bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of α4β1-Integrin Binding Sequences of Fibronectin on Growth of Cells From Human Hematopoietic Progenitors
Authors: Schofield Karen P. , Humphries Martin J. , de Wynter Erika , Testa Nydia , Gallagher John T.
Published: October 2019

Highly regulated interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. We have examined the relationship between α4β1-integrin–mediated adhesion and growth of CD34+ cells by assessing their adhesive and migratory patterns of proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant fragments of theHepII/IIICS region of fibronectin. CD34+ cells were isolated from cord blood and placed in culture wells containing serum-free medium and growth factors. Wells were precoated with either the H120 fragment of fibronectin, which contains three α4β1-integrin binding sites, or the H0 fragment, which lacks the two highest affinity α4β1 binding sequences. Proliferation of single cells of CD34+38+DR+and CD34+38−DR+ phenotypes occurred in contact with the H120 substrate and was associated with migration. Larger numbers of cells were used to quantitate proliferative responses. Cells growing in wells coated with H120 formed attachments to the base of the wells throughout the culture period. Higher total cell counts were consistently found in wells coated with H120 compared with H0 and bovine serum albumin controls. The difference was first apparent at day 8 of culture and reached a maximum at days 11 through 13, when expansion with H120 was a mean of 1.8-fold higher than that seen with H0 (P≤ .0001). The greatest expansion (2.25-fold) with H120 compared with H0 was seen when the growth factor concentrations were reduced to 1/16 of the standard levels (P ≤ .001). The increase in total cell numbers was not at the expense of CD34+ cells as numbers of these were similar in H120 and control cultures. These results provide evidence for synergy between growth factors and integrins that may be relevant to understanding hematopoiesis in marrow stroma.
Telomerase Activity in Candidate Stem Cells From Fetal Liver and Adult Bone Marrow
Authors: Yui Jane , Chiu Choy-Pik , Lansdorp Peter M.
Published: October 2019

Telomerase is a ribonucleoprotein polymerase that synthesizes telomeric repeats onto the 3′ ends of eukaryotic chromosomes. Activation of telomerase may prevent telomeric shortening and correlates with cell immortality in the germline and certain tumor cells. Candidate hematopoietic stem cells (HSC) from adult bone marrow express low levels of telomerase, which is upregulated with proliferation and/or differentiation. To address this issue, we stimulated purified candidate HSC from human adult bone marrow with stem cell factor (SCF), interleukin-3 (IL-3), and Flt3-ligand (FL). After 5 days in culture, activity was detected in total cell extracts from IL-3–, SCF + FL–, SCF + IL-3–, FL + IL-3–, and SCF + IL-3 + FL–stimulated cultures, but not from cells cultured in SCF or FL alone. Within the CD34+fraction of the cultured cells, significant activity was found in the CD34+CD71+ fraction. In addition, PKH26 staining confirmed that detectable telomerase activity was present in dividing PKH26lo cells, whereas nondividing PKH26hi cells were telomerase negative. Because in these experiments no distinction could be made between cycling “candidate” stem cells that had retained or had lost self-renewal properties, fetal liver cells with a CD34+CD38− phenotype, highly enriched for cycling stem cells, were also examined and found to express readily detectable levels of telomerase activity. Given the replication-dependent loss of telomeric DNA in hematopoietic cells, these observations suggest that the observed telomerase activity in candidate stem cells is either expressed in a minor subset of stem cells or, more likely, is not sufficient to prevent telomere shortening.
Hypoxia Stimulates Urokinase Receptor Expression Through a Heme Protein-Dependent Pathway
Authors: Graham Charles H. , Fitzpatrick Tania E. , McCrae Keith R.
Published: October 2019

