Antiallergic Effects of N,N-dicoumaroylspermidine Isolated from Lithospermum erythrorhizon on Mast Cells and Ovalbumin-Induced Allergic Rhinitis.

In East Asia, the dried root of Lithospermum erythrorhizon has been utilized as an anti-inflammatory, antipyretic, detoxifying, and anti-inflammatory agent. Recently, we reported that L. erythrorhizon protects against allergic rhinitis; however, the component within L. erythrorhizon that exerts antiallergic activity remains unknown. The purpose of the current study was to isolate and characterize the antiallergic active components in an ethanolic extract of L. erythrorhizon roots. We examined the antiallergic effects of L. erythrorhizon reflux ethanol extracts in an ovalbumin (OVA)-induced allergic rhinitis mouse model, and compared the chemical compounds extracted using the hot reflux and cold extraction methods. Chromatographic separation identified two novel anthraquinones, erythrin A and B, one newly discovered compound from the Lithospermum genus, N1″,N3″-dicoumaroylspermidine, and nineteen other recognized compounds. Their chemical structures were elucidated by single (1D) and 2D analysis of nuclear magnetic resonance (NMR) spectroscopic data, as well as high resolution mass spectrometry. Among the identified compounds, N,N'-dicoumaroylspermidine strongly inhibited the release of ß-hexosaminidase, as well as the production of IL-3, IL-4, and IL-13 by IgE-sensitized and BSA-stimulated RBL-2H3 cells. Using the OVA-induced allergic rhinitis mouse model, we showed that N,N'-dicoumaroylspermidine reduced the production of serum OVA-specific IgE and the number of inflammatory cells in nasal lavage fluid. N,N'-dicoumaroylspermidine isolated from L. erythrorhizon exhibits antiallergic properties, making it potentially effective for allergic rhinitis.

Effect of fetal liver condition media derived cytokines(IL-6 and Flt-3) on human bone marrow stem cells colony formation.

Earlier research from our laboratory demonstrated the presence of stimulatory activity of different growth factors in the fetal liver (FL) extracts when collected in a medium known as fetal liver conditioned medium (FLCM) using Enzyme-linked Immunosorbent Assay (ELISA). In the present study, we have assessed two other cytokines viz. IL-6 and FMS like tyrosine kinase-3 (Flt-3) with the help of bioneutralization assay. FLCM was prepared by incubating fetal liver cells with Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum (FBS) and 10% Phytohemagglutinin and collected after 24hrs, 48hrs, 72 hrs. and on the 7th day of incubation. Clonal cultures were established for 1 X 105 normal bone marrow (BM) mononuclear cells (NBM MNC) per plate with methylcellulose medium containing cytokines SCF and EPO. Mean Colony forming units-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were assessed with and without the addition of FLCM. It was found that FLCM enhanced the number of colonies made by NBM MNCs. Further, cytokines IL-6 and Flt-3, present in FLCM, were bioneutralized with respective anti-cytokine antibodies. Neutralized FLCM was evaluated for the colony-forming potential of CFU-GEMM colonies. The maximum reduction of 42% was seen with 20 ng/ml of anti-IL-6 antibody. Maximum suppression up to 20% was observed with 0.7 ng/ml of anti Flt-3 antibody for CFU-GEMM colonies. Presence of cytokines IL-6 and Flt-3 in FL extracts and their colony stimulatory activity suggests that fetal liver infusion (FLI) may be a valuable alternative for managing BM recovery in certain clinical conditions such as AA.

Characterisation of the porcine cytokines which activate the CD131ßc common sub-unit, for potential immune-augmentation.

Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.

[Effect of total saponins from Sanguisorba officinalis on megakaryocyte progenitor cells proliferation, differentiation and relative receptor expression].

