresistance [
36,
37]. Here, we showed that knockdown of Notch1 prevented the protective effect of hypoxia/HIF-1α against dexamethasone-induced apoptosis. This sensitization correlated with loss of the effect of hypoxia/HIF-1α on Bcl-2 and Bcl-xL expression.
Rohwer et al. reported that HIF-1α suppressed gastric cancer chemosensitivity via modulation of p53, indicating that hypoxia-induced chemoresistance was dependent on a functional p53 pathway [
38]. Because the leukemic T-cell lines used in our study do not have functional p53, our results provide evidence that activation of the Notch1 pathway may represent an alternative mechanism for hypoxia-induced chemoresistance in mutant p53 cell lines.
HIF-1α knockdown by siRNA or antisense techniques has been shown to suppress cell growth, proliferation and migration in both normal human cells and malignant tumor cells, including umbilical vascular endothelial cells, medulloblastoma, prostate cancer and glioma [
39‐
42]. Silencing HIF-1α also has been shown to reverse chemotherapy resistance in tumor cells [
42]. Our finding, that blocking HIF-1α sensitized T-ALL cells to dexamethasone treatment, suggests that HIF-1α may be a potential target for gene therapy in T-ALL cells.
Conclusions
The present study shows that, in T-ALL cells, proliferation, invasion and resistance to chemotherapy were stimulated when cells were exposed to hypoxic conditions, due in part to activation of Notch1 signalling. Moreover, we show that in hypoxic conditions Notch1 signalling is required to activate genes regulating cellular proliferation, invasion and chemoresistance, increasing the aggressiveness of T-ALL and its likelihood for progression. Our work is the first to characterize the interaction between Notch1 and HIF-1α in T-ALL. These results suggest that pharmacological inhibition of Notch1 or HIF-1α signalling might have potential for improving T-ALL therapy.
Acknowledgments
This study was partially supported by grants from the National Natural Science Foundation of China (81070422, 30871088, 81070407), SRFDP of Educational Ministry (20100131110060), the Shandong Technological Development Project (2009GG20002020, 2008GJHZ10202, 2009HD012, BS2009SW014, ZR2010HQ030), and Short-Term Scientist Exchange Program (STSEP) from the National Cancer Institute (NCI). Editorial assistance was provided by Helen Kim, MD.
Competing interests
The authors reported no potential competing interest.
Authors’ contributions
JZ carried out the cellular biologic studies and drafted the manuscript. PL designed the primers and performed qRT-PCR. FL carried out Western blots. NL participated in the flow cytometry. JjD designed siRNAs and performed the siRNA transfection. JjY carried out the reporter gene assay. XQ provided the reagents for research. XlS provided the analysis tools. DxM participated in design and statistical analysis of the study. JP carried out the statistical analysis. CyJ conceptualized the study, participated in its design and coordination and provided vital subjects for research. All authors read and approved the final manuscript.