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PubMed1998

Purification of human anti-TF (Thomsen-Friedenreich) and anti-Tn antibodies by affinity chromatography on glycophorin A derivatives and characterization of the antibodies by microtiter plate ELISA.

The TF and Tn antigens were obtained from glycophorin A (GPA) by desialylation under mild acidic conditions and by desialylation followed by Smith degradation, respectively. A method of purification of anti-TF and anti-Tn antibodies from human sera by affinity chromatography on the immobilized asialoGPA (TF antigen) and on asialo-agalactoGPA (Tn antigen), respectively, is described. Purity of the antibodies was demonstrated by SDS-polyacrylamide gel electrophoresis and their specific reactivity with TF or Tn antigens was shown using hemagglutination and the microtiter plate ELISA. A high unspecific binding of human immunoglobulins to the ELISA plates was encountered, therefore optimal conditions for the most specific binding of the antibodies to the target antigens were selected. Problems of the unspecific binding of immunoglobulins were more difficult to overcome when the antibodies were determined in whole sera by their binding to antigen-coated ELISA plates.

Antibodies↗
PubMed1998

MDM2 protein overexpression inhibits apoptosis of TF-1 granulocyte-macrophage colony-stimulating factor-dependent acute myeloblastic leukemia cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for acute myeloblastic leukemia (AML) cells. Murine double minute 2 (MDM2) oncoprotein, a potent inhibitor of wild-type p53 (wtp53), can function both to induce cell proliferation and enhance cell survival, and is frequently overexpressed in leukemias. Therefore, we focused on the importance of MDM2 protein in GM-CSF-dependent versus GM-CSF- independent growth of AML cells. The TF-1 AML cell line, which has both wtp53 and mutant p53 genes, showed GM-CSF-dependent growth; deprivation of GM-CSF resulted in G1 growth arrest and apoptosis. MDM2 mRNA and protein were highly expressed in proliferating TF-1 cells in the presence of GM-CSF and decreased significantly with deprivation of GM-CSF. In contrast, p53 protein increased with GM-CSF deprivation. Ectopic overexpression of MDM2 in TF-1 AML cells conferred resistance to GM-CSF deprivation, and is associated with decreased p53 protein expression. Moreover, a variant of TF-1 cells that grows in a GM-CSF-independent fashion also expressed high levels of MDM2 and low levels of p53. These results suggest that GM-CSF-independent growth of AML cells is associated with overexpression of MDM2 protein and related modulation of p53 expression.

Animals↗
PubMed1997

Lack of correlation between growth of TF-1 cells and tyrosine phosphorylation signals in response to IL-3, IL-5 and GM-CSF.

The human cell line, TF-1, was used to compare responses to interleukin 3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF). TF-1 cells grew well in the presence of any one of the cytokines in early passages. However, the level of tyrosine phosphorylation was minimal in response to IL-5, and detection of a tyrosine phosphorylation signal required high concentrations of IL-5. When grown for longer periods of time in the presence of one of the cytokines, there were dramatic difference in the cells' responses. IL-3 or GM-CSF-grown cells showed only half of the original bioassay response to IL-5. However, cells grown in IL-5 alone kept the same response, and all cells showed the same response to IL-3 and GM-CSF. IL-5-grown cells also had an increased tyrosine phosphorylation signal, along with increased sensitivity to IL-5, yet there was no difference in an IL-5 bioassay. The relative level of detection of tyrosine phosphorylated JAK-2, STAT-5, SHC, and other substrates corresponded to the overall tyrosine phosphorylation signal. IL-5-grown cells had approximately 10-fold more IL-5 receptor alpha subunit message compared to IL-3-grown. These results suggest that response of TF-1 cells to IL-5 may be deceiving in that a good response in a bioassay can be observed with relatively little tyrosine phosphorylation, but an increase in tyrosine phosphorylation can be correlated with an increase in the expression of IL-5 receptor alpha subunit.

Cell Division↗
PubMed1983

Isoelectric focusing with immobilized pH gradients for the analysis of transferrin (Tf) subtypes and variants.

The human serum transferrin (Tf) system was analyzed by isoelectric focusing (IEF) with immobilized pH gradients. For the demonstration of the genetic variability the Fe1-Tf region was chosen. The pH range suitable for analysis of the Tf system was pH 5.20 to pH 5.75. The phenotypes of the common six TfC subtypes are described. No further heterogeneity among TfC1, TfC2, and TfC3 was noted. Aslo presented are the phenotypes of the TfC6 subtype, and of three different TfB and three different TfD variants. IEF with immobilized pH gradients appears to be a suitable method for the analysis of the inherited transferrin polymorphism.