OBJECTIVE: To investigate the effects of total saponins from Sanguisorba officinalis (DYS) on hematopoietic cell proliferation, differentiation and the expression level of IL-3R and c-kit. METHOD: Baf3 and 32D cells were cultured with or without IL-3, then the cells were exposed to DYS in different concentrations of 5, 10, 20, 30 and 40 mg x L(-1) for 24, 48, 72 and 96 hours separately. After that, the cell proliferation and differentiation capacity were determinated by the methods of CCK8 and Giemsa staining separately. The effects of DYS on the expression level of IL-3 receptor in Baf3 cells and the expression level of c-kit in 32D cells were determinated using RT-PCR. RESULT: DYS promotes alone proliferation of Baf3 cells and 32D cells after 48 h. In contrast to control cells, 32D cells containing DYS without IL-3 form many large clusters. DYS also increases the proliferation when cultured with IL-3. High concentration of DYS induce alone the differentiation of 32D cells and increase alone the number of the polyploidy megakaryocyte. Moreover, DYS increases alone the expression level of IL-3R in Baf3 cells and the expression level of c-kit in 32D cells separately. CONCLUSION: Our data shows DYS can promote alone proliferation and differentiation of megakaryocyte progenitor cells. The proliferative and differentiative effect of DYS on megakaryocyte progenitor cells is correlated to the up-regulation of IL-3 receptor and c-kit expression.

A novel and simple hollow-fiber assay for in vivo evaluation of nonpeptidyl thrombopoietin receptor agonists.

Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult because of the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to preclinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for nonpeptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. Cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a nonpeptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signaling transducers and activators of transcription in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.

Cytotoxic action of Ganoderma lucidum on interleukin-3 dependent lymphoma DA-1 cells: involvement of apoptosis proteins.

Aqueous extracts and a semipurified fraction obtained by methanol extraction and column chromatography were isolated from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] and their effects on interleukin 3-dependent lymphoma cells (DA-1) were studied. Cell viability was reduced by the action of unboiled aqueous extract and by the methanol-extracted column-chromatography semipurified fraction, producing DNA fragmentation in DA-1 cells. Treatments with aqueous extracts showed increments of Bax after 13 h, increments of p53 and Mdm2 after 19 h and a reduction of these three proteins after 24 h. The methanol-extracted semipurified fraction also induced increments of p53 and Mdm2 factors at 19 h with a reduction after 24 h. The methanol-extracted column-chromatography semipurified fraction from Ganoderma lucidum produced minor changes in the level of Akt after treatments for 19 h in DA-1 cells with a slight reduction in the levels of NFkB-p65 factor. Both the unboiled aqueous extract and the methanol-extracted column-chromatography semipurified fraction produced cleavage of inactive caspase 3, as a clear indication of induction of apoptosis by compounds present in Ganoderma lucidum.

Characterization of variant diphtheria toxin-interleukin-3 fusion protein, DTIL3K116W, for phase I clinical trials.

We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin-interleukin-3 fusion protein, for treatment of acute myeloid leukemia. The product was filter sterilized, aseptically vialed, and stored at -80 degrees C. It was characterized by Coomassie-stained SDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy, receptor binding affinity, ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography-HPLC, sequencing, and immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296EU/mg. Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies. Potency study revealed a 48-h EC(50) of 0.5 pM on TF1/H-ras cell. Its binding properties were confirmed by competitive experiments showing IC(50) of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at -80 degrees C. Further, the drug was stable at 4 and 25 degrees C in the plastic syringe and administration tubing for 48 h.

Licochalcone A is a potent inhibitor of TEL-Jak2-mediated transformation through the specific inhibition of Stat3 activation.