Hypoxia underlies a number of biologic processes in which cellular migration and invasion occur. Because earlier studies have shown that the receptor for urokinase-type plasminogen activator (uPAR) may facilitate such events, we studied the effect of hypoxia on the expression of uPAR by first trimester human trophoblasts (HTR-8/SVneo) and human umbilical vein endothelial cells (HUVEC). Compared with control cells cultured under standard conditions (20% O2), HTR-8/SVneo cells and HUVEC cultured in 1% O2 expressed more uPAR, as determined by flow cytometric and [125I]-prourokinase ligand binding analyses. Increased uPAR expression paralleled increases in uPAR mRNA. The involvement of a heme protein in the hypoxia-induced expression of uPAR was suggested by the observations that culture of cells with cobalt chloride, or sodium 4,5-dihydroxybenzene-1,3-disulfonate (Tiron), an iron-chelating agent, also stimulated uPAR expression, and that the hypoxia-induced uPAR expression was inhibited by adding carbon monoxide to the hypoxic atmosphere. Culture of HTR-8/SVneo cells with vascular endothelial growth factor (VEGF) did not increase uPAR mRNA levels, suggesting that the hypoxia-mediated effect on uPAR expression by these cells did not occur through a VEGF-dependent mechanism. The functional importance of these findings is suggested by the fact that HTR-8/SVneo cells cultured under hypoxia displayed higher levels of cell surface plasminogen activator activity and greater invasion through a reconstituted basement membrane. These results suggest that hypoxia may promote cellular invasion by stimulating the expression of uPAR through a heme protein-dependent pathway.
Myc Is Essential for Transformation by TEL/Platelet-Derived Growth Factor Receptor β (PDGFRβ)
Authors: Bourgeade Marie-Françoise , Défachelles Anne-Sophie , Cayre Yvon E.
Published: October 2019

The t(5;12) translocation identified in patients with chronic myelomonocytic leukemia (CMML) encodes a TEL/platelet-derived growth factor receptor β (PDGFRβ) fusion protein. A key hypothesis for how the TEL/PDGFRβ fusion protein would function as an oncogene is that it represents a constitutively active version of the normal PDGFRβ. A link between the function of the t(5;12)-encoded TEL/PDGFRβ fusion protein and Myc expression is suggested by the fact that Myc is induced by PDGF and is essential for entry of cells into the S phase of the cell cycle. We here show that the kinase activity of TEL/PDGFRβ is necessary for Ba/F3 cells to acquire interleukin-3 (IL-3) independence and that, in contrast to their untransfected counterpart, Ba/F3 cells stably transfected with TEL/PDGFRβ maintain a high level of Myc expression after removal of IL-3. Using dominant negative mutants of Myc, we show that a threshold of active Myc is essential for TEL/PDGFRβ to transform Ba/F3 and Rat-1 cells. The findings that the kinase activity of TEL/PDGFRβ and a threshold of active Myc are involved in TEL/PDGFRβ transformation may allow for the development of therapeutic strategies in patients with t(5;12)+ CMML using specific inhibitors of the PDGFRβ kinase as well as compounds designed to interfere specifically with Myc.
Expression of Apoptosis-Regulating Proteins in Chronic Lymphocytic Leukemia: Correlations With In Vitro and In Vivo Chemoresponses
Authors: Kitada Shinichi , Andersen Janet , Akar Sophie , Zapata Juan M. , Takayama Shinichi , Krajewski Stanislaw , Wang Hong-Gang , Zhang Xin , Bullrich Florencia , Croce Carlo M. , Rai Kanti , Hines John , Reed John C.
Published: October 2019

B-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD), as opposed to increased cell proliferation. Defects in the PCD pathway also contribute to chemoresistance. The expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2–binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was evaluated by immunoblotting using 58 peripheral blood B-CLL specimens from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1, Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells, whereas the Bcl-XL and BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil) (P = .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious interpretation of these observations. Higher levels of the anti-apoptotic protein BAG-1 were also marginally associated with failure to achieve CR (P = .04). Apoptosis-regulating proteins were not associated with patient age, sex, Rai stage, platelet count, hemoglobin (Hb) concentration, or lymph node involvement, although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were correlated with high numbers (>105/μL) of white blood cells (WBC) (P = .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at 13q14 (P = .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various apoptosis-regulating proteins. Although larger prospective studies are required before firm conclusions can be reached, these studies show the expression in B-CLLs of multiple apoptosis-regulating proteins and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro chemosensitivity data, however, do not appear to be particularly useful in predicting responses in B-CLL.
Expression of the T-Cell–Specific Tyrosine Kinase Lck in Normal B-1 Cells and in Chronic Lymphocytic Leukemia B Cells
Authors: Majolini M. Bernardetta , D'Elios Mario M. , Galieni Piero , Boncristiano Marianna , Lauria Francesco , Del Prete Gianfranco , Telford John L. , Baldari Cosima T.
Published: October 2019

