bluuebunny/crossref_metadata_2025 huggingface dataset:
Fas-Mediated Modulation of Bcr/Abl in Chronic Myelogenous Leukemia Results in Differential Effects on Apoptosis
Authors: Selleri Carmine , Maciejewski Jaroslaw P. , Pane Fabrizio , Luciano Luigia , Raiola Anna Maria , Mostarda Ilaria , Salvatore Francesco , Rotoli Bruno
Published: October 2019

Fas-R is expressed constitutively in CD34+ cells of patients with chronic myelogenous leukemia (CML); Fas-R triggering results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that α-interferon (IFN-α) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN-α can prime CML cells for the effects of Fas, the response to IFN-α in vivo is not a constant feature in CML patients. We studied the mechanisms of Fas-mediated apoptosis in 11 patients suffering from CML in chronic phase and tried to see whether there was a correlation between in vitro inducibility of apoptosis in CD34+ CML cells after Fas-R triggering and the clinical response to IFN-α. After priming with IFN-α, Fas triggering resulted in in vitro suppression of hematopoietic cell growth in seven of eight patients who had optimal hematologic response to IFN-α; in the same conditions, no inhibitory response to Fas-R agonist was observed in cells from three of three patients who proved to be poor responders to IFN-α. In responders to IFN-α, Fas-R agonist induced dose-dependent apoptosis of CD34+ cells; this effect was associated with a decrease in the bcr/abl protein level. In cells derived from patients with a poor response to IFN-α, the rate of apoptosis in culture remained unchanged in the presence of Fas-R agonist and nobcr/abl downmodulation was observed. Finally, we measuredbcr/abl mRNA by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and found that decreased bcr/ablprotein after Fas triggering was not associated with decreased amounts of specific mRNA, a finding which is consistent with a posttranscriptional regulation of the bcr/abl protein expression. It appears that Fas-mediated downmodulation of p210bcr/abl restores susceptibility to apoptosis of CML cells; in addition, in vitro studies on CML cells may predict response to IFN-α treatment.

© 1998 by The American Society of Hematology.
Ex Vivo Expansion of Genetically Marked Rhesus Peripheral Blood Progenitor Cells Results in Diminished Long-Term Repopulating Ability
Authors: Tisdale J.F. , Hanazono Y. , Sellers S.E. , Agricola B.A. , Metzger M.E. , Donahue R.E. , Dunbar C.E.
Published: October 2019

The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)–mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with 0.01% in the original IL-3/IL-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells.

This is a US government work. There are no restrictions on its use.
The Distal Cytoplasmic Domain of the Erythropoietin Receptor Induces Granulocytic Differentiation in 32D Cells
Authors: Harris Kevin W. , Hu Xian-Jue , Schultz Suzanne , Arcasoy Murat O. , Forget Bernard G. , Clare Nanette
Published: October 2019

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)–dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation.

© 1998 by The American Society of Hematology.
A Novel Human Actin-Binding Protein Homologue That Binds to Platelet Glycoprotein Ib
Authors: Xu Wen-feng , Xie Zhi-wei , Chung Dominic W. , Davie Earl W.
Published: October 2019

Glycoprotein (GP)Ib-IX-V is one of the major transmembrane complexes present on the platelet surface. Its extracellular domain binds von Willebrand factor (vWF) and thrombin, while its intracellular domain associates tightly with the cytoskeleton through the actin-binding protein (ABP)-280, also known as filamin. In the present study, a full-length cDNA coding for a human ABP homologue has been cloned and sequenced. This protein was identified by the yeast two-hybrid screening procedure via its interaction with the intracellular domain of GPIb. Initially, a 1.3-kb partial cDNA was isolated from a megakaryocyte-like cell line (K562) cDNA library followed by a full-length cDNA of 9.4 kb that was identified in a human placenta library. The full-length cDNA encoded a protein of 2,578 amino acids with a calculated molecular weight of 276 kD (ABP-276). The amino terminal 248 amino acids contained an apparent actin binding domain followed by 24 tandem repeats each containing about 96 amino acids. The amino acid sequence of the protein shared a high degree of homology with human endothelial ABP-280 (70% identity) and chicken filamin (83% identity). However, the 32 amino acid Hinge I region in ABP-280 that contains a calpain cleavage site conferring flexibility on the molecule, was absent in the homologue. An isoform containing a 24 amino acid insertion with a unique sequence at the missing Hinge I region was also identified (ABP-278). This isoform resulted from alternative RNA splicing. ABP-276 and/or ABP-278 were present in all tissues examined, but the relative amount varied in that some tissue contained both forms, while other tissue contained predominately one or the other.