Aberrant activation of Jak/Stat signaling causes a number of hematopoietic disorders and oncogenesis, and therefore the effective inhibitors of the Jak/Stat signaling pathway may be therapeutically useful. TEL-Jak2 gene fusion, which has been identified in human leukemia, encodes a chimeric protein endowed with constitutive tyrosine kinase activity. Expression of TEL-Jak2 protects Ba/F3 cells from IL-3 withdrawal-induced apoptotic cell death and leads to IL-3-independent growth. However, its mechanisms remain to be only partially understood. Here, we first found that Licochalcone A, one of the flavonoids isolated from the root of Glycyrrhiza inflate, inhibited TEL-Jak2-mediated cell proliferation and survival in the absence of IL-3. Licochalcone A failed to inhibit the activity of TEL-Jak2, however, this induced apoptosis of TEL-Jak2-transformed cells with a much lower concentration in the absence of IL-3 than in the presence of IL-3. Interestingly, Licochalcone A significantly inhibited the phosphorylation and nuclear localization of Stat3, which is essential for TEL-Jak2-induced cell transformation. These data suggest that Licochalcone A is a specific inhibitor for Stat3 and would be employed for the treatment of various diseases caused by disorders of the Jak/Stat pathway.

CFU-megakaryocytic progenitors expanded ex vivo from cord blood maintain their in vitro homing potential and express matrix metalloproteinases.

BACKGROUND: In patients transplanted with cord blood (CB), prolonged thrombocytopenia is a major complication. However, this could be alleviated by supplementing the CB graft with ex vivo-expanded megakaryocytic progenitors (CFU-Meg), provided that the homing properties of these cells are not affected negatively by expansion. METHODS AND RESULTS: We assessed the in vitro homing potential of CFU-Meg progenitors expanded from CB and showed that the combination of thrombopoietin (TPO) with interleukin-3 (IL-3) used for expansion not only results in optimal proliferation of CFU-Meg but also protects these cells from apoptosis. Moreover, we found that ex vivo-expanded CFU-Meg maintained expression of the CXCR4 receptor throughout a 9-day culture and were chemoattracted towards a stromal cell-derived factor-1 (SDF-1) gradient. They also expressed matrix metalloproteinase-9 (MMP-9) and membrane-type (MT) 1-MMP, and transmigrated across the reconstituted basement membrane Matrigel. Finally, we observed that SDF-1 up-regulated the expression of both MMP-9 and MT1-MMP in CB CD34(+) cells and ex vivo-expanded CFU-Meg. DISCUSSION: We suggest that CB-expanded CFU-Meg, in particular those from day 3 of expansion, when their proliferation and in vitro homing potential are maximal, could be employed to supplement CB grafts and speed up platelet recovery in transplant recipients.

FHL3 negatively regulates human high-affinity IgE receptor beta-chain gene expression by acting as a transcriptional co-repressor of MZF-1.

The high-affinity IgE receptor FcepsilonRI plays a key role in triggering allergic reactions. We recently reported that human FcepsilonRI beta-chain gene expression was down-regulated by a transcription factor, MZF-1, through an element in the fourth intron. In the present study, we found that this transcriptional repression by MZF-1 required FHL3 (four and a half LIM domain protein 3) as a cofactor. Yeast two-hybrid and immunoprecipitation assays demonstrated that FHL3 bound MZF-1 in vitro and in vivo. Overexpression of FHL3 in KU812 cells suppressed the beta-chain promoter activity through the element in the fourth intron in an MZF-1-dependent manner. Furthermore, results from pull-down assays and gel-filtration chromatography employing nuclear extracts indicated that MZF-1 and FHL3 formed a complex of high molecular mass with some additional proteins in the nucleus. Granulocyte-macrophage colony-stimulating factor, which was reported to decrease FcepsilonRI expression, induced the accumulation of FHL3 in the nucleus, in accordance with the repressive role of FHL3 in beta-chain gene expression.

Soluble c-kit receptor mobilizes hematopoietic stem cells to peripheral blood in mice.