Src family kinases play a key role in mitogenesis. The exquisitely tissue-specific distribution of different Src family members suggests that a fine tuning of their expression might be a key prerequisite for cell homeostasis. We tested B cells from patients affected by B-cell chronic lymphocytic leukemia (B-CLL) for expression of Src family kinases. The T-cell–specific tyrosine kinase Lck was found to be expressed at significant levels in CLL B-cells. This finding could be accounted for either by ectopic expression of Lck in B-CLL or by specific expression of this kinase in normal B-1 cells, which are believed to be the normal counterpart of CLL B cells. To answer this question B cells from different sources, characterized by a different size of the B-1 subpopulation, were tested for Lck expression. The results show that Lck expression is a feature of CD5+, B-1 cells, suggesting a potential role for Lck in the self-renewal capacity of this B-cell subpopulation and supporting the notion that B-1 cells are the subset undergoing oncogenic transformation in B-CLL. Furthermore, we show that the CD5−, B-2 subpopulation, while normally lacking Lck expression, acquires the capacity to express Lck ectopically upon transformation by EBV.
6q Deletions in Acute Lymphoblastic Leukemia and Non-Hodgkin's Lymphomas
Authors: Merup Mats , Moreno Teresa Calero , Heyman Mats , Rönnberg Kristina , Grandér Dan , Detlofsson Rut , Rasool Omid , Liu Yie , Söderhäll Stefan , Juliusson Gunnar , Gahrton Gösta , Einhorn Stefan
Published: October 2019

Deletions on the long arm of chromosome 6 are frequently found in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphomas (NHL). We have used polymerase chain reaction analysis to study loss of heterozygosity of 16 microsatellite markers on chromosome 6 in 74 ALL and 54 NHL patients. Our results show that deletions of 6q in ALL are more frequent than what has been reported in previous studies, occurring in at least 32% of the patients. The corresponding figure for NHL patients is 7%. Our results define a region of minimal deletion in ALL of less than 500 kb between markers D6S1709 and D6S434. The common region of deletion in NHL is located telomeric of this region. Thus, two different tumor suppressor genes on chromosome 6q seem to be relevant for the development of lymphoid malignancies.
Reduced Ex Vivo Interleukin-8 Production by Neutrophils in Septic and Nonseptic Systemic Inflammatory Response Syndrome
Authors: Marie Christelle , Muret Jane , Fitting Catherine , Losser Marie-Reine , Payen Didier , Cavaillon Jean-Marc
Published: October 2019

Ex vivo cytokine production by circulating lymphocytes and monocytes is reduced in patients with infectious or noninfectious systemic inflammatory response syndrome. Very few studies have addressed the reactivity of polymorphonuclear cells (PMN). To analyze further the relative contribution of systemic inflammatory response syndrome alone or in combination with infection we studied the interleukin-8 (IL-8) production by PMN isolated from patients who had undergone cardiac surgery with cardiopulmonary bypass (CPB) and patients with sepsis. Cells were activated with either lipopolysaccharide (LPS) or heat-killed streptococci. Compared with healthy controls, the release of IL-8 by PMN in both groups of patients was significantly reduced whether activated by LPS, independently of its concentration and origin, or by heat-killed streptococci. These observations suggest that stressful conditions related to inflammation, independently of infection, rapidly dampened the reactivity of circulating PMN. We investigated whether the observed diminished reactivity of PMN might reflect an endotoxin tolerance phenomenon. Our in vitro experiments with PMN from healthy controls indicated that PMN could not be rendered tolerant stricto sensu. However, our data suggested that LPS-induced mediators such as IL-10 may be responsible for the observed anergy in patients.
Mutations in the E-Domain of RARα Portion of the PML/RARα Chimeric Gene May Confer Clinical Resistance to All-transRetinoic Acid in Acute Promyelocytic Leukemia
Authors: Imaizumi Masue , Suzuki Hoshiro , Yoshinari Miyako , Sato Atsushi , Saito Toshiaki , Sugawara Akira , Tsuchiya Shigeru , Hatae Yoshiro , Fujimoto Takeo , Kakizuka Akira , Konno Tasuke , Iinuma Kazuie
Published: October 2019