Acrylic Resins↗
PubMed2000

COUP-TF orphan nuclear receptors in development and differentiation.

Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are orphan members of the steroid/thyroid hormone receptor superfamily. They have been shown to negatively regulate the activation function of vitamin D, thyroid hormone, retinoic acid, the retinoid X and the peroxisome proliferator-activated receptors. COUP-TF genes have been cloned from many species and their sequences are exceptionally conserved through evolution. This suggests a critical role for the COUP-TFs in these organisms. Indeed, the Drosophila COUP-TF, seven-up and mouse COUP-TFII are essential for development and differentiation during embryogenesis. Our current understanding of COUP-TF function suggests that they serve vital physiological roles during development despite extensive overlaps of expression. This defines the COUP-TFs as important factors in regulation of development and differentiation in multiple organisms.

Animals↗
PubMed1992

The COUP transcription factor (COUP-TF) is directly involved in the regulation of oxytocin gene expression in luteinizing bovine granulosa cells.

Competition with specific oligonucleotides in DNA-binding experiments, polyacrylamide gel electrophoresis, and recognition by specific antibodies have identified the ubiquitous transcription factor COUP as one of the nuclear proteins binding to the promoter region of the bovine oxytocin gene in endogenously expressing bovine granulosa cells. PCR cloning of partial cDNA sequences for bovine COUP-TF I and II and development of RNase protection assays demonstrated the up-regulation of COUP-TF in bovine granulosa cells and corpus luteum under conditions where the oxytocin gene is switched off. These experimental results from in vitro and in vivo studies point to the direct involvement of COUP-TF in oxytocin gene down-regulation during luteinization of bovine granulosa cells.

Animals↗
PubMed2006

Preanalytical aspects of quantitative TaqMan real-time RT-PCR: applications for TF and VEGF mRNA quantification.

OBJECTIVES: The present paper focuses on preanalytical aspects of tissue factor (TF) and vascular endothelial growth factor (VEGF) mRNA quantification: the choice of blood collection tubes and defining the time frame allowed before processing the sample. DESIGN AND METHODS: Blood was collected from healthy volunteers in K(3) EDTA tubes, CPT, endotoxin-free EndoTube tubes and in PAXgene tubes. Total RNA concentration was determined by absorbance readings at 260 nm with a GeneQuantII UV spectrophotometer. RNA quantity and quality were also determined by the Lab on a Chip technique (Agilent 2100 Bioanalyzer). Real-time RT-PCR assays were performed by the TaqMan technology. RESULTS: The more expensive PAXgene and CPT tubes and the Endo tubes did not give superior results from those obtained in inexpensive routine K(3) EDTA tubes. The PAXgene tubes preserved high molecular mass rRNA better than the other tubes. CONCLUSION: Both the PAXgene system and routine EDTA tubes are suitable for clinical purposes aimed at quantitation of mRNA for TF and VEGF. PAXgene yielded rRNA that was less degraded but had lower mRNA per microg extracted RNA. A time frame up to 24 h until sample processing is acceptable for TF and VEGF mRNA.

DNA, Complementary↗
PubMed2005

Cytokine mediated suppression of TF-1 apoptosis requires PI3K activation and inhibition of Bim expression.

Phosphatidylinositol 3-kinase (PI3K) and its effector protein kinase B (PKB/c-akt) have been implicated as critical mediators of cytokine-induced survival signals. In this study, we have utilized an IL-5 dependent hematopoietic cell line (TF-1) to investigate the signaling events involved in cytokine-dependent erythroblast survival. We demonstrate that IL-5 rescues TF-1 cells from apoptosis through a PI3K/PKB-dependent signaling pathway. Cytokine-withdrawal leads to activation of the Forkhead transcription factor FOXO3a and subsequent expression of the pro-apoptotic Bcl-2 family member Bim. Bim is itself sufficient to induce apoptosis in these cells. Importantly, activation of an inducible active FOXO3a mutant is alone sufficient for upregulation of Bim expression and induction of apoptosis. These data define a mechanism by which survival factors inhibit the default apoptotic pathway and can regulate TF-1 erythroblast survival.

