| Background and ObjectiveHypoxic microenvironment exists in the bone marrow due to poor blood perfusion. Direct measurement of oxygen levels has revealed that the bone marrow (BM), in general, is hypoxic (1-6%oxygen tension). This hypoxic microenvironment in which the hematopoietic stem cells (HSCs) are supported, also provides a protection for the leukemia cells of minimal residual disease which leads to the relapse.HSCs resides in specific stem cell niches of the bone marrow. Compared to the low oxygen tension in the niche, the oxygen consuming of HSCs is pretty high. So HSCs grow under the condition of seriously low oxygen tension. Hypoxia probably plays a role on the self-renewel and differentiation of HSCs. Nowadays HSCs are used in clinic for the treatment of hematological malignancies, some solid tumors and autoimmune diseases. However, normally HSCs are expanded under environmental oxygen tension(21%) in vitro. Exposed in relatively high oxygen tension, HSCs will have an accumulation of reactive oxygen species in mitochondria which leads to oxidative damage and result in the poor expansion of HSCs.Leukemia is a malignant clonal disorder of the stem/precursor cells in the bone marrow. Combination chemotherapy is still the main treatment for leukemia. Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. In AML, complete remission (CR) rate after chemotherapy is only50-80%. Multidrug resistance (MDR) is the main cause of chemotherapy failure. Leukemia cells resistant to drugs survive the chemotherapy and hide in tne bone marrow as minimal residual disease which leads to the relapse. The hypoxic microenvironment in bone marrow may be a sanctuary for these leukemia cells.This research is to investigate the effect of hypoxia on the self-renewal and differentiation ability of human HSCs in vitro and explore the possibility of promoting the expansion potential of HSCs in hypoxia. Moreover, this research also investigates the effect of hypoxia on the stem cell-like properties, which relate to MDR in leukemia cells and aims to exploit the critical relationship between MDR and hypoxic BM microenvironment.Materials and methods:Mononuclear cells (MNCs) were separated by leukapheresis. CD34-positive cells were isolated by magnetic activated cell sorting (MACS). CD34-enriched cells were cultured in growth factors under1%O2(hypoxia) and21%O2(normoxia) for7days. The phenotypes of expanded cells (CD34, CD45and CD61) were analyzed by flowcytometry and the expression of hypoxia inducible factor1α (HIF-1α) was analyzed by real time RT-PCR. After incubation in different oxygen tension, the expanded cells were plated in methyl cellulose conlony assay medium and colony-forming units (BFU-E, CFU-GM and CFU-GEMM) were counted on day14.Multidrug resistant K562/DOX cell line was built up by stepwise increasing the concentrations of doxorubicin in the medium. Then the resistance of the K562/DOX cells to different drugs was testified by MTT assay. The K562/DOX cells and its wild type K562/WT cells were exposed in hypoxia (1%O2) and normoxia(21%) for a period. Drug resistance of these cells in different oxygen tension was measured by MTT assay. The growth curves of the two cell lines under hypoxia and normoxia were observed too. Hypoxia inducible factor1α (HIF-1α), hypoxia inducible factor2a (HIF-2a) as well as stem cell markers Oct4and CD133expression were measured by Western blot. ABC transporter ABCG2and stem cell phenotype CD34were analysed by flow cytometry. In addition, Smad2phosphorylation was measured by Western blot and after TGF-β-RI kinase inhibitor treatment, Oct4and CD133expression in the K562/DOX cells was evaluated by Western blot.Results1. The total nucleated cell count after normoxia culture (12.1±5.4)×106was significantly higher than in hypoxia (9.2±3.37)×106(t=2.749, P<0.05). But in hypoxic culture, the percentage of CD34+cells (3.0±2.71)%was significantly higher than normoxic culture (0.9±1.95)%(t=4.805, P<0.05). The absolute value of CD34+cells after hypoxic culture (0.37±0.3)×106was significantly higher than in normoxia (0.1±0.23)×106too (t=3.513, P<0.05). But no significant difference was found in CD45and CD61phenotype (P>0.05).2. Hypoxia significantly upregulated the mRNA level of HIF-1α by (39±18)%(t=6.280, P<0.05).3. In the colony-forming unit assay, the total colonies were increased slightly by hypoxia, but the difference between hypoxia (43.5±22.9/103cells) and normoxia (30.5±27.9/103cells) was not significant (P>0.05). But hypoxia enhanced the colony-forming ability of erythroid progenitor cells significantly shown by increased BFU-E in hypoxia (17.5±11.2/103cells) compared to normoxia (6.5±16.1/103cells)(t=2.327, P<0.05). The colony forming unit assay also suggested the cells expanded in1%oxygen tension (1±1.6/103cells) generated significantly higher number of CFU-GEMM than in normal oxygen tension (0±0.7/103cells)(t=2.764,P<0.05). But for