© 1998 by The American Society of Hematology.
Two Signaling Pathways Can Increase Fas Expression in Human Thymocytes
Authors: Moulian Nathalie , Bidault Jocelyne , Planché Claude , Berrih-Aknin Sonia
Published: October 2019

Fas, a cell surface receptor, can induce apoptosis after cross-linking with its ligand. Fewer than 3% of human thymocytes strongly express Fas. We report that Fas antigen expression can be upregulated by two signaling pathways in vitro, one mediated by anti-CD3 and the other by interleukin-7 + interferon-γ. The two signaling pathways differed in several respects. (1) Fas expression increased in all thymic subsets after cytokine activation, but only in the CD4 lineage after anti-CD3 activation. (2) Fas upregulation was inhibited by cyclosporin A (a calcineurin inhibitor) in anti-CD3–activated but not in cytokine-activated thymocytes. (3) Cycloheximide (a metabolic inhibitor) inhibited Fas upregulation in cytokine-activated thymocytes but not in anti-CD3–activated thymocytes. (4) Cytokine-activated thymocytes were more susceptible than anti-CD3–activated thymocytes to Fas-induced apoptosis, a difference mainly accounted for by CD4+ cells. The nature of the stimulus might thus influence the susceptibility of human thymocytes to Fas-induced apoptosis.

© 1998 by The American Society of Hematology.
Tissue Inhibitor of Metalloproteinase (TIMP)-1 Induces Differentiation and an Antiapoptotic Phenotype in Germinal Center B Cells
Authors: Guedez Liliana , Courtemanch Laurel , Stetler-Stevenson Maryalice
Published: October 2019

Tissue inhibitors of metalloproteinases (TIMPs) have been shown to be multifunctional factors. Contrasting with their enzyme-inhibitory activity, TIMPs also promote cell growth. Previously, we have reported an enhanced expression of TIMP-1 by normal reactive B cells and high-grade lymphomas. In the present study, a series of Burkitt’s lymphoma (BL) cell lines were analyzed for their expression of TIMP-1. TIMP-1 expression correlates with upregulation of activation and survival markers. TIMP-1–negative cells express the phenotype associated with group I BL lines and Epstein-Barr virus (EBV)-negative, nonendemic BLs (CD10+, CD38+, sIg+, and CD77+). However, TIMP-1+ BL lines showed group II/III BL phenotype, downregulation of the above markers, and upregulation and secretion of the activation marker CD23. Also, TIMP-1+ cells have high levels of CD40 expression. To determine whether TIMP-1 is directly involved in the BL phenotype, an EBV-negative BL line JD38 was infected with timp-1–expressing retrovirus and analyzed. In the absence of EBV, upregulation of TIMP-1 is sufficient to induce the same phenotype seen in TIMP-1+, EBV+ BL lines (CD10−, CD38−, sIg−, CD77−, CD23+, CD40 bright). This study not only suggests a role for TIMP-1 in BLs, but also supports its value as a prognostic factor.

This is a US government work. There are no restrictions on its use.
On the Role of the Proform-Conformation for Processing and Intracellular Sorting of Human Cathepsin G
Authors: Garwicz Daniel , Lindmark Anders , Persson Ann-Maj , Gullberg Urban
Published: October 2019

The serine protease cathepsin G is synthesized during the promyelomonocytic stage of neutrophil and monocyte differentiation. After processing, including removal of an amino-terminal propeptide from the catalytically inactive proform, the active protease acquires a mature conformation and is stored in azurophil granules. To investigate the importance of the proform-conformation for targeting to granules, a cDNA encoding a double-mutant form of human preprocathepsin G lacking functional catalytic site and amino-terminal prodipeptide (CatG/Gly201/▵Gly19Glu20) was constructed, because we were not able to stably express a mutant lacking only the propeptide. Transfection of the cDNA to the rat basophilic leukemia RBL-1 and the murine myeloblast-like 32D cl3 cell lines resulted in stable, protein-expressing clones. In contrast to wild-type proenzyme, CatG/Gly201/▵Gly19Glu20 adopted a mature conformation cotranslationally, as judged by the early acquisition of affinity to the serine protease inhibitor aprotinin, appearing before the carboxyl-terminal processing and also in the presence of the Golgi-disrupting agent brefeldin A. The presence of a mature amino-terminus was confirmed by amino-terminal radiosequencing. As with wild-type proenzyme, CatG/Gly201/▵Gly19Glu20 was proteolytically processed carboxyl-terminally and glycosylated with asparagine-linked carbohydrates that were converted into complex forms. Furthermore, it was targeted to granules, as determined by subcellular fractionation. Our results show that the initial proform-conformation is not critical for intracellular sorting of human cathepsin G. Moreover, we demonstrate that double-mutant cathepsin G can achieve a mature conformation before carboxyl-terminal processing of the proform.

