Sequential administration of delta-tocotrienol ameliorates radiation-induced myelosuppression in mice and non-human primates through inducing G-CSF production.

To date only four recombinant growth factors, including Filgrastim (rhG-CSF), have been approved by FDA as radiomitigators to ameliorate hematopoietic acute radiation syndrome (H-ARS). These approved agents are not stable under room-temperature, needing to be stored at 2-8 °C, and would not be feasible in a mass casualty scenario where rapid and cost-effective intervention is crucial. Delta-tocotrienol (δ-T3H), the most potent G-CSF-inducing agent among vitamin E isoforms, exhibited efficiency and selectivity on G-CSF production in comparison with TLR and STING agonists in mice. Five-dose δ-T3H was utilized as the optimal therapeutic regimen due to long-term G-CSF production and the best peripheral blood (PB) recovery of irradiated mice. Comparable with rhG-CSF, sequential administration of δ-T3H post-irradiation improved hematologic recovery and accelerated the regeneration of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow (BM) and spleen of 6.5Gy irradiated mice; and consistently enhanced repopulation of BM-HSCs. In 4.0Gy irradiated nonhuman primates, δ-T3H exhibited comparable efficacy as rhG-CSF to promote PB recovery and colony-formation of BM-HPCs. Altogether, we demonstrated that sequential administration of delta-tocotrienol ameliorates radiation-induced myelosuppression in mice and non-human primates through inducing G-CSF production, indicated δ-T3H as a promising radiomitigator for the management of H-ARS, particularly in a mass casualty scenario.

Vitamin B complex blocks the dust fall PM2 .5 -induced acute lung injury through DNA methylation in rats.

This study aimed to explore whether vitamin B complex (folic acid, B6 , and B12 ) could avert DNA methylation changes associated with inflammation induced by acute PM2.5 exposure. Sprague-Dawley rats were administered by gavage with different concentrations of vitamin B complex once a day for 28 days, and then by intratracheal instillation with saline or PM2.5 once every 2 days for three times. Vitamin B continued to be taken during the PM2.5 exposure. Rats were sacrificed 24 h after the last exposure. The results showed that vitamin B complex could block the pathological changes and injury in lungs induced by PM2.5 . Meanwhile, vitamin B complex could prevent the abnormal DNA methylation of IL-4 and IFN-γ to antagonize the imbalance of IL-4/IFN-γ associated with inflammation. It was further found that vitamin B complex could regulate DNA methyltransferases (DNMTs) and increase the S-adenosylmethionine (SAM)/S-Adenosyl-L-homocysteine (SAH) ratio to reverse the hypomethylation of genomic DNA and the abnormal DNA methylation of IL-4 and IFN-γ. In conclusion, vitamin B complex has a protective effect on acute lung injury by attenuating abnormal DNA methylation induced by PM2.5 in rats. This study may provide a new insight into the physiological function of vitamin B to prevent the health effects induced by PM2.5 .

Identification of a new Mpl-interacting protein, Atp5d.

Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the δ subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling.

Pennogenin tetraglycoside induces rat myometrial contraction and MLC20 phosphorylation via PLC-IP(3) and RhoA/Rho kinase signaling pathways.

BACKGROUND: Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been widely used in China for the treatment of abnormal uterine bleeding. We previously studied the main active constituents of TSSPs and their structure-activity relationships with respect to rat myometrial contractions. Tg (pennogenin tetraglycoside) was identified as one of the active ingredients in TSSPs able to induce rat myometrial contractions. However, the mechanisms underlying the pharmacological actions on uterine activity have not been described clearly. METHODS: Here Tg was screened for effects on contractile activity in isolated uterine strips from estrogen-primed rats and on MLC20 phosphorylation and related signaling pathways in cultured rat myometrial cells as determined by Western blot. Intracellular calcium ([Ca(2+)](i)) was monitored under a confocal microscope using Fluo-4 AM-loaded myometrial cells. RESULTS: Tg dose-dependently stimulated rat myometrial contractions as well as MLC20 phosphorylation in vitro, which could be completely suppressed by an inhibitor of myosin light chain kinase (MLCK). Use of Ca(2+) channel blockers and kinase inhibitors demonstrated that Tg-induced myometrial contractions are mediated by activation of the phospholipase C (PLC)-inositol triphosphate (IP3) signaling pathway, resulting in increased MLC20 phosphorylation. Furthermore, Y27632, a specific inhibitor of Rho kinase (ROK), notably suppressed Tg-stimulated myometrial contractions and decreased MLC20 phosphorylation. CONCLUSIONS: These data provide evidence that rat myometrial contractility induced by Tg results from enhanced MLC20 phosphorylation, while both PLC-IP3 and RhoA/ROK signaling pathways mediate the process. These mechanisms may be responsible for the therapeutic effects of TSSPs on abnormal uterine bleeding.