the CFU-GM, no significant difference was observed between the two groups (P>0.05).4. The K562/DOX cells which were induced by doxorubicin showed higher IC50for various drugs. The ICsovalue of doxorubicin in the K562/DOX cells (78.53±15.25μM) was significantly higher than in the K562/WT cells (1.24±0.24μM)(t=9.852, P<0.001); the IC50value of vincristine in the K562/DOX cells (1.636±0.232μM) was significantly higher than in the K562/WT cells (0.021±0.001μM)(t=12.037, P<0.001); the IC50value of cytosine arabinoside in the K562/DOX cells (109.94±16.85μM) was significantly higher than the K562/WT cells (63.02±4.68μM)(t=4.648, P<0.01). The results from flow cytometry showed significantly higher ABCG2expression in hypoxia (6.2±1.7%) than in normoxia (2.6±1.8%)(t=3.195, P<0.05).5. In the K562/WT cells, the IC50value of doxorubicin was enhanced significantly in hypoxia (3.32±0.26μM) compared to normoxia (1.24±0.24μM)0=10.211, P<0.05). But for the K562/DOX cells, no significantly different IC50value of doxorubicin was observed between hypoxic incubation (88.98±15.42μM) and normoxic incubation (78.53±15.25μM)(P>0.05). The effect of hypoxia on drug resistance between the two cell lines was significantly (P<.05) different. Hypoxia also significantly upregulated ABCG2expression both in the K562/WT (from2.6±1.8%tol5.9±4.8%)(t=4.766, P<0.05) and in the K562/DOX cells (from6.2±1.7%to20.6±2.3%)0=8.804, P<0.05).6. Hypoxia induced significant growth inhibition in the K562/WT cells (tds=3.286, P<0.01;td6=2.512, P<0.05;td7=12.84, P<0.001), but for the K562/DOX cells no difference between hypoxic and normoxic cultures (P>0.05). Consistently, HIF-2α in the K562/WT cells was induced significantly in hypoxia (t=2.717, P<0.05), but only slight upregulation in K562/DOX (P>0.05).7. The results from flow cytometry showed that CD34expression which was significantly higher in the K562/DOX cells (1.6±0.6%) than in the K562/WT cells (0.4±0.1%)(t=3.816, P<0.05). When the K562/DOX cells were transferred into hypoxia, CD34protein was upregulated significantly from1.6±0.6%to12.4±1.9%(t=9.265, P<0.05). In the K562/WT cells, CD34protein was also upregulated significantly from0.4±0.1%to9.1±2.4%(t=6.368, P<0.05). The upregulation of CD34in the K562/WT cells was significantly higher than in the K562/DOX cells (t=3.084, P<0.05). Moreover, hypoxia significantly upregulated CD133and Oct4expression in the K562/WT cells (tOct4=3.229,tCD133=2.569, P<0.05). But in the K562/DOX cells, no significant upregulation was detected (P>0.05). For the K562/DOX cells, the hypoxia-induced upregulation of CD133and Oct4was significantly lower than in the wild type cells (tOct4=2.876, tCD133=3.617, P<0.05). 8. The protein level of phospho-Smad2in the K562/DOX cells was significantly higher than in the K562/WT cells (t=5.364,P<0.05). Hypoxia induced the phosphorylation of Smad2in both cell lines (F=5.132, P<0.05).9. After the K562/DOX cells were treated with SD-208, an inhibitor of the TGF-β receptor I kinase, significantly dose-dependent downregulation of CD133and Oct4in hypoxia was detected following the inhibition of TGF-β/Smad signal pathway (FOct4=8.103, FCD133=17.43, P<0.05).Conclusion1. Hypoxia due to poor perfusion in the bone marrow plays an important role on the maintenance of hematopoietic stem cells. In this reseach, purified CD34+cells were cultured in1%oxygen tension with some cytokines and the expansion of these stem/progenitor cells was evaluated after hypoxic incubation. This research verifies hypoxia enhances the self-renewal ability of the hematopoietic stem/progenitor cells, although inhibiting the cell proliferation to some extent. So it can be speculated that in the bone marrow niche with lower oxygen tension maitains the stem/progenitor cells in a quiescent and undifferentiated status, but higher oxygen tension promoting greater differentiation to mature blood cells.2. Hypoxia also contributes to the development of erythroid lineage.3. Hypoxia takes part in the modulation of the self-renewal and differentiation of stem/progenitor cells and the activation of HIF-1α may be involved in this mechanism.4. This study illustrates the importance of hypoxia in stem/progenitor cell expansion in vitro and suggests that we need to take hypoxia into account while thinking about effective expansion.5. Hypoxia existing in this microenvironment has an effect on the modulation of multidrug resistance in leukemic cells. Hypoxic stress enhances the stem cell-like properties in leukemic cells, futhermore contributing to the developing of multidrug resistance. 6. The activation of the TGF-β/Smad signaling pathway may be involved in the regulation of this pathophysiological process.7. This research about hpoxia suggests a new mechanism of MDR and the treatment targeting stem cell phenotypes and TGF-β/Smad signaling pathway may be a new method to reverse MDR. |
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