OBJECTIVE: The mechanisms of mobilization of hematopoietic stem cells (HSC) from bone marrow to peripheral blood (PB) by cytokines are poorly understood. One hypothesis is that cytokines disrupt cytoadhesive interactions of stem cells with bone marrow stroma. The soluble portion of c-kit (s-kit) binds stem cell factor (SCF) and can specifically block the ability of SCF to bind HSC. MATERIALS AND METHODS: To examine stem cell mobilization by s-kit, we prepared PB mononuclear cells from s-kit- or granulocyte colony-stimulating factor (G-CSF)-treated mice and assayed their colony-forming abilities and their long-term reconstituting abilities by transplantation into lethally irradiated Ly-5.2 congenic mice. RESULTS: We confirmed the published findings that human recombinant s-kit can block SCF-stimulated hematopoietic colony growing. We then found that s-kit could mobilize colony-forming cells from bone marrow to PB, and we found long-term reconstitution cells in the PB from s-kit-treated mice. The majority of s-kit-mobilized stem cells were in the CD34(+) cell population. We also tested the additive effect between G-CSF and s-kit. The mean percentages of donor cells in the mice transplanted with Lin(-) cells from the G-CSF-treated mice and the G-CSF/s-kit-treated mice were 44.6% and 64.8%, respectively (p=0.028). CONCLUSIONS: These findings demonstrate that stem cells with long-term engraftment capabilities can be mobilized by s-kit, and that s-kit combined with G-CSF treatment leads to significant enhancement of engraftment efficiency, suggesting mobilization via disruption between c-kit and SCF as the mechanism.

The TEL/ARG leukemia oncogene promotes viability and hyperresponsiveness to hematopoietic growth factors.

The TEL/ARG oncogene associated with acute myeloid leukemia is formed by the t(1;12)(q25;p13) reciprocal translocation, which fuses part of the TEL gene to the tyrosine kinase, c-ARG. In an effort to determine the biological effects and investigate signaling of the TEL/ARG fusion protein, multiple sublines of Ba/F3 cells were generated in which a TEL/ARG complementary DNA was expressed under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline, a tetracycline analogue, rapidly induced expression of the TEL/ARG protein. TEL/ARG was heavily phosphorylated on tyrosine residues and was also found to rapidly induce tyrosine phosphorylation of multiple cellular proteins, including rasGAP, CBL, STAT5, PI3K, SHP2, Dok, and SHC. The Ba/F3-tet-TEL/ARG cells remained interleukin (IL)-3 dependent without doxycycline but with doxycycline displayed a marked reduction in cell death in the absence of IL-3. TEL/ ARG cells also displayed an enhanced proliferative response to IL-3 and to insulin-like growth factor 1. At least in Ba/F3 cells, although the growth rate was much lower compared to that with IL-3, TEL/ARG appeared to induce some cell proliferation as an immediate consequence. Nonetheless, the hyperresponsiveness to growth factors reported here is more likely to contribute to the pathogenesis of leukemia.

Diphtheria toxin fused to variant interleukin-3 provides enhanced binding to the interleukin-3 receptor and more potent leukemia cell cytotoxicity.

Chemoresistance is a common cause of treatment failure in patients with acute myeloid leukemia (AML). We generated a diphtheria toxin (DT) fusion protein composed of the catalytic and translocation domains of DT (DT388) fused to interleukin-3 (IL-3). IL-3 receptors (IL-3R) are overexpressed on blasts from many AML patients. DT388IL-3 showed cytotoxicity to leukemic blasts in vitro and in vivo and minimal damage to normal tissues in nonhuman primate models. However, only a fraction of patient leukemic samples were sensitive to the agent. To enhance the potency and specificity of the DT388IL-3 molecule, we constructed variants with altered residues in the IL-3 moiety. Two of these variants, DT388IL-3[K116W] and DT388IL-3[Delta125-133], were produced and partially purified from Escherichia coli with excellent yields. They showed enhanced binding to the human IL-3R and greater cytotoxicity to human leukemia cell lines relative to wild-type DT388IL-3. Interestingly, the results support a previously hypothesized model for interaction of the C-terminal residues of IL-3 with a hydrophobic patch on the alpha-subunit of IL-3R. Rational modification of the targeting domain based on structural analysis can produce a fusion toxin with increased ability to kill tumor cells. One or both of these variant fusion proteins merit further development for therapy of chemotherapy refractory AML.