Apoptosis↗
PubMed1998

Cytokine dependent growth of human TF-1 leukemic cell line in human GM-CSF and IL-3 producing transgenic SCID mice.

Although severe combined immunodeficient (SCID) mice are considered useful as an animal model for human hematopoietic diseases, the complete reconstruction of human hematopoietic cells can not be established even in these mice. This appears to be because human cytokines, adhesion molecules and extracellular matrices which support differentiation and growth of human hematopoietic cells differ from those in animals. To improve this animal model, we attempted to produce transgenic (Tg) mice producing human interleukin 3 (hIL-3) and human granulocyte macrophage colony stimulating factor (hGM-CSF) with the homozygote of the scid gene. We established two Tg mouse lines, one releasing both 0.5-1 ng/ml of hIL-3 and 0.05-0.2 ng/ml of hGM-CSF in their sera and another releasing only high (2-10 ng/ml) levels of hGM-CSF. When human cytokine-dependent myeloid cell line, TF-1, was subcutaneously transplanted into these two Tg-SCID mouse lines, TF-1 could be successfully engrafted and grew in all lines of Tg-SCID mice but not in control mice. We also observed that TF-1 grows in GM-CSF Tg-SCID mice in a dose dependent manner in vivo and IL-3 shows an additive effect on its growth. These results indicated that these Tg-SCID mice were an useful in vivo model for investigating human leukemogenesis, especially the role of IL-3 and GM-CSF in leukemogenesis.

Animals↗
PubMed2005

Tf-lipoplex-mediated NGF gene transfer to the CNS: neuronal protection and recovery in an excitotoxic model of brain injury.

The development of efficient systems for in vivo gene transfer to the central nervous system (CNS) may provide a useful therapeutic strategy for the alleviation of several neurological disorders. In this study, we evaluated the feasibility of nonviral gene therapy to the CNS mediated by cationic liposomes. We present evidence of the successful delivery and expression of both a reporter and a therapeutic gene in the rodent brain, as evaluated by immunohistochemical assays. Our results indicate that transferrin-associated cationic liposome/DNA complexes (Tf-lipoplexes) allow a significant enhancement of transfection activity as compared to plain complexes, and that 8/1 (+/-) Tf-lipoplexes constitute the best formulation to mediate in vivo gene transfer. We demonstrated that Tf-lipoplex-mediated nerve growth factor transgene expression attenuates the morphological damages of the kainic acid-induced lesion as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) vital staining. These findings suggest the usefulness of these lipid-based vectors in mediating the delivery of therapeutic genes to the CNS.

Animals↗
PubMed2002

Transforming growth factor-beta1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell-cycle effects.

A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-beta1 has been shown to activate a TGF-beta-activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-beta1 and CD34 overexpression. Our data demonstrate that TGF-beta1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents.

Antigens, CD34↗
PubMed2004

Hydroquinone modulates the GM-CSF signaling pathway in TF-1 cells.

Human leukemogens, including alkylating chemotherapeutic agents and benzene, enhance granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent proliferation of human CD34+ bone marrow (BM) cells. The extracellular signal-regulated kinase (ERK) pathway plays an important role in GM-CSF-dependent proliferation and also has been implicated in the pathogenesis of acute myelogenous leukemia. Therefore, we investigated the effects of the benzene metabolite, hydroquinone (HQ), on alterations in the GM-CSF signaling pathway in TF-1 erythroleukemia cells and human CD34+ BM cells. HQ treatment in TF-1 cells results in a strong proliferative response that is dependent on ERK activation and GM-CSF production. HQ also induces ERK-dependent AP-1 activation with concomitant increased transcriptional activity of AP-1 reporter gene. However, the kinetics of ERK activation are different between rhGM-CSF and HQ in TF-1 cells: rhGM-CSF results in immediate activation of ERK, whereas HQ activation of ERK is delayed. Further, HQ and rhGM-CSF together produce an immediate increase in ERK phosphorylation, which is sustained for over 48 h. HQ also stimulates colony formation, AP-1 DNA binding and GM-CSF production in human CD34+ BM cells. These results suggest that HQ stimulates proliferation via activation of ERK/AP-1 and is at least partially mediated via the production of GM-CSF.

Antigens, CD34↗
PubMed2003

An increase in the expression and total activity of endogenous p60(c-Src) in several factor-independent mutants of a human GM-CSF-dependent leukemia cell line (TF-1).