© 1998 by The American Society of Hematology.
Infusion of Cytotoxic T Cells for the Prevention and Treatment of Epstein-Barr Virus–Induced Lymphoma in Allogeneic Transplant Recipients
Authors: Rooney Cliona M. , Smith Colton A. , Ng Catherine Y.C. , Loftin Susan K. , Sixbey John W. , Gan Yanjun , Srivastava Deo-Kumar , Bowman Laura C. , Krance Robert A. , Brenner Malcolm K. , Heslop Helen E.
Published: October 2019

Epstein-Barr virus (EBV) causes potentially lethal immunoblastic lymphoma in up to 25% of children receiving bone marrow transplants from unrelated or HLA-mismatched donors. Because this complication appears to stem from a deficiency of EBV-specific cytotoxic T cells, we assessed the safety and efficacy of donor-derived polyclonal (CD4+ and CD8+) T-cell lines as immunoprophylaxis and treatment for EBV-related lymphoma. Thirty-nine patients considered to be at high risk for EBV-induced lymphoma each received 2 to 4 intravenous infusions of donor-derived EBV-specific T lymphocytes, after they had received T-cell–depleted bone marrow from HLA-matched unrelated donors (n = 33) or mismatched family members (n = 6). The immunologic effects of this therapy were monitored during and after the infusions. Infused cells were identified by detection of the neo marker gene. EBV-specific T cells bearing theneo marker were identified in all but 1 of the patients. Serial analysis of DNA detected the marker gene for as long as 18 weeks in unmanipulated peripheral blood mononuclear cells and for as long as 38 months in regenerated lines of EBV-specific cytotoxic T cells. Six patients (15.5%) had greatly increased amounts of EBV-DNA on study entry (>2,000 genome copies/106 mononuclear cells), indicating uncontrolled EBV replication, a complication that has had a high correlation with subsequent development of overt lymphoma. All of these patients showed 2 to 4 log decreases in viral DNA levels within 2 to 3 weeks after infusion and none developed lymphoma, confirming the antiviral activity of the donor-derived cells. There were no toxic effects that could be attributed to prophylactic T-cell therapy. Two additional patients who did not receive prophylaxis and developed overt immunoblastic lymphoma responded fully to T-cell infusion. Polyclonal donor-derived T-cell lines specific for EBV proteins can thus be used safely to prevent EBV-related immunoblastic lymphoma after allogeneic marrow transplantation and may also be effective in the treatment of established disease.

© 1998 by The American Society of Hematology.
Proliferation in Monocyte-Derived Dendritic Cell Cultures Is Caused by Progenitor Cells Capable of Myeloid Differentiation
Authors: Cavanagh Lois L. , Saal Russell J. , Grimmett Karen L. , Thomas Ranjeny
Published: October 2019

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [3H]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor- led to maturation of the DC, but did not inhibit proliferation. Ki67+ cells were observed in cytospins of these DC, and by double staining were CD3−CD19−CD11c−CD40−and myeloperoxidase+, suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34+CD33−CD14− progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes.

© 1998 by The American Society of Hematology.
A Janus Kinase Inhibitor, JAB, Is an Interferon-γ–Inducible Gene and Confers Resistance to Interferons
Authors: Sakamoto Hiroshi , Yasukawa Hideo , Masuhara Masaaki , Tanimura Shyu , Sasaki Atsuo , Yuge Kentaro , Ohtsubo Motoaki , Ohtsuka Akira , Fujita Takasi , Ohta Tsunetaka , Furukawa Yusuke , Iwase Satsuki , Yamada Hisashi , Yoshimura Akihiko
Published: October 2019

It has been shown that interferons (IFNs) exert their signals through receptor-associated Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). However, molecular mechanism of regulation of IFN signaling has not been fully understood. We have reported novel cytokine-inducible SH2 protein (CIS) and JAK binding protein (JAB) family genes that can potentially modulate cytokine signaling. Here we report that JAB is strongly induced by IFN-γ but not by IFN-β in mouse myeloid leukemia M1 cells and NIH-3T3 fibroblasts. NIH-3T3 cells ectopically expressing JAB but not CIS3 lost responsiveness to the antiviral effect of IFN-β and IFN-γ. M1 leukemic cells stably expressing JAB were also resistant to IFN-γ and IFN-β–induced growth arrest. In both NIH-3T3 and M1 transformants expressing JAB, IFN-γ did not induce tyrosine phosphorylation and DNA binding activity of STAT1. Moreover, IFN-γ–induced activation of JAK1 and JAK2 and IFN-β–induced JAK1 and Tyk2 activation were inhibited in NIH-3T3 JAB transformants. These results suggest that JAB inhibits IFN signaling by blocking JAK activity. We also found that IFN-resistant clones derived from LoVo cells and Daudi cells expressed high levels of JAB without stimulation. In IFN-resistant Daudi cells, IFN-induced STAT1 and JAK phosphorylation was partially reduced. Therefore, overexpression of JAB could be, at least in part, a mechanism of IFN resistance.

© 1998 by The American Society of Hematology.
Human Herpesvirus 7 Infection Induces Profound Cell Cycle Perturbations Coupled to Disregulation of cdc2 and Cyclin B and Polyploidization of CD4+ T Cells
Authors: Secchiero Paola , Bertolaso Lucia , Casareto Luca , Gibellini Davide , Vitale Marco , Bemis Kristi , Aleotti Arrigo , Capitani Silvano , Franchini Genoveffa , Gallo Robert C. , Zauli Giorgio
Published: October 2019

Human herpesvirus 7 (HHV-7) infection of both primary CD4+ T lymphocytes and SupT1 lymphoblastoid T-cell line induced a progressive accumulation of cells exibiting a gap 2/mitosis (G2/M) and polyploid content coupled to an increased cell size. The expression of both cyclin-dependent kinase cdc2 and cyclin B was increased in HHV-7–infected cells with respect to the uninfected ones. Moreover, the simultaneous flow cytometric analysis of cyclin B and DNA content showed that cyclin B expression was not only increased but also unscheduled with respect to its usual cell cycle pattern. However, the levels of kinase activity associated to cdc2 were decreased in HHV-7–infected cells with respect to uninfected cultures. To elucidate the origin of the enlarged HHV-7–infected cells, extensive electron and confocal microscopy analyses were performed. Membrane fusion events associated to cytoplasmic bridges, which characterize the formation of syncytia, were never observed. On the other hand, analysis of serial sections of the same cells strongly suggested that enlarged HHV-7–infected cells contained a single polylobated nucleus. This was confirmed by flow cytometry analysis performed on nuclei isolated from HHV-7–infected cells, which showed multiple peaks with a DNA content >4n. Taken together, these data indicate that giant cells, which represent the hallmark of in vitro HHV-7 infection, arise from single CD4+ T cells undergoing a process of polyploidization.

© 1998 by The American Society of Hematology.
IL-2 Adenovector-Transduced Autologous Tumor Cells Induce Antitumor Immune Responses in Patients With Neuroblastoma
Authors: Bowman Laura , Grossmann Michael , Rill Donna , Brown Michael , Zhong Wan-yun , Alexander Barbara , Leimig Thasia , Coustan-Smith Elaine , Campana Dario , Jenkins Jesse , Woods Diane , Kitchingman Geoffrey , Vanin Elio , Brenner Malcolm
Published: October 2019

In many different murine models, the immunogenicity of tumor cells can be increased by transduction with a range of immunostimulatory genes, inducing an immune response that causes regression of pre-existing unmodified tumor cells. To investigate the relevance of these animal models to pediatric malignancy, we used autologous unirradiated tumor cells transduced with an adenovirus-IL-2 to immunize 10 children with advanced neuroblastoma. In a dose-escalation study, we found that this tumor immunogen induced a moderate local inflammatory response consisting predominantly of CD4+ T lymphocytes, and a systemic response, with a rise in circulating CD25+and DR+ CD3+ T cells. Patients also made a specific antitumor response, manifest by an IgG antitumor antibody and increased cytotoxic T-cell killing of autologous tumor cells. Clinically, five patients had tumor responses after the tumor immunogen alone (one complete tumor response, one partial response, and three with stable disease). Four of these five patients were shown to have coexisting antitumor cytotoxic activity, as opposed to only one of the patients with nonresponsive disease. These results show a promising correlation between preclinical observations and clinical outcome in this disease, and support further exploration of the approach for malignant diseases of children.

© 1998 by The American Society of Hematology.
Interleukin-10 Inhibits Erythropoietin-Independent Growth of Erythroid Bursts in Patients With Polycythemia Vera
Authors: Geissler Klaus , Öhler Leopold , Födinger Manuela , Kabrna Eva , Kollars Marietta , Skoupy Sonja , Lechner Klaus
Published: October 2019

In polycythemia vera (PV) erythroid colonies that grow in vitro in the absence of exogenous erythropoietin (EPO) arise from the abnormal clone that is responsible for overproduction of red blood cells. Although the mechanism of autonomous formation of burst-forming units-erythroid (BFU-E) is not fully understood, a spontaneous release of growth regulatory molecules by PV cells and/or by accessory cells is likely to be involved. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate abnormal erythropoiesis in PV. We studied the effect of recombinant human IL-10 on the EPO-independent growth of erythroid bursts derived from peripheral blood mononuclear cells (PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent, and specific inhibitory effect on autonomous BFU-E formation. Ten nanograms per milliliter of IL-10 significantly suppressed spontaneous growth of erythroid colonies in methylcellulose in five of five PV patients tested with a mean inhibition by 81% (range, 72-94). To elucidate the possible mechanism of the inhibitory action of IL-10 we further studied the effect of anticytokine antibodies on autonomous BFU-E growth and the ability of exogenous cytokines to restore IL-10–induced suppression of erythroid colony growth. Among a panel of growth regulatory factors tested (granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-3, granulocyte colony-stimulating factor, stem cell factor, and insulin-like growth factor-1) GM-CSF was the only molecule for which both an inhibition of spontaneous BFU-E formation by its respective antibody as well as a significant restimulation of erythroid colonies in IL-10-treated cultures by exogenous addition was found. Moreover, inhibition of GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level. Our data indicate that autonomous BFU-E growth in PV can be profoundly inhibited by IL-10 and that this inhibitory effect seems to be at least in part secondary to suppression of endogenous GM-CSF production.

© 1998 by The American Society of Hematology.
Impaired Expression of Integrin -4 Subunit in Cultured Mast Cells Derived From Mutant Mice of mi/mi Genotype
Authors: Kim Dae-Ki , Morii Eiichi , Ogihara Hideki , Hashimoto Koji , Oritani Kenji , Lee Young-Mi , Jippo Tomoko , Adachi Shiro , Kanakura Yuzuru , Kitamura Yukihiko
Published: October 2019

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called MITF). We have reported that expression of several genes was impaired in cultured mast cells (CMCs) ofmi/mi mice due to a defective transactivation ability of mutant MITF (mi-MITF). Because attachment of mi/mi CMCs to fibroblasts is impaired, we examined the expression of integrin genes in mi/mi CMCs in the present study. Among the integrin genes examined, the expression of integrin 4 subunit was barely detectable in mi/mi CMCs, and the 4 protein was not detected by flow cytometry either. The specific adhesion to vascular cell adhesion molecule-1 (VCAM-1), the ligand for 4 subunit, was observed in +/+ CMCs but not in mi/mi CMCs, indicating that the expression of integrin 4 subunit at a functional level did not occur in mi/mi CMCs. In the promoter region of the 4 subunit gene, there was a CACTTG motif to which normal MITF (+- MITF) bound. The coexpression of +-MITF but not of mi-MITF transactivated the promoter of the 4 subunit gene. The deletion or mutation of the CACTTG motif abolished the transactivation by +-MITF, suggesting that +-MITF directly transactivated the gene encoding 4 subunit of integrin.

© 1998 by The American Society of Hematology.
Saturation Mutagenesis of the β Subunit of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor Shows Clustering of Constitutive Mutations, Activation of ERK MAP Kinase and STAT Pathways, and Differential β Subunit Tyrosine Phosphorylation
Authors: Jenkins Brendan J. , Blake Timothy J. , Gonda Thomas J.
Published: October 2019

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific  subunits and a common signal-transducing β subunit (hβc). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hβc. We report here a comprehensive screen of the entire hβc molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hβc that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hβc mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant β subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hβc activation, dissociate hβc tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hβc.

© 1998 by The American Society of Hematology.
Ultrastructural Analysis of Bone Marrow Hematopoiesis in Mice Transgenic for the Thymidine Kinase Gene Driven by the IIb Promoter
Authors: Poujol Christel , Roux Diana Tronik-Le , Tropel Philippe , Roullot Valérie , Nurden Alan , Marguerie Gérard , Nurden Paquita
Published: October 2019