Supplementation of conventional freezing medium with a combination of catalase and trehalose results in better protection of surface molecules and functionality of hematopoietic cells.

Our previous studies had shown that a combination of the bio-antioxidant catalase and the membrane stabilizer trehalose in the conventional freezing mixture affords better cryoprotection to hematopoietic cells as judged by clonogenic assays. In the present investigation, we extended these studies using several parameters like responsiveness to growth factors, expression of growth factor receptors, adhesion assays, adhesion molecule expression, and long-term culture-forming ability. Cells were frozen with (test cells) or without additives (control cells) in the conventional medium containing 10% dimethylsulfoxide (DMSO). Experiments were done on mononuclear cells (MNC) from cord blood/fetal liver hematopoietic cells (CB/FL) and CD34(+) cells isolated from frozen MNC. Our results showed that the responsiveness of test cells to the two early-acting cytokines, viz. interleukin-3 (IL-3) and stem cell factor (SCF) in CFU assays was better than control cells as seen by higher colony formation at limiting concentrations of these cytokines. We, therefore, analyzed the expression of these two growth factor receptors by flow cytometry. We found that in cryopreserved test MNC, as well as CD34(+) cells isolated from them, the expression of both cytokine receptors was two- to three-fold higher than control MNC and CD34(+) cells isolated from them. Adhesion assays carried out with CB/FL-derived CD34(+) cells and KG1a cells showed significantly higher adherence of test cells to M210B4 than respective control cells. Cryopreserved test MNC as well as CD34(+) cells isolated from them showed increased expression of adhesion molecules like CD43, CD44, CD49d, and CD49e. On isolated CD34(+) cells and KG1a cells, there was a two- to three-fold increase in a double-positive population expressing CD34/L-selectin in test cells as compared to control cells. Long-term cultures (LTC) were set up with frozen MNC as well as with CD34(+) cells. Clonogenic cells from LTC were enumerated at the end of the fifth week. There was a significantly increased formation of CFU from test cells than from control cells, indicating better preservation of early progenitors in test cells. Our results suggest that use of a combination of catalase and trehalose as a supplement in the conventional freezing medium results in better protection of growth factor receptors, adhesion molecules, and functionality of hematopoietic cells, yielding a better graft quality.

Isolation of (+)-catechin and (-)-epicatechin from Actinidia arguta as bone marrow cell proliferation promoting compounds.

The MeOH extract of stems of Actinidia arguta promoted proliferation of cultured bone marrow cells and stimulated formation of myeloid colonies from bone marrow cells. (+)-Catechin ( 1) and (-)-epicatechin ( 2) were isolated as active compounds from the MeOH extract. Compounds 1 and 2 stimulated the cell proliferation in a concentration-dependent manner in the range of 1 to 100 mg/mL. Compounds 1 and 2 also stimulated formation of myeloid colonies and enhanced the effect of interleukin-3 (IL-3) to increase the number of colony forming-units in culture (CFU-c). In an ex vivo experiment using a model mouse of decreasing bone marrow functions, orally administrated 1 (100 mg/kg/day) stimulated IL-3-induced CFU-c formation of the bone marrow cells.

Mapping gene expression patterns during myeloid differentiation using the EML hematopoietic progenitor cell line.

OBJECTIVE: The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line. METHODS: Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML. RESULTS: Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes. CONCLUSION: Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.

Serum suppresses myeloid progenitor apoptosis by regulating iron homeostasis.