Growth factor independence of hematopoietic cells can be induced by ectopic expression of a variety of oncogenes encoding receptor or cytoplasmic tyrosine kinases. To examine whether the activation of tyrosine kinases occurs in factor-independent mutants in vivo, the tyrosine-phosphorylated proteins from 14 factor-independent mutants of a GM-CSF-dependent cell line (TF-1) were analysed. These mutants did not secrete any growth-stimulating activity for TF-1 cells, suggesting that activation of intracellular signaling rather than an autocrine stimulation by secreted growth factors is responsible for their factor-independent growth. In 11 out of 14 GM-CSF-independent mutants analysed, a constitutively tyrosine-phosphorylated protein of 60 kDa was detected, which was subsequently identified as p60(c-Src). The kinase activity of p60(c-Src) was increased up to 12-fold in these mutants, which was at least in part due to overexpression of the c-src gene on the RNA and protein level. The Src substrate Sam68 showed an increased phosphorylation in mutants with high Src activity, suggesting that p60(c-Src) triggers downstream signaling in these cells. Treatment of the factor-independent mutants with the Src kinase inhibitor PP2 resulted in a reduced proliferation, demonstrating that Src kinases are essential for these cells for maximal proliferation. Further analysis of factor-independent mutants with low or undetectable Src activity revealed a constitutive phosphorylation of the common beta chain of the GM-CSF receptor and STAT5. Our data indicate an increase in the expression and total activity of endogenous p60(c-Src) in several GM-CSF-independent TF-1 mutants, further underlining the role of Src in the process of autonomous growth of hematopoietic cells.

Base Sequence↗
PubMed2005

Acetyl nitrate nitrations in [bmpy][N(Tf)2] and [bmpy][OTf], and the recycling of ionic liquids.

In this work we have examined the nitration by acetyl nitrate of a range of activated and deactivated aromatic substrates in two ionic liquids and compared the results to the same reaction in dichloromethane. Both ionic liquids are stable to the reaction conditions, and in both ionic liquids the yields of reaction are higher after unit time than the same reactions in dichloromethane, although the regioselectivity is little affected by solvent choice. This result gives further support to the suggestion that in the ionic liquid, acetyl nitrate dissociates to give the nitronium ion, and that this is the effective nitrating agent here. However, it is shown that [bmpy][N(Tf)(2)] is a better solvent for aromatic nitration than [bmpy][OTf]. This is due to the ease of formation of nitronium ion in the former ionic liquid, and is consistent with the fact that [bmpy][N(Tf)(2)] is a weaker hydrogen bond acceptor solvent than [bmpy][OTf]. Finally, a method by which [bmpy][N(Tf)(2)] may be recovered and reused for aromatic nitration has been demonstrated.

Journal Article↗
PubMed2001

Lysophosphatidic acid promotes phorbol-ester-induced apoptosis in TF-1 cells by interfering with adhesion.

When exposed to PMA, the erythroblastic cell line TF-1 and its cytokine-independent variant D2 cells can be induced to undergo differentiation and apoptosis. In this study we investigated the mechanism responsible for the differential responses to PMA induction and show that serum present in the medium has a major role in promoting PMA-induced apoptosis in TF-1 and D2 cells. Interestingly, lysophosphatidic acid (LPA) could replace serum to co-operate with PMA in inducing apoptosis via the Rho-dependent pathway. The expression of a constitutively active form of RhoA also increased PMA-induced apoptosis. However, by inhibiting adhesion, most cells underwent PMA-induced apoptosis even in the absence of LPA or serum, indicating that adhesion is required for PMA-induced differentiation. Given that LPA could prevent adhesion for cells maintained in the serum-free medium, here we propose that RhoA has a switching role in determining whether TF-1 and D2 cells undergo differentiation or apoptosis in response to PMA, by modulating cell adhesion.

Apoptosis↗
PubMed1996

Proteoglycan synthesis in human and murine haematopoietic progenitor cell lines: isolation and characterization of a heparan sulphate proteoglycan as a major proteoglycan from the human haematopoietic cell line TF-1.

Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the microenvironment of haematopoietic tissues. Proteoglycans might also be involved in the interaction of haematopoietic stem and stromal cells. Recently, several studies have been reported on the proteoglycan synthesis of stromal cells, but little is known about the proteoglycan synthesis of haematopoietic stem or progenitor cells. Here we report on the isolation and characterization of proteoglycans from two haematopoietic progenitor cell lines, the murine FDCP-Mix A4 and the human TF-1 cell line. Proteoglycans were isolated from metabolically labelled cells and purified by several chromatographic steps, including anion-exchange and size-exclusion chromatography. Biochemical characterization was performed by electrophoresis or gel-filtration chromatography before and after digestion with glycosaminoglycan-specific enzymes or HNO2 treatment. Whereas FDCP-Mix A4 cells synthesize a homogeneous chondroitin 4-sulphate proteoglycan, isolation and characterization of proteoglycans from the human cell line TF-1 revealed, that TF-1 cells synthesize, in addition to a chondroitin sulphate proteoglycan, a heparan sulphate proteoglycan as major proteoglycan. For this heparan sulphate proteoglycan a core protein size of approx. 59 kDa was determined. Immunochemical analysis of this heparan sulphate proteoglycan revealed that it is not related to the syndecan family nor to glypican.

Animals↗
PubMed2001

Molecular mechanisms involved in CD43-mediated apoptosis of TF-1 cells. Roles of transcription Daxx expression, and adhesion molecules.

CD43 (leukosialin, sialophorin), an abundant leukocyte surface sialoglycoprotein, regulates leukocyte adhesion and transmits activating signals in T cells and dendritic cells. Immobilized anti-CD43 monoclonal antibody (mAb) MEM-59 has been previously shown to induce apoptosis of hematopoietic progenitors. In this study we show that it also triggers apoptosis of the myeloid progenitor-derived cell line TF-1. The kinetics of the MEM-59-induced apoptosis were unusually slow, with the first apoptotic cells appearing 36-48 h after their contact with the immobilized antibody; in 5 days, 90% of the cells were dead. CD43-mediated apoptosis was enhanced by coimmobilized anti-CD45 mAb and partly suppressed by coimmobilized anti-CD50 (ICAM-3) or anti-CD99 mAb. The MEM-59-triggered apoptosis of TF-1 cells was also inhibited by the overexpression of an apoptotic regulator, Daxx. CD43-mediated apoptosis was preceded by the repression of the DNA binding activity of the transcription factor AP-1. DNA array screening revealed that the expression of several genes encoding apoptosis-regulating proteins, including 14-3-3 proteins and the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor beta-subunit, was repressed in TF-1 cells bound to immobilized MEM-59. The down-regulation of 14-3-3 proteins and GM-CSF receptor beta was accompanied by translocation of the proapoptotic protein Bad to the mitochondria. These results suggest that engagement of CD43 may, presumably through the repressing transcription, initiate a Bad-dependent apoptotic pathway.

12E7 Antigen↗
PubMed2003

Calcium induces cell survival and proliferation through the activation of the MAPK pathway in a human hormone-dependent leukemia cell line, TF-1.

Survival and proliferation of cells of a human myelo-erythroid CD34+ leukemia cell line (TF-1) depend on the presence of granulocyte-macrophage colony-stimulating factor or interleukin-3. Upon hormone withdrawal these cells stop proliferating and undergo apoptotic process. In this report we demonstrate that a controlled increase in [Ca2+]i induces hormone-independent survival and proliferation of TF-1 cells. We found that moderate elevation of [Ca2+]i by the addition of cyclopiasonic-acid protected TF1 cells from apoptosis. Furthermore, a higher, but transient elevation of [Ca2+]i by ionomycin treatment induced cell proliferation. In both cases caspase-3 activity was reduced, and Bcl-2 was up-regulated. Higher elevation of [Ca2+]i by ionomycin induced MEK-dependent biphasic ERK1/2 activation, sufficient to move the cells from G0/G1 to S/M phases. Meanwhile, activation of ERK1/2, phosphorylation of the Elk-1 transcription factor, and, consequently, a substantial elevation of Egr-1 and c-Fos levels and AP-1 DNA binding were observed. Moderate elevation of [Ca2+]i, on the other hand, caused a delayed monophasic activation of ERK1/2 and Elk-1 that was accompanied with only a small increase of Egr-1 and c-Fos levels and AP-1 DNA binding. The specific MEK-1 kinase inhibitor, PD98059, inhibited all the effects of increasing [Ca2+]i, indicating that the MAPK/ERK pathway activation is essential for TF-1 cell survival and proliferation. Based on these results we suggest that the elevation of the [Ca2+]i may influence the cytokine dependence of hemopoietic progenitors and may contribute to pathological hematopoiesis.

Antigens, CD34↗