Pennogenin tetraglycoside stimulates secretion-dependent activation of rat platelets: evidence for critical roles of adenosine diphosphate receptor signal pathways.

BACKGROUND: Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been demonstrated to promote hemostasis in vivo and induce platelet aggregation in vitro. Pennogenin tetraglycoside (Tg) has been identified as one of the active ingredients in TSSPs and can induce rat platelet aggregation. OBJECTIVE: To investigate the functional role of Tg in platelets and the signaling pathway mechanisms which mediate Tg-induced platelet aggregation. METHODS AND RESULTS: Using scanning electron microscopy, the turbidimetric method and flow cytometry, we demonstrated that Tg induces shape change and concentration-dependently induces aggregation, dense granule secretion and a-granule secretion in rat platelets. The activation characteristics were comprehensively confirmed using transmission electron microscopy. Apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12, completely inhibited Tg-induced platelet aggregation, which was not sensitive to indomethacin or SQ29548 inhibition. Furthermore, ADP receptor antagonists inhibited Tg-induced a-granule secretion, and blockade of the P2Y1 receptor prevented Tg-induced platelet shape changes. Tg-induced dense granule secretion was not affected by ADP receptor antagonists or various various pharmacological inhibitors of the intracellular effectors involved in dense granule secretion signaling pathways. CONCLUSION: We identified that Tg directly induces platelet activation and demonstrated that Tg-induced platelet activation depends on dense granule secretion of ADP, which in turn activates the P2Y1 and P2Y12 receptor signaling pathways.

High dose granulocyte colony-stimulating factor enhances survival and hematopoietic reconstruction in canines irradiated by 2.3 Gy mixed fission neutron and gamma ray.

This study was purposed to evaluate the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on hematopoietic reconstruction and survival in beagles exposed to mixed fission neutron and γ-ray. 13 beagles were unilaterally exposed to single dose of 2.3 Gy 90% neutrons. The experiments were divided into 3 groups: irradiation control group (no any treatment, n = 4), supportive care group (n = 5) and rhG-CSF plus supportive care group (n = 4, abbreviated as rhG-CSF group) in which the beagles were subcutaneously injected with 200 µg/kg of rhG-CSF early at half an hour and 24 hours post-irradiation respectively. The results showed that 2.3 Gy 90% neutron irradiation induced a severe acute radiation sickness of bone marrow type. The administration of rhG-CSF increased the survival rate from 60% in supportive care group to 100%. Twice injection of rhG-CSF in the first 24 hours reduced duration of neutropenia, enhanced neutrophil nadir and promoted neutrophil recovery when compared with control cohort administered clinical support. The number of colony-forming cells (CFU-GM, CFU-E, and BFU-E) in peripheral blood of rhG-CSF treated canines increased 2-to 5-fold relative to those of the supportive care group on day 3. All canines treated with rhG-CSF achieved hematopoietic reconstruction as evidenced by the pathological section of sternum while severe shortage of hemopoietic cells remained in the cohorts given supportive care alone. It is concluded that the combination of supportive care and high-dose rhG-CSF can accelerate hematopoietic recovery and enhance survival of dogs exposed to 2.3 Gy mixed neutron and gamma ray.

RhG-CSF improves radiation-induced myelosuppression and survival in the canine exposed to fission neutron irradiation.

Fission-neutron radiation damage is hard to treat due to its critical injuries to hematopoietic and gastrointestinal systems, and so far few data are available on the therapeutic measures for neutron-radiation syndrome. This study was designed to test the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in dogs which had received 2.3 Gy mixed fission-neutron-γ irradiation with a high ratio of neutrons (~90%). Following irradiation, rhG-CSF treatment induced 100% survival versus 60% in controls. Only two of five rhG-CSF-treated dogs experienced leukopenia (white blood cells [WBC] count < 1.0 × 10(9)/L) and neutropenia (neutrophil [ANC] count < 0.5 × 10(9)/L), whereas all irradiated controls displayed a profound period of leukopenia and neutropenia. Furthermore, administration of rhG-CSF significantly delayed the onset of leukopenia and reduced the duration of leucopenia as compared with controls. In addition, individual dogs in the rhG-CSF-treated group exhibited evident differences in rhG-CSF responsiveness after neutron-irradiation. Finally, histopathological evaluation of the surviving dogs revealed that the incidence and severity of bone marrow, thymus and spleen damage decreased in rhG-CSF-treated dogs as compared with surviving controls. Thus, these results demonstrated that rhG-CSF administration enhanced recovery of myelopoiesis and survival after neutron-irradiation.

[Effect of rhG-CSF on blood coagulation in beagles irradiated by 2.3 Gy neutron].

The aim of this study was to investigate the effect of recombinant human granulocyte stimulating factor (rhG-CSF) on blood coagulation of beagles irradiated by 2.3 Gy neutron so as to provide new therapy for blood coagulation disorder after neutron irradiation. 10 beagles were exposed to 2.3 Gy neutron, and then randomly assigned into supportive care group and rhG-CSF-treated group. The rhG-CSF-treated cohorts were injected subcutaneously with rhG-CSF (10 µg/kg·d) beginning at the day of exposure for 21 consecutive days. Peripheral blood platelet counts were examined once every two days. In vitro platelet aggregation test, thromboelastography and blood clotting tetrachoric tests were also performed. The results indicated that the blood clotting system of irradiated dogs was in hypercoagulable state in the early days after 2.3 Gy neutron irradiation, and became hypocoagulable at crisis later and were mainly on intrinsic coagulation pathway. Blood fibrinogen increased markedly during the course of disease, while platelet counts and aggregation function were decreased remarkably. rhG-CSF administered daily could correct hypercoagulable state induced by 2.3 Gy neutron irradiation at the early time post exposure, shortened the thromboplastin generation time and clotting formation, down-regulated the abnormal high fibrinogen in blood, and improved platelet aggregation function. It is concluded that rhG-CSF can improve coagulation disorders of irradiated dogs.

Structural requirement of spirostanol glycosides for rat uterine contractility and mode of their synergism.

OBJECTIVES: Total steroidal saponins extracted from the rhizome of Paris polyphylla (TSSP) have been used in China for the treatment of abnormal uterine bleeding. The aim of this study was to analyse the structure-activity relationship of steroidal saponins purified from P. polyphylla Sm. var. yunnanensis on rat myometrial contractions, and investigate the synergism among themselves as well as with known inherent agonists, such as Prostaglandin F(2alpha) (PGF-2alpha). METHODS: In this study, 22 steroidal saponins purified from TSSP were screened for their contractile activity in isolated uterine strips from estrogen-primed rats. KEY FINDINGS: It was shown that spirostanol glycosides exhibited inducible or inhibitory activity in rat uterine contraction based on the difference of their structures, which was not only attributed in part to the number, the length and the position of sugar side chains attached by a glycoside, but also related to the structure of the aglycone. Furthermore, synergistic actions were observed among pennogenin or diosgenin glycosides as well as with the known inherent agonist PGF-2alpha, indicating they may share, at least in part, similar pathways with PGF-2alpha in stimulating myometrial contractions. Finally, the contractile response of rat myometrium to spirostanol glycosides was significantly enhanced with advancing pregnancy. CONCLUSIONS: Together, these data support the possibility that some spirostanol glycosides may represent a new type of contractile agonist for the uterus and their synergism may be responsible for the therapeutic effect of TSSP on abnormal uterine bleeding.

Fluacrypyrim, a novel STAT3 activation inhibitor, induces cell cycle arrest and apoptosis in cancer cells harboring constitutively-active STAT3.

STAT3 protein has an important role in oncogenesis and is a promising anticancer target. Herein, we demonstrate that a novel small molecule fluacrypyrim (FAPM) inhibits the growth of leukemia cells by a predominant G1 arrest with significant decrease of the protein and mRNA levels of cyclin D1. As cyclin D1 is transcriptionally regulated by STAT3, FAPM is then shown to markedly inhibit the STAT3 phosphorylation with marginal effect on the other signal transducers and activators of transcription, and without effect on phosphoinositide-3-kinase and mitogen-activated protein kinase pathways. Further analysis shows that FAPM significantly increases the protein tyrosine phosphatases (PTPs) activity in a dose-dependent manner, and the inhibition of PTP activation by sodium pervanadate reverses FAPM-induced suppression of STAT3 tyrosine phosphorylation, indicating an important role of PTP in the action of FAPM. Finally, FAPM treatment results in selective suppression of STAT3-mediated transcriptional activity and its downstream effectors, and subsequent induction of growth arrest and apoptosis in STAT3-dependent cancer cell lines. This study therefore identifies FAPM as a potent STAT3 activation inhibitor with possible therapeutic potential against malignancies with constitutive STAT3 activation.

Melissoidesin G, a diterpenoid purified from Isodon melissoides, induces leukemic-cell apoptosis through induction of redox imbalance and exhibits synergy with other anticancer agents.

Melissoidesin G (MOG) is a new diterpenoid purified from Isodon melissoides, a plant used in Chinese traditional medicine as antitumor and anti-inflammatory agents. In our study, MOG was shown to specifically inhibit the growth of human leukemia cell lines and primary acute myeloid leukemia (AML) blasts via induction of apoptosis, with the evidence of mitochondrial DeltaPsim loss, reactive oxygen species production, caspases activation and nuclear fragmentation. Furthermore, it was shown that thiol-containing antioxidants completely blocked MOG-induced mitochondrial DeltaPsim loss and subsequent cell apoptosis, while the inhibition of apoptosis by benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone only partially attenuated mitochondrial DeltaPsim loss, indicating that MOG-induced redox imbalance is an early event upstream to mitochondrial DeltaPsim loss and caspase-3 activation. Consistently, it was found that MOG rapidly decreased the intracellular glutathione (GSH) content in a dose-dependent manner and the significance of GSH depletion in MOG-induced apoptosis was further supported by the protective effects of tert-butylhydroquinone (tBHQ) and the facilitative effects of DL-buthionine (S,R)-sulfoximine (BSO). Furthermore, it was showed that GSH depletion induced by MOG rendered some leukemia cell lines more sensitive to arsenic trioxide (As2O3), doxorubicin or cisplatin. Additionally, the synergistic apoptotic effects of MOG with As2O3 were detected in HL-60 and primary AML cells, but not in normal cells, suggesting the selective toxicity of their combination to the malignant cells. Together, we proposed that MOG alone or administered with other anticancer agents may provide a novel therapeutic strategy for leukemia.

G1 phase cell cycle arrest induced by SARS-CoV 3a protein via the cyclin D3/pRb pathway.

SARS-CoV 3a is a structural protein, mainly localizing to Golgi apparatus and co-localizing with SARS-CoV M in co-transfected cells. Here we observed that transient expression of 3a inhibited cell growth and prevented 5-bromodeoxyuridine incorporation, suggesting that 3a deregulated cell cycle progression. Cell cycle analysis demonstrated that 3a expression was associated with blockage of cell cycle progression at G1 phase in HEK 293, COS-7, and Vero cells 24-60 h after transfection. Mutation analysis of 3a revealed that C-terminal region (176 aa approximately 274 aa), including a potential calcium ATPase motif, was essential for induction of cell cycle arrest. Topological analysis showed that 3a predominantly located in Golgi apparatus, with its N-terminus residing in the lumen (Nlum) and C-terminus in the cytosol (Ccyt). Analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that 3a expression was correlated with a significant reduction of cyclin D3 level and phosphorylation of retinoblastoma (Rb) protein at Ser-795 and Ser-809/811, not with the expression of cyclin D1, D2, cdk4, and cdk6 in 293 cells. Increases in p53 phosphorylation on Ser-15 were observed in both SARS-CoV M and 3a transfected cells, suggesting that it might not correlate with the 3a-induced G0/G1 phase arrest. The reduction of cyclin D3 level and phosphorylation of Rb were further confirmed in SARS-CoV infected Vero cells. These results indicate that SARS-CoV 3a protein, through limiting the expression of cyclin D3, may inhibit Rb phosphorylation, which in turn leads to a block in the G1 phase of the cell cycle and an inhibition of cell proliferation.