The growth and survival of committed hematopoietic progenitors is dependent upon cytokine signaling. However, serum is also required for optimal growth of these progenitors in culture ex vivo. Here we report that serum withdrawal leads to myeloid progenitor cell apoptosis. Although serum deprivation-induced cell death has many hallmarks typical of apoptosis, these cell deaths were not inhibited by hemopoietins, survival factors such as IGF-I, or treatment with a broad-spectrum caspase inhibitor. Rather, apoptosis due to serum withdrawal was associated with damage to mitochondria. Surprisingly the serum factor required for myeloid cell survival was identified as iron, and loss of iron led to marked reductions in ATP production. Furthermore, supplementing serum-deprived myeloid cells with bound or free iron promoted cell survival and prevented mitochondrial damage. Therefore, serum suppresses hematopoietic cell apoptosis by providing an obligate source of iron and iron homeostasis is critical for proper myeloid cell metabolism and survival.

[The expression of human telomerase reverse transcriptase gene in cord blood hematopoietic stem cells and its significance].

OBJECTIVE: To investigate the expression and significance of human telomerase reverse transcriptase (hTERT) gene in cord blood hematopoietic stem cells. METHODS: Using in situ hybridization techniques, we detect the expression of hTERT gene in cord blood hematopoietic stem cells in different condition during different culture time. RESULTS: hTERT gene was lowly expressed in freshly isolated cord blood CD34+ cells; the positive rate was 13%. It could be increased in 5-7 days when the cells were cultured in vitro, especially in the presence of stem cell factor, interleukin-3(IL-3), IL-6 and Flt-3 Ligand together; the positive rate reached 48%. Transform growth factor-beta 1 and all-trans retinoic acid could repress hTERT gene. CONCLUSION: hTERT gene was lowly expressed in the cord blood CD34+ cells; it could be upregulated in the culture in vitro along with optimal cytokines, but was downregulated by negative regulator and induction differentiation.

Stroma-derived factor 1alpha induces a selective inhibition of human erythroid development via the functional upregulation of Fas/CD95 ligand.

CXC chemokine receptor 4 (CXCR4), the high-affinity receptor for stroma-derived factor 1alpha (SDF-1alpha), shows distinct patterns of expression in human CD34+ haematopoietic progenitor cells induced to differentiate in vitro along the granulocytic and erythroid lineages. In serum-free liquid cultures supplemented with stem cell factor (SCF), interleukin 3 (IL-3) and granulocyte colony-stimulating factor, the expression of surface CXCR4 progressively increased in cells differentiating along the granulocytic lineage. The addition in culture of 200 ng/ml of SDF-1alpha, a concentration which maximally activated intracellular Ca2+ flux, only modestly affected the expression levels of CD15 and CD11b granulocytic antigens, as well as the total number of viable cells. On the other hand, in liquid cultures supplemented with SCF, IL-3 and erythropoietin, SDF-1alpha induced the downregulation of glycophorin A erythroid antigen, accompanied by a progressive decline in the number of viable erythroblasts. Moreover, in semisolid assays, SDF-1alpha significantly reduced the number of plurifocal erythroid colonies (erythroid blast-forming units; BFU-E), whereas it did not affect granulocyte-macrophage colony-forming units (CFU-GM). We also demonstrated that the inhibitory effect of SDF-1alpha on glycophorin A+ erythroid cell development was mediated by the functional upregulation of CD95L in erythroid cultures. These data indicate that SDF-1alpha plays a role as a negative regulator of erythropoiesis.

Effects of deregulated Raf activation on integrin, cytokine-receptor expression and the induction of apoptosis in hematopoietic cells.

The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines. The conditionally active Raf proteins were regulated by beta-estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (deltaRaf:ER). Continuous deltaRaf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/deltaRaf:ER cells as they grew in large aggregated masses (>100 cells) whereas the parental cytokine-dependent FDC-P1 cells grew in smaller grape-like clusters (< 10 cells). FD/deltaRaf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and beta-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of deltaRaf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited 3H-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation.