Hematopoietic stem cells (HSC) are the source of all blood cells, which can differentiate into various hematopoietic hierarchy cells. Physiological level of reactive oxygen species (ROS) plays an important role in regulating functions of HSC as excessive ROS is harmful to HSC. Oxidative reductases and antioxidants can eliminate cellular ROS to maintain ROS homeostasis and thus avoid excessive ROS-caused damages. There are several types of oxidative reductases in cells such as catalase, manganese superoxide dismutase (MnSOD), glutathione peroxidase 1 (GPX1), thioredoxin reductase 1 (Txrnd1) and Nqo1 [NAD(P)H dehydrogenase quinone 1]. However, the functional roles of various oxidative reductases in regulating ROS level in hematopoietic cells remain unclear. This study was to investigate the expression patterns of these oxidative reductases in mouse hematopoietic cells that were sorted out via flow cytometry and to find out important oxidative reductases involving in HSC ROS regulation. The expression of various oxidative reductases was detected by semi-quantitative real-time PCR. The results showed that the expression level of catalase in T cell population was 0.14 times that in LT-HSC population (P < 0.05). The expression levels of MnSOD in CLP population and myeloid cells were 0.56 and 0.47 times that in LT-HSC population respectively (P < 0.05). The expression levels of GPX1 in ST-HSC, GMP, Myeloid cells, MEP, T lymphocytes and B lymphocytes were 1.79, 2.96, 2.07, 0.58, 0.10, 0.6 times that in LT-HSC population respectively (P < 0.05). The expression levels of Txrnd1 in ST-HSC, MPP, CMP, GMP, Myeloid cells, T lymphocytes and B lymphocytes were 3.36, 3.18, 4.19, 6.39, 4.27, 0.016, 0.56 time that in LT-HSC population, respectively (P < 0.05). The expression levels of Nqo1 in ST-HSC, MPP, CMP, GMP, CLP and B cell were 0.30, 0.17, 0.25, 0.10, 0.04, 0.01 times that in LT-HSC population, respectively (P < 0.05). It is concluded that the expression levels of oxidative reductases (catalase, MnSOD, GPX1, Txrnd1 and Nqo1) in hematopoietic hierarchy cells are cell-type specific. It suggests that reductases may play divergent roles in various hematopoietic cell populations. More importantly, the expression level of Nqo1 in LT-HSC population significantly increased as compared with other cell populations, thereby suggesting its unique regulatory role in HSC.
Animals ; Hematopoietic Stem Cells ; enzymology ; Mice ; Mice, Inbred C57BL ; Myeloid Cells ; enzymology ; Oxidation-Reduction ; Oxidative Stress ; Oxidoreductases ; metabolism ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the changes of apoptosis and the activity of caspases 3 and 9 in bone marrow hematopoietic progenitor cells in myelodysplastic syndromes (MDS).

METHODSBone marrow hematopoietic progenitor cells (including both CD(34)(+) and CD(34)(-) cells) were collected by negative selection in 34 patients with MDS. Apoptosis was measured with Annexin V assay and activities of caspases 3 and 9 by spectrophotometer.

RESULTS1. Apoptosis was significantly increased in MDS-RA (39.5%, P < 0.01) and MDS-RAEB (31.0%, P < 0.05), but was not different statistically in MDS-RAEBt/AML (18.8%) compared with that of control. 2. Activities of caspases 3 and 9 increased 45 and 20 fold in MDS-RA, increased 14 and 2 fold in MDS-RAEB, respectively and was not increased in MDS-RAEBt/AML compared with that of control. 3. Apoptosis and activities of caspases 3 and 9 reduced in 3 cases of MDS-RAEB group who progressed into AML.

CONCLUSIONIncreased activities of caspases 3 and 9 may be one of causes of excessive apoptosis in MDS. With progress to AML, activities of caspases 3 and 9 and apoptosis reduced. Reduced activity of caspase 9 may result in apoptosis "escape" and progression into AML.

Apoptosis ; Arsenicals ; pharmacology ; Caspase 3 ; Caspase 9 ; Caspases ; metabolism ; Hematopoietic Stem Cells ; enzymology ; physiology ; Humans ; Myelodysplastic Syndromes ; enzymology ; pathology ; Oxides ; pharmacology

OBJECTIVETo explore the relationship of telomerase RNA component (hTERC) and the telomerase reverse transcriptase (hTERT) with telomerase activity in the marrow hemopoietic stem cells of children with aplastic anemia (AA).

METHODSFifty-two children with chronic AA, 13 children with acute AA and 21 normal controls were enrolled in the study. Telomerase activity and the expression of mRNA of hTERT and hTERC were detected by Telomeric Repeat Amplification Protocol (TRAP) with silver staining and real-time Q-PCR respectively.

RESULTSLevels of telomerase activity in both the chronic and acute AA groups were higher than in the control group (P<0.01). The AA groups had significantly higher expression of hTERT mRNA than the control group (P<0.01). The chronic AA group had higher expression of hTERT mRNA and telomerase activity than the acute AA group (P<0.05). There was no significant difference in the expression of hTERC mRNA among the three groups (P=0.812). There was a significant correlation between the expression of hTERT mRNA and telomerase activity (r=0.660, P<0.01).

CONCLUSIONSExpression of telomerase activity may be involved in the pathophysiology and development of AA, and hTERT plays a crucial role in expression of telomerase activity.

Adolescent ; Anemia, Aplastic ; enzymology ; Child ; Child, Preschool ; Female ; Hematopoietic Stem Cells ; enzymology ; Humans ; Infant ; Male ; RNA ; genetics ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism

To evaluate expression of caspase-3 protein in CD34(+) cells from cord blood (CB) and its significance during culture in vitro with various growth factors, RT-PCR, Western blot and flow cytometry were used to determine cell proliferation, apoptosis and expression of caspase-3 in CD34(+) cells during culture in vitro. The results demonstrated that caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34(+) cells. Expression of caspase-3 mRNA and protein was upregulated when these cells were expanded in suspension culture with growth factors for 3 days. However, only the 32 kD inactive caspase-3 proenzyme was detected in the freshly isolated CD34(+) cells and those cells after 3 days expansion with cytokines. Along with the culture time extension (7 days) in vitro, especially without early-effective cytokines SCF or FL existence, caspase-3 was activated and a cleavage 20 kD was detected. It is concluded that the caspase-3 is involved in apoptosis of primitive CD34(+) cells during expansion in vitro, and early-effective cytokines, SCF and FL, could conserve hematopoietic stem cells and suppress apoptosis.
Antigens, CD34 ; blood ; Apoptosis ; Caspase 3 ; Caspases ; analysis ; physiology ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; enzymology ; physiology ; Humans ; Infant, Newborn ; Reverse Transcriptase Polymerase Chain Reaction

Dipeptidylpeptidase (DPP) 4, also known as CD26, is an enzyme present on the surface of a number of different cell types. It is also found within cells and as a soluble protein in body fluids. It can specifically truncate proteins at the penultimate N-terminus residue for some amino acids, such as alanine, proline, serine, and perhaps others. DPP4 has been implicated in regulating the in vitro and in vivo functional activities of a number of hematopoietically active molecules, and this information, along with that on inhibition of DPP4, has been studied in efforts to enhance hematopoietic cell transplantation (HCT), hematopoiesis after stress in mouse models, and in the clinical setting of single-unit cord blood (CB) HCT. This article reviews the current status of this compound's effects on regulatory proteins, the field of CB HCT, a potential role for modulating DPP4 activity in enhancing single-unit CB HCT in adults, and future aspects in context of other cellular therapies and the area of regenerative medicine.
Animals ; *Cord Blood Stem Cell Transplantation ; Dipeptidyl Peptidase 4/*metabolism ; Dipeptidyl-Peptidase IV Inhibitors/*therapeutic use ; Hematopoiesis/*drug effects ; Hematopoietic Stem Cells/*drug effects/enzymology ; Humans ; Regenerative Medicine/*methods ; Signal Transduction/drug effects

To investigate the expression of telomerase in cord blood hematopoietic stem/progenitor cells during their committed differentiation in vitro and provide an index of monitoring the proliferating potential of the hematopoietic stem/progenitor cells and security for clinical application. Human CD34 positive cells were isolated from umbilical cord blood by using magnetic cell sorting system (MACS), and were induced to differentiation with hematopoietic growth factors (SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO) in a liquid culture system. The telomerase activity and the cytalytic subunit of telomerase (hTERT) of the cells were analysed during different periods of culture by using TRAP-PCR, TRAP-ELISA, Western blot and RT-PCR techniques, respectively. The results showed that a peak of cell growth was achieved on day 14 - 21 during induction of differentiation in vitro. Total cell number could increase 1006.4 +/- 103.2 times and could not increase there after. Telomerase activity and hTERT expression were low in freshly isolated cord blood CD34(+) cells and increased after about 7 days of culture in addition of cytokine combinations of SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO, respectively. The telomerase activity and hTERT decreased after 14 days of culture and were not detected after 28 days of culture. It was concluded that the hematopoietic stem/progenitor cells can be expanded in large number in vitro and do not have the character of immortality and the telomerase activity could be a useful index in hematopoiesis regulation.
Antigens, CD34 ; blood ; Blotting, Western ; Cell Differentiation ; DNA-Binding Proteins ; Enzyme-Linked Immunosorbent Assay ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; enzymology ; Humans ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism

Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway- specific inhibitors for PI3K or FRAP/TOR, two upstream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.
1-Phosphatidylinositol 3-Kinase/metabolism ; Drug Synergism ; Enzyme Activation ; Granulocyte-Macrophage Colony-Stimulating Factor/*pharmacology ; Hematopoietic Stem Cells/*enzymology ; Human ; Phosphorylation/drug effects ; Protein-Serine-Threonine Kinases/metabolism ; Ribosomal Protein S6 Kinases, 70kD/antagonists & inhibitors/*metabolism ; Stem Cell Factor/*pharmacology ; Tacrolimus Binding Protein 1A/metabolism

OBJECTIVETo investigate the effects of panax notoginseng saponins (PNS) on expression, regulation and phosphorylation of multiple protein kinases in mitogen activated protein kinase (MAPK) intracellular signal pathway and GATA transcription factors in hematopoietic cells, so as to explore its mechanism of proliferation and differentiation activity on hematopoiesis.

METHODSThe human granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF-288 and Meg-01 cell lines were treated by PNS, the positive control of K562, CHRF-288 cells treated by recombination human erythropoietin (Epo) and thrombopoietin (Tpo) respectively. The total cell lysate and nuclei protein were extracted after being treated by PNS, subsequently, analyzed by both Western blot and immune-precipitation. Meanwhile, the nuclei extract was performed for electrophoretic mobility shift assay (EMSA) by using (32)P radio labeled double-stranded GATA consensus oligonucleotide.

RESULTSThe expression levels of kinase MEK-1, MEK-2, ERK-1, ERK-2, AKT-1, AKT-2 and PI-3K were increased by PNS treatment to different extent in four cell lines, depending on cellular heterogeneity and sensitivity to PNS, also phosphorylation of MEK-1, ERK-1 was differentially promoted by PNS respectively P<0.05, 0.01, 0.001). The expression levels of transcription factors GATA-1 and GATA-2 were increased, moreover, their DNA binding activities were raised dramatically in PNS treated K562, CHRF-288 and Meg-01 cells compared with the controls respectively (P<0.05, 0.01, 0.001). The positive control of K562, CHRF-288 cells treated by Epo or Tpo respectively also displayed up-regulation of protein kinases and GATA transcription factors respectively (P<0.05, 0.01, 0.001).

CONCLUSIONThe results indicated that intracellular signal pathway initiated by PNS was involved in MAPK pathway and transcription factors of GATA family in hematopoietic cells. PNS displayed the role to promote proliferation and differentiation, by means of increasing expression level and phosphorylation status of multiple protein kinases, also inducing synthesis of GATA transcription factors and upregulation its DNA binding activity.

Blotting, Western ; Cell Line, Tumor ; DNA ; metabolism ; Electrophoretic Mobility Shift Assay ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; GATA Transcription Factors ; metabolism ; Hematopoietic Stem Cells ; drug effects ; enzymology ; Humans ; Immunoprecipitation ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Panax notoginseng ; chemistry ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Protein Binding ; drug effects ; Protein Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Saponins ; pharmacology ; Up-Regulation ; drug effects