The binding of all-trans retinoic acid (ATRA) to the ligand-binding region in the E-domain of retinoic acid receptor-α (RARα) modifies the transcriptional activity of RARα protein. ATRA probably induces differentiation of acute promyelocytic leukemia (APL) cells by binding to the E-domain of the RARα portion (RARα/E-domain) of PML/RARα chimeric protein. Therefore, molecular alteration in the RARα/E-domain of the chimeric gene is one mechanism by which patients with APL may acquire resistance to ATRA therapy. In this study using reverse transcription-polymerase chain reaction and single-strand conformation polymorphism, DNA segments amplified from the RARα/E-domain in fresh APL cells of 23 APL patients (8 males and 15 females from 4 to 76 years of age) were screened for mutations. Of those patients, 3 patients (1 with de novo and 2 with relapse) had clinical resistance to ATRA therapy. We found mutations in the RARα/E-domain of PML/RARα chimeric gene exclusively in the 2 patients who exhibited ATRA-resistance at relapse, whereas the mutations were not detected at their initial onset. Interestingly, these patients received a prolonged or intermittent administration of ATRA before relapse with ATRA-resistance. The mutations lead to the change of amino acid in the ligand-binding region of RARα/E-domain, Arg272Gln, or Met297Leu according to the amino acid sequence of RARα, respectively. Further study demonstrated that the in vitro ligand-dependent transcriptional activity of the mutant PML/RARα protein was significantly decreased as compared with that of wild-type PML/RARα. These findings suggest that mutations in the RARα/E-domain of the PML/RARα chimeric gene may confer clinical resistance to ATRA therapy in patients with APL.
Granulocyte-Macrophage Colony-Stimulating Factor Rescues TF-1 Leukemia Cells From Ionizing Radiation-Induced Apoptosis Through a Pathway Mediated by Protein Kinase Cα
Authors: Kelly Mary L. , Tang Yan , Rosensweig Nitsa , Clejan Sanda , Beckman Barbara S.
Published: October 2019

Protein kinase C (PKC) activity has a recognized role in mediating apoptosis. However, the role of individual PKC isoforms in apoptosis is poorly defined. Therefore, we investigated the translocation of individual PKC isoforms during radiation-induced apoptosis with and without rescue from apoptosis by granulocyte-macrophage colony-stimulating factor (GM-CSF) in the human erythroleukemia cell line TF-1. PKCα was translocated from the particulate to cytosolic fraction of TF-1 cells within 5 minutes of treatment with apoptosis-inducing levels of ionizing radiation. However, this postirradiation translocation did not occur when cells were rescued from apoptosis by GM-CSF. Furthermore, treatment of cells with Gö6976, an inhibitor of classical PKC isoforms, abrogated the rescue effect of GM-CSF. The calcium-independent novel PKC isoform, PKCδ appeared to be degraded in both the particulate and cytosolic fractions of TF-1 cells after treatment with apoptosis-inducing levels of ionizing radiation in either the presence or absence of GM-CSF rescue. Levels of ceramide, a lipid mediator of apoptosis, were measured at 2, 4, 8, 10, and 60 minutes after treatment with ionizing radiation and were substantially reduced in TF-1 cells rescued from apoptosis by GM-CSF compared with apoptotic TF-1 cells. The largest decrease in ceramide production seen was at 4 minutes postirradiation, with a 46% reduction in ceramide levels in TF-1 cells rescued from apoptosis by GM-CSF compared with those in apoptotic TF-1 cells. Because ceramide has been shown to affect PKCα subcellular distribution, these data implicate a role for ceramide in mediating the rapid postirradiation translocation and inhibition of PKCα in TF-1 cells not rescued from apoptosis by GM-CSF. Expression of the antiapoptotic protein Bcl-2 doubled in TF-1 cells rescued from apoptosis by GM-CSF, but did not increase in unrescued cells. Our findings suggest that activated PKCα and increased expression of Bcl-2 after γ irradiation determine survival in TF-1 cells rescued from apoptosis with GM-CSF and that PKCδ plays a role in mediating signals involved in sensing cellular damage and/or regulation of cell damage repair.
Role of GATA-1 in Proliferation and Differentiation of Definitive Erythroid and Megakaryocytic Cells In Vivo
Authors: Takahashi Satoru , Komeno Takuya , Suwabe Naruyoshi , Yoh Keigyo , Nakajima Osamu , Nishimura Sigeko , Kuroha Takashi , Nagasawa Toshiro , Yamamoto Masayuki
Published: October 2019

To elucidate the contributions of GATA-1 to definitive hematopoiesis in vivo, we have examined adult mice that were rendered genetically defective in GATA-1 synthesis (Takahashi et al, J Biol Chem272:12611, 1997). Because the GATA-1 gene is located on the X chromosome, which is randomly inactivated in every cell, heterozygous females can bear either an active wild-type or mutant (referred to asGATA-1.05) GATA-1 allele, consequently leading to variable anemic severity. These heterozygous mutant mice usually developed normally, but they began to die after 5 months. These affected animals displayed marked splenomegaly, anemia, and thrombocytopenia. Proerythroblasts and megakaryocytes massively accumulated in the spleens of the heterozygotes, and we showed that the neomycin resistance gene (which is the positive selection marker in ES cells) was expressed profusely in the abnormally abundant cells generated in the GATA-1.05 mutant females. We also observed hematopoiesis outside of the bone marrow in the affected mutant mice. These data suggest that a small number of GATA-1.05 mutant hematopoietic progenitor cells begin to proliferate vigorously during early adulthood, but because the cells are unable to terminally differentiate, this leads to progenitor proliferation in the spleen and consequently death. Thus, GATA-1 plays important in vivo roles for directing definitive hematopoietic progenitors to differentiate along both the erythroid and megakaryocytic pathways. The GATA-1 heterozygous mutant mouse shows a phenotype that is analogous to human myelodysplastic syndrome and thus may serve as a useful model for this disorder.
The Induction of Megakaryocyte Differentiation Is Accompanied by Selective Ser133 Phosphorylation of the Transcription Factor CREB in Both HEL Cell Line and Primary CD34+Cells
Authors: Zauli Giorgio , Gibellini Davide , Vitale Marco , Secchiero Paola , Celeghini Claudio , Bassini Alessandra , Pierpaoli Sabina , Marchisio Marco , Guidotti Lia , Capitani Silvano
Published: October 2019

The addition of thrombopoietin (TPO) to HEL cells, cultured in a chemically defined serum-free medium, induced a rapid and dose-dependent phosphorylation of the transcription factor CREB on serine133 (PSer133), as detected by Western blot analysis. TPO also significantly increased the transactivation of CRE-dependent promoter, as determined in transient transfection experiments. On the other hand, neither erythropoietin (Epo; 1 to 10 U) nor hemin (10−7 mol/L) were able to significantly stimulate CREB-PSer133 or to activate CRE-promoter in HEL cells. Although pharmacological inhibitors of protein kinase C (chelerytrine and BIM) and protein kinase A (H-89) failed to block the TPO-mediated CREB phosphorylation, a specific inhibitor of the mitogen-activated protein kinases (PD98059) completely blocked the ability of TPO to stimulate CREB-PSer133. Moreover, PD98059 significantly decreased the ability of TPO to upregulate the surface expression of the αIIbβ3 megakaryocytic marker in HEL cells. In parallel, primary CD34+ hematopoietic cells were seeded in liquid cultures supplemented with 100 ng/mL of TPO and examined by immunofluorescence for the coexpression of αIIbβ3 and CREB-PSer133 at various time points. High levels of nuclear CREB-PSer133 were unequivocally demonstrated in αIIbβ3+cells, including morphologically recognizable megakaryocytes. Taken together, these data suggest that CREB plays a role in modulating the expression of genes critical for megakaryocyte differentiation and that the TPO-mediated CREB phosphorylation seems to be regulated via mitogen-activated protein kinases.
Mechanism and Kinetics of Factor VIII Inactivation: Study With an IgG4 Monoclonal Antibody Derived From a Hemophilia A Patient With Inhibitor
Authors: Jacquemin Marc G. , Desqueper Benoı̂t G. , Benhida Abdellah , Vander Elst Luc , Hoylaerts Marc F. , Bakkus Marleen , Thielemans Kris , Arnout Jef , Peerlinck Kathelijne , Gilles Jean Guy G. , Vermylen Jos , Saint-Remy Jean-Marie R.
Published: October 2019