Transgenic mice have been generated with expression of the herpes virus thymidine kinase gene directed by a 2.7-kb fragment of the IIb murine promoter of the gene encoding the IIb-subunit of the platelet integrin IIbβ3 (Tropel et al, Blood90:2995, 1997). Administration of ganciclovir (GCV) to these mice resulted not only in an acute cessation of platelet production due to the depletion of the megakaryocytic lineage, but also a decrease in erythrocyte and leukocyte numbers. Immunogold staining on ultrathin frozen sections and electron microscopy has now shown that the remaining population of immature hematopoietic cells contain a high proportion of Sca-1+ and CD34+ cells, with CD45R+ cells of the lymphopoietic lineage being maintained. Stromal cells were also preserved. Blood thrombopoietin levels were high. At 4 days of the recovery phase, Sca-1 and CD34 antigen expression decreased with intense proliferation of cells of the three lineages, with megakaryocyte (MK) progenitors being identified by their positivity for glycoprotein IIb-IIIa. These results suggest that transcriptional activity for the IIb gene promoter was present on pluripotent hematopoietic stem cells. At 6 to 8 days after cessation of GCV, numerous mature MK were observed, some of them with deformed shapes crossing the endothelial barrier through thin apertures. Proplatelet production was visualized in the vascular sinus. After 15 days, circulating platelet levels had increased to approximately 65% of normal. Transgenic IIb-tk mice constitute a valuable model to study in vivo megakaryocytopoiesis.

© 1998 by The American Society of Hematology.
Allelotype Analysis of Adult T-Cell Leukemia
Authors: Hatta Yoshihiro , Yamada Yasuaki , Tomonaga Masao , Said Jonathan W. , Miyosi Isao , Koeffler H. Phillip
Published: October 2019

Allelotype analysis of adult T-cell leukemia (ATL) was undertaken for the first time to identify chromosomal loci relevant to the development of acute/lymphomatous ATL. Loss of heterozygosity (LOH) was screened using 94 highly polymorphic microsatellite markers, distributed among all nonacrocentric, autosomal chromosomes. In each of the 22 cases, DNA obtained from their leukemic cells in acute/lymphomatous phase was compared with their constitutional DNA from mononuclear cells in chronic or remission phase. Allelic losses of at least on one chromosome arm occurred in 91% of the cases (20 individuals). Among 39 chromosome arms, allelic losses were observed on 31 arms at least for one sample. A high frequency of allelic loss (>30%) was seen on chromosome arms 6q (41%) and 17p (48%). The mean fractional allelic loss (FAL) was 0.109. These findings suggest that a novel tumor suppressor gene on chromosome arm 6q, as well as the p53 gene on chromosome arm 17p, probably have an important role in the development of acute/lymphomatous ATL.

© 1998 by The American Society of Hematology.
The Fanconi Anemia Proteins FAA and FAC Function in Different Cellular Compartments to Protect Against Cross-Linking Agent Cytotoxicity
Authors: Kruyt Frank A.E. , Youssoufian Hagop
Published: October 2019

Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, bone marrow failure, and a high risk of developing malignancies. Although the disorder is genetically heterogeneous, all FA cells are defined by their sensitivity to the apoptosis-inducing effect of cross-linking agents, such as mitomycin C (MMC). The cloned FA disease genes, FAC and FAA, encode proteins with no homology to each other or to any known protein. We generated a highly specific antibody against FAA and found the protein in both the cytoplasm and nucleus of mammalian cells. By subcellular fractionation, FAA is also associated with intracellular membranes. To identify the subcellular compartment that is relevant for FAA activity, we appended nuclear export and nuclear localization signals to the carboxy terminus of FAA and enriched its localization in either the cytoplasm or the nucleus. Nuclear localization of FAA was both necessary and sufficient to correct MMC sensitivity in FA-A cells. In addition, we found no evidence for an interaction between FAA and FAC either in vivo or in vitro. Together with a previous finding that FAC is active in the cytoplasm but not in the nucleus, our results indicate that FAA and FAC function in separate subcellular compartments. Thus, FAA and FAC, if functionally linked, are more likely to be in a linear pathway rather than form a macromolecular complex to protect against cross-linker cytotoxicity.
Quinine-Dependent Antibodies Bind a Restricted Set of Epitopes on the Glycoprotein Ib-IX Complex: Characterization of the Epitopes
Authors: Burgess Janette K. , Lopez Jose A. , Berndt Michael C. , Dawes Ian , Chesterman Colin N. , Chong Beng H.
Published: October 2019

Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb-IIIa or both epitopes has also been reported. The objective of this study was to characterize the binding site of GPIb-IX–specific quinine-dependent antibodies. Antibody binding to Chinese hamster ovary cells or mouse L cells stably transfected with various combinations of the three genes (Ibα, Ibβ, or IX) that encode this complex was detected using flow cytometry, monoclonal antibody–specific immobilization of platelet antigens assay, and differential adsorption studies. IgG in sera from 15 patients with quinine-induced thrombocytopenia binding to the cells, in the presence of quinine, showed three distinct patterns. Group 1 sera contained at least two antibody populations, one which binds to GPIbα and another which recognizes GPIX. Group 2 sera contained an antibody which binds drug dependently to GPIX, and Group 3 sera contained an antibody which recognizes a quinine-dependent epitope on GPIbα. Thus, the quinine-dependent antibodies fall into two distinct populations that bind to GPIbα and GPIX independently. Using proteases which cleave GPIbα at specific sites, we have shown that the GPIbα-specific antibody binds to an 11–amino acid (283 to 293) region. Peptide inhibition studies provide confirmatory evidence that this region contains the epitope for the GPIbα-specific quinine-dependent antibody.
Salicylates Inhibit Adhesion and Transmigration of T Lymphocytes by Preventing Integrin Activation Induced by Contact With Endothelial Cells
Authors: Gerli Roberto , Paolucci Cristina , Gresele Paolo , Bistoni Onelia , Fiorucci Stefano , Muscat Christopher , Belia Silvia , Bertotto Alberto , Costantini Vincenzo
Published: October 2019

The inhibition of cyclooxygenase does not fully account for the spectrum of activities of nonsteroidal antiinflammatory drugs. It is evident, indeed, that regulation of inflammatory cell function may contribute in explaining some of the effects of these drugs. Tissue recruitment of T cells plays a key role in the development of chronic inflammation. Therefore, the effects of salicylates on T-cell adhesion to and migration through endothelial cell monolayers on collagen were analyzed in an in vitro static system. Aspirin and sodium salicylate reduced the ability of unstimulated T cells to adhere to and transmigrate through cytokine-activated endothelium. Although salicylates did not modify the expression of integrins on T cells, they blunted the increased adherence induced by the anti-β2monoclonal antibody (MoAb) KIM127 and prevented the appearance of an activation-dependent epitope of the CD11/CD18 complex, recognized by the MoAb 24, induced by contact with endothelial cells. Salicylates also induced an increase of intracellular calcium ([Ca2+]i) and activation of protein kinase C (PKC) in T cells, but not cell proliferation and interleukin (IL)-2 synthesis. The reduction of T-cell adhesiveness appears to be dependent on the increase in[Ca2+]i levels, as it could be reversed by blocking Ca2+ influx, but not by inhibiting PKC. Moreover, ionomycin at concentrations giving an increase in [Ca2+]i similar to that triggered by aspirin, strictly reproduced the T-cell phenotypic and functional changes induced by salicylates. Aspirin reduced T-cell adhesion and migration also ex vivo after infusion to healthy volunteers. These data suggest that the antiinflammatory activity of salicylates may be due, at least in part, to an interference with the integrin-mediated binding of resting T lymphocytes to activated endothelium with consequent reduction of a specific T-cell recruitment into inflammatory sites.
Age-Related Decline of Perforin Expression in Human Cytotoxic T Lymphocytes and Natural Killer Cells
Authors: Rukavina Daniel , Laskarin Gordana , Rubesa Gordana , Strbo Natasa , Bedenicki Ivica , Manestar Darko , Glavas Mario , Christmas Stephen E. , Podack Eckhard R.
Published: October 2019

In this study a flow cytometric technique for detecting cytoplasmic perforin (P) has been used to quantify age-related changes in perforin expression in human peripheral blood lymphocytes (PBL). Proportions of P+ lymphocytes increased after birth, but declined rapidly after the age of 70 years. This was true for both T cells and CD16+ and CD56+ natural killer (NK) cells. Children showed in addition to high levels of perforin positive CD8+ cells a much higher proportion of CD4+P+ cells than the other age groups. In elderly individuals there was also a highly significant reduction in mean levels of perforin per cell as compared with all other groups (P < .05 to .001). Adult women had consistently higher mean levels of perforin per cell than adult men for all P+cell phenotypes. Functional tests clearly showed the deficiency in early spontaneous cytotoxic potential of PBL from elderly persons due to relative P deficiency, which can be corrected by stimulation of cytolytic cells with target cells and interleukin-2 (IL-2). The deficiency in cytolytic activity on the contact with target cells may have implications for antiviral and antitumor immunity in elderly persons.
Mutations of the CD40 Ligand Gene and Its Effect on CD40 Ligand Expression in Patients With X-Linked Hyper IgM Syndrome
Authors: Seyama Kuniaki , Nonoyama Shigeaki , Gangsaas Ingvild , Hollenbaugh Diane , Pabst Henry F. , Aruffo Alejandro , Ochs Hans D.
Published: October 2019

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.
Regulation of the c-jun Gene in p210 BCR-ABL Transformed Cells Corresponds With Activity of JNK, the c-jun N-Terminal Kinase
Authors: Burgess Gem S. , Williamson Elizabeth A. , Cripe Larry D. , Litz-Jackson Sara , Bhatt Jay A. , Stanley Kurt , Stewart Mark J. , Kraft Andrew S. , Nakshatri Harikrishna , Boswell H. Scott
Published: October 2019

Activity of the c-jun N-terminal kinase (JNK) has been shown in hematopoietic cells transformed by p210 BCR-ABL. However, analysis has not been reported for hematopoietic cells on the consequences of this activity for c-jun promoter regulation within its distinctive proximal 8-base consensus CRE-like element, an element linked to JNK-mediated increase in c-jun transcription. In the present study, regulation of the proximal c-jun promoter was studied in murine myeloid cells transformed by p210 BCR-ABL. Promoter regulation in p210 BCR-ABL transformed cells was compared with regulation of the promoter in nontransformed interleukin-3 (IL-3)–dependent parental cells. The composition of nuclear AP-1 proteins contained within cells with p210 BCR-ABL, and their binding to the c-jun promoter proximal CRE-like element, was compared with the composition and binding of AP-1 proteins in IL-3–treated parental cells without p210 BCR-ABL. The present analysis found fivefold increased c-jun transcription occurring in p210 BCR-ABL transformed murine myeloid cells possessing a corresponding magnitude of increased kinase activity of JNK, compared with IL-3–stimulated parental cells. Augmented JNK activity was accompanied by increased nuclear abundance of c-jun and c-fos proteins that bound specifically to the proximal c-jun promoter CRE element. Also, representative human leukemic cell lines expressing p210 BCR-ABL and possessing abundant kinase activity of JNK, when compared with parental cells that were deficient in JNK activity, had increased c-jun and c-fosproteins. Finally, to show the relevance of these observations in model systems, we studied blast cells from patients with Philadelphia chromosome–positive acute leukemic transformation, and observed comparable activities of JNK catalysis and c-jun/AP-1 protein relative to the cell lines that possessed p210 BCR-ABL and JNK activity. These studies provide a basis for investigating the set of downstream genes which augmented c-jun/AP-1 activity enlists in the process of transformation by p210 BCR-ABL.
Stem Cell Factor as a Single Agent Induces Selective Proliferation of the Philadelphia Chromosome Positive Fraction of Chronic Myeloid Leukemia CD34+ Cells
Authors: Moore Sarah , Haylock David N. , Lévesque Jean-Pierre , McDiarmid Louise A. , Samels Leanne M. , To L. Bik , Simmons Paul J. , Hughes Timothy P.
Published: October 2019

The interaction between p145c-KIT and p210bcr-abl in transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34+ cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34+ cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34+ cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit–granulocyte-macrophage (CFU-GM) from CML CD34+CD38+ cells and the more primitive CML CD34+CD38− cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34+ cells showed exclusive growth of Ph+cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of β1-integrin–mediated adhesion of CML CD34+cells to fibronectin was not increased when compared with the effect on normal CD34+ cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML.
Nutritional Folate Status Influences the Efficacy and Toxicity of Chemotherapy in Rats
Authors: Branda Richard F. , Nigels Elizabeth , Lafayette Amy R. , Hacker Miles
Published: October 2019

The effect of folate status on the efficacy and toxicity of chemotherapy was investigated in weanling Fischer 344 rats maintained on diets of varying folate content or supplemented with daily injections of folic acid, 50 mg/kg, for 6 to 7 weeks. MADB106 rat mammary tumor growth rate was the same in folate replete and supplemented rats, but retarded in the low folate groups. The tumor growth inhibitions in low folate, replete and high folate rats treated with cyclophosphamide were: 53%, 98%, and 97% (P = .048); with 5-fluorouracil (5-FU): 46%, 49%, and 66%; and with doxorubicin: 25%, 55%, and 61%. Significant differences in survival were observed for cyclophosphamide (P = .0084) and 5-FU (P = .025) related to dietary folate content. Thus, folate deficiency impedes tumor growth rate, but supplementation does not accelerate it in folate replete animals. Correction of folate deficiency approximately doubles the efficacy of cyclophosphamide in rats with much less host toxicity. Folate repletion improves survival in 5-FU–treated animals. These studies indicate that nutritional folate status has an important influence on the efficacy and toxicity of some commonly used cancer chemotherapeutic drugs.