SARS coronavirus 7a protein blocks cell cycle progression at G0/G1 phase via the cyclin D3/pRb pathway.

The genome of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains four structural genes that are homologous to genes found in other coronaviruses, and also contains six subgroup-specific open reading frames (ORFs). Expression of one of these subgroup-specific genes, ORF7a, resulted in apoptosis via a caspase-dependent pathway. Here, we observed that transient expression of ORF7a protein fused with myc or GFP tags at its N or C terminus inhibited cell growth and prevented BrdU incorporation in different cultural cells, suggesting that ORF7a expression may regulate cell cycle progression. Analysis by flow cytometry demonstrated that ORF7a expression was associated with blockage of cell cycle progression at G0/G1 phase in HEK 293 cells after 24 to 60 h post-transfection. Similar results were observed in COS-7 and Vero cells. Mutation analysis of ORF7a revealed that the domain spanning aa 44-82 of 7a protein was essential for its cytoplasmic localization and for induction of the cell cycle arrest. After analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that ORF7a expression was correlated with a significant reduction of cyclin D3 level of mRNA transcription and expression, and phosphorylation of retinoblastoma (Rb) protein at ser795 and ser809/811, not with the expression of cyclin D1, D2, cdk4 and cdk6 in HEK 293 cells. These results suggest that the insufficient expression of cyclin D3 may cause a decreased activity of cyclin D/cdk4/6, resulting in the inhibition of Rb phosphorylation. Accumulation of hypo- or non-phosphorylated pRb thus prevents cell cycle progression at G0/G1 phase.

Subcellular localization and membrane association of SARS-CoV 3a protein.

SARS-CoV 3a protein was a unique protein of SARS coronavirus (SARS-CoV), which was identified in SARS-CoV infected cells and SARS patients' specimen. Recent studies revealed that 3a could interact specifically with many SARS-CoV structural proteins, such as M, E and S protein. Expressed 3a protein was reported to localize to Golgi complex in SARS-CoV infected cells. In this study, it was shown that 3a protein was mainly located in Golgi apparatus with different tags at N- or C-terminus. The localization pattern was similar in different transfected cells. With the assay of truncated 3a protein, it was shown that 3a might contain three transmembrane regions, and the second or third region was properly responsible for Golgi localization. By ultra-centrifugation experiment with different extraction buffers, it was confirmed that 3a was an integral membrane protein and embedded in the phospholipid bilayer. Immunofluorescence assay indicated that 3a was co-localized with M protein in Golgi complex in co-transfected cells. These results provide a new insight for further study of the 3a protein on the pathogenesis of SARS-CoV.

Regulation of LIP level and ROS formation through interaction of H-ferritin with G-CSF receptor.

A variety of hematopoietic factors including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3) and thrombopoietin (TPO) induce a rapid increase of intracellular reactive oxygen species (ROS). ROS induces the activation of many signaling molecules, including Shc, Lck, syk, PKC, MAPK, STAT3, through inhibition of protein phosphatase. Each growth factor has a specific cell-surface receptor, which activates both unique and shared signal transduction pathways. The processes of signal transduction linking cell-surface receptor to the formation of intracellular ROS have not been elucidated fully. Ferritins are composed of two subunit types, H and L, and made of 24 subunits that sequester up to 4500 atoms of iron. When the stored iron atoms are released from H-ferritin, through iron-catalyzed reaction, they have the capacity to promote the formation of ROS. Here, the interaction of G-CSFR and H-ferritin was confirmed by yeast two-hybrid screen, mammalian two-hybrid assays, glutathione-S-transferase (GST) pull-down experiments and immunoprecipitation studies in vitro and in vivo. Additional immunofluorescence assay showed that the two proteins colocalized along the plasma membrane and partly in the cytoplasm. The binding site for H-ferritin was demonstrated to locate to the box3 motif on the C-terminal region of granulocyte colony-stimulating factor receptor (G-CSFR). Furthermore, we found the interaction of full-length G-CSFR with H-ferritin was dissociated at 30 minutes after G-CSF induction and then began to assemble at 45 minutes. The labile iron pool (LIP) is a pool of redox-active iron complexes, which is regulated tightly by the expression of H-ferritin. Experiments showed that the level of LIP increased significantly at 30 minutes after G-CSF stimulation and intracellular ROS formation changed in a pattern similar to LIP response to G-CSF in bone-marrow hematopoietic cells. G-CSF-induced changes in the level of LIP and ROS formation could be blocked by pretreatment with iron chelators that repressed the expression of H-ferritin. In addition, the phosphorylation of STAT3 induced by G-CSF was decreased in iron chelator-treated hematopoietic cells. These data suggested that LIP may be released from the dissociated H-ferritin, and then induce intracellular ROS formation in the bone-marrow hematopoietic cells. ROS, acting as a second messenger, might take part in G-CSF receptor signal transduction. So, here, a new G-CSFR-H-ferritin-LIP-ROS pathway is proposed for regulation of intracellular ROS formation in bone-marrow hematopoietic cells.

[The effects of rhG-CSF and rhSCF on peripheral blood leukocytes and CFU-GM in rhesus monkeys].

To evaluate the effects of rhG-CSF and rhSCF on mobilization of the peripheral blood stem cells, 15 monkeys were divided into control, rhG-CSF 10 micro g/(kg x day) and rhG-CSF 10 micro g/(kg x day) + rhSCF 50 micro g/(kg x day) treated groups. Monkeys were administered with vehicle, rhG-CSF and rhG-CSF + rhSCF subcutaneously once daily for 14 days, respectively. The results showed that the highest counts of leukocyte of rhG-CSF treated group were 411% of baseline value on day 7 after administration, compared with that of rhG-CSF + rhSCF treated group which were 538% on day 9. The highest counts of leukocytes lasted for 3 days in combined treated group. CFU-GM from peripheral blood in the two groups were 8.37 and 11.75 times higher at 5 and 9 days respectively after the mobilization. It is concluded that rhG-CSF significantly increases the number of peripheral blood leukocytes and CFU-GM, and a better effect can be obtained by rhSCF + rhG-CSF combined administration.

[Identification of differentially expressed genes of acute hypoxia-treated HepG2 cells and hypoxia-acclimatized HepG2 cells].

To provide necessary information for further understanding of molecular mechanism of hypoxia acclimatization, the differentially expressed genes of HepG2 cells exposed to normoxia, acute hypoxia-treated cells which were exposed to 1% oxygen for 48 h, and hypoxia-acclimatized HepG2 cells which were cultured for 6 circles of alternate low oxygen (1% oxygen for 24 h) and normal oxygen (21% oxygen for 24 h), were identified respectively by combining the suppression subtractive hybridization (SSH) and cDNA microarray. Thirty-seven genes were expressed differentially in cells exposed to 1% oxygen for 48 h compared with those in cells exposed to normoxia. The expression of all these 37 genes was down-regulated, including the genes participating in cell cycle, cell response to stimulus, and cell signal transduction, and cell cytoskeleton formation, the genes associated with transcription and cell metabolism, 4 expressed sequence tags (ESTs), and 12 genes of which the functions are not known. There is a novel gene sequence, which has not been found in existing databases. There were only 6 genes differentially expressed in the hypoxia-acclimatized cells compared with cells exposed to normoxia, including two mitochondrion genes, metalloprotease-1 gene, ferritin gene, thymosin beta-4 and TPT1 genes. The expressions of mitochondrion ND4, ferritin, thymosin beta-4 and TPT1 were up-regulated, while the expressions of mitochondrion ND1 gene and metalloproease-1 gene were down-regulated. Cell tolerance to hypoxia increased after the cells were hypoxia-acclimatized. The different gene expression patterns of the acute hypoxia-treated cells and the hypoxia-acclimatized cells may be related to the increased tolerance of the cells to hypoxia.