The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4κ monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass ∼7.4 × 105M−1 s−1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss ≤1 × 10−5s−1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.
Expression of P-Selectin on Hepatic Endothelia and Platelets Promoting Neutrophil Removal by Liver Macrophages
Authors: Shi Jialan , Kokubo Yoshihiro , Wake Kenjiro
Published: October 2019

The role of P-selectin on polymorphonuclear leukocyte (PMN) adhesion-induced PMN elimination in the liver is unclear. Our objectives were to show the expression and distribution of P-selectin in rat liver, as well as to evaluate the changes in the modulation of the expression of P-selectin and its role in the accumulation and sequestration of PMNs. The intravenous administration of endotoxin markedly increased the expression of P-selectin on the venous and sinusoidal endothelial cells, as well as on the platelets trapped in the liver. Its expression peaked at 6 hours postinjection and was associated with a rapid increase in the aggregation and elimination of PMNs in the hepatic sinusoids. Combined treatment with an antibody to P-selectin or with low molecular weight heparin, a P-selectin antagonist, blocked the P-selectin, significantly reduced the arrest of PMNs, and delayed their removal in the liver. Pretreatment with gadolinium chloride inhibited phagocytosis of PMNs by the Kupffer cells, decreased the expression of P-selectin, and limited the hepatic accumulation of PMNs. Thus, P-selectin played a role in accumulation and elimination of PMNs from the liver. Results also suggest that activated Kupffer cells can modulate the expression of P-selectin in the liver and influence the homeostasis of PMNs in the circulation during acute inflammation.
Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Translocations and Chromosomal Aberrations in Acute Leukemia
Authors: Pallisgaard Niels , Hokland Peter , Riishøj Dorthe C. , Pedersen Bent , Jørgensen Poul
Published: October 2019

We have developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) reaction, which enables us to detect 29 translocations/chromosomal aberrations in patients with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). Through the construction and optimization of specific primers for each translocation, we have been able to reduce the set-up to 8 parallel multiplex PCR reactions, thus greatly decreasing the amount of work and reagents. We show the value of our set-up in a retrospective analysis on cryopreserved material from 102 AML and 62 ALL patients. The multiplex RT-PCR detected a hybrid mRNA resulting from a structural chromosomal aberration in 45 of 102 (44%) of the AML and in 28 of 62 (45%) of the pediatric ALL cases. Importantly, in 33% of AML and in 47% of the ALL cases with cytogenetic data, submicroscopic chromosomal aberrations or masked translocations were shown that were not detected in the cytogenetic analysis either for structural reasons or because of an insufficient number of metaphases obtained. This multiplex RT-PCR system, which can handle up to 10 patients with a response time of 2 working days, is thus an important tool that complements cytogenetic analysis in the up-front screening of acute leukemia patients and should provide a rapid and efficient characterization of leukemia cells, even in situations with sparse patient material.
Impaired Granulocytic Differentiation In Vitro in Hematopoietic Cells Lacking Retinoic Acid Receptors α1 and γ
Authors: Labrecque Jean , Allan Deborah , Chambon Pierre , Iscove Norman N. , Lohnes David , Hoang Trang
Published: October 2019

Transcripts for the retinoic acid receptors (RARs) α1, α2, γ1, and γ2 were found in the granulocytic lineage (Gr-1+cells) through semiquantitative polymerase chain reaction (PCR) analysis. The screening of single cell cDNA libraries derived from hematopoietic progenitors also showed the presence of RARα and, to a lesser extent, RARγ transcripts in committed granulocyte (colony-forming unit-granulocyte [CFU-G]) or granulocyte-macrophage (CFU-GM) colony-forming cells. The contribution of RARα1 and γ to hematopoietic cell differentiation was therefore investigated in mice bearing targeted disruption of either one or both of these loci. Because RARγ and RARα1γ compound null mutants die shortly after birth, bone marrow cells were collected from fetuses at 18.5 days postcoitum (dpc) and evaluated for growth and differentiation in culture in the presence of Steel factor (SF), interleukin-3 (IL-3), and erythropoietin (Epo). The frequency of colony-forming cells from bone marrow populations derived from RARα1/γ double null mice was not significantly different from that of RARγ or RARα1 single nulls or from wild-type controls. In addition, the distribution of erythroid, granulocyte, and macrophage colonies was comparable between hematopoietic cells from all groups, suggesting that lineage commitment was not affected by the lack of RARα1 and/or RARγ. Colony cells were then harvested individually and evaluated by morphologic criteria. While terminal granulocyte differentiation was evident in wild-type cells and colonies from either single null mutant, colonies derived from RARα1−/−γ−/− bone marrow populations were blocked at the myelocyte and, to a lesser extent, at the metamyelocyte stages, whereas erythroid and macrophage differentiation was not affected. Together, these results indicate that both RARα1 and γ are required for terminal maturation in the granulocytic lineage in vitro, but appear to be dispensable for the early stages of hematopoietic cell development. Our results raise the possibility that in acute promyelocytic leukemia (APL), the different RARα fusion proteins cause differentiation arrest at a stage when further maturation requires not only RARα, but also RARγ. Finally, bone marrow cells appear to differentiate normally in vivo, suggesting an effective compensation mechanism in the RARα1/γ double null mice.
Role of Class-II Major Histocompatibility Complex (MHC)-Antigen–Positive Donor Leukocytes in Transfusion-Induced Alloimmunization to Donor Class-I MHC Antigens
Authors: Kao K.J. , del Rosario M.L.U.
Published: October 2019

It has been shown that peripheral-blood mononuclear leukocytes (MNL) are responsible for transfusion-induced alloimmunization to donor major histocompatability complex (MHC) antigens. However, it is not known which subset of MNL is responsible for this immune response. Because elimination of class-II MHC antigen-positive passenger leukocytes effectively prolongs the survival of allografts, it has been hypothesized that class-II positive MNL are responsible for immunizing transfusion recipients to donor MHC antigens. To test this hypothesis, two different approaches were used. First, we compared the alloantigenicity of BALB/c mice (H-2d) peripheral blood MNL before and after depletion of class-II positive cells. CBA mice (H-2k) were used as transfusion recipients. Antibody development to donor class-I H-2 antigens was determined by flow cytometry and enzyme-linked immunoassay. After four weekly transfusions of MNL depleted for class-II positive cells, only 25% of recipient mice developed antibodies to donor H-2d antigens. In contrast, all mice transfused with control MNL became immunized. Second, we studied the alloantigenicity of peripheral MNL from C57BL/6 mice (H-2b) with homozygous deficiency of class-II MHC molecules in H-2 disparate recipient mice. After transfusions with class-II MHC molecule-deficient MNL, 0% of BALB/c, 40% of C57BR, and 25% of CBA-recipient mice developed antibodies to donor H-2b antigen. All control recipient mice were immunized. The antibody activities of the controls were also higher than those in the treatment group who became immunized. Thus, our study shows that class-II MHC antigen-positive MNL play a significant role in transfusion-induced alloimmunization to donor class-I MHC antigens. The results also support the hypothesis that direct antigen presentation by donor class-II positive MNL to the immune system of transfusion recipients is critical for the initiation of humoral immune response to donor MHC antigens.
The α-Chemokine Receptor CXCR4 Is Expressed on the Megakaryocytic Lineage From Progenitor to Platelets and Modulates Migration and Adhesion
Authors: Wang Jian-Feng , Liu Zhong-Ying , Groopman Jerome E.
Published: October 2019

CXCR4 is the receptor for the α-chemokine stromal cell-derived factor 1 (SDF-1) and has been shown to be expressed on a diversity of leukocytes. In this report, the expression of the CXCR4 receptor in cells of megakaryocytic lineage and the role of SDF-1 in megakaryocytopoiesis were investigated. Using flow cytometry in combination with reverse transcriptase-polymerase chain reaction (RT-PCR), we observed that bone marrow CD34+, CD61+ cells, blood platelets, and megakaryocytic leukemia cell lines all expressed the CXCR4 receptor. To examine the expression of the CXCR4 receptor on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-Meg]), CXCR4-positive and -negative CD34+ populations were separated from bone marrow and cultured in a plasma clot culture system. A subpopulation of the CFU-Meg was found in the CXCR4-positive fraction. The functional significance of CXCR4 expression on cells of the megakaryocytic lineage was examined by studying the effects of SDF-1α on migration and proliferation of megakaryocyte progenitor cells in vitro. We found that SDF-1α potently induced megakaryocyte progenitor migration and significantly enhanced adhesion of mature marrow megakaryocytes to endothelium. No marked effects of SDF-1α alone or in combination with thrombopoietin and stem cell factor/kit ligand on megakaryocyte production in vitro were noted. These results demonstrate for the first time that the CXCR4 α-chemokine receptor is expressed on cells of the megakaryocytic lineage from progenitors to platelets and that its ligand SDF-1α may modulate several aspects of megakaryocytopoiesis.

© 1998 by The American Society of Hematology.
Expression of Cyclin E and the Cyclin-Dependent Kinase Inhibitor p27 in Malignant Lymphomas—Prognostic Implications
Authors: Erlanson Martin , Portin Cajsa , Linderholm Barbro , Lindh Jack , Roos Göran , Landberg Göran
Published: October 2019

Cyclin E and the cyclin-dependent kinase inhibitor p27 are two important regulators of the G1-S transition modulating the activity of cyclin-dependent kinases. Aberrations in the cell cycle control are often observed in tumors and might even be mandatory in tumor development. To investigate the importance of cell-cycle defects in malignant lymphomas we have characterized the expression of cyclin E and p27 in 105 newly diagnosed lymphomas using immunohistochemistry. A significant, inverse correlation between p27 and cyclin E expression was observed (rs = −.24, P = .02) and both proteins correlated with the S-phase fraction (rs = −.35, P < .001 andrs = .45, P < .001, respectively). The inverse relationship between p27 expression and proliferation was abrogated in some lymphomas, suggesting that p27 downregulation can represent a genuine aberration. Survival analysis was performed in 105 patients with a median observation time of 86 months. Low p27 and high cyclin E expression were significantly associated with a poor prognosis (P = .0001 and .03, respectively). In a multivariate Cox analysis, p27 expression, stage, serum lactate dehydrogenase level, grade, and age were independent prognostic factors, in contrast to S-phase fraction and cyclin E expression. This is the first report showing that p27 expression in malignant lymphomas has independent prognostic significance, which necessitates future studies regarding its more precise biological role in lymphoid tumorogenesis.

© 1998 by The American Society of Hematology.
Mutual Education Between Hematopoietic Cells and Bone Marrow Stromal Cells Through Direct Cell-to-Cell Contact: Factors That Determine the Growth of Bone Marrow Stroma-Dependent Leukemic (HB-1) Cells
Authors: Jiang Huijie , Sugimoto Kenkichi , Sawada Hitoshi , Takashita Emi , Tohma Maki , Gonda Hiroyuki , Mori Kazuhiro John
Published: October 2019

A stroma-dependent cell line (HB-1) was established from myelogenous leukemic cells of CBA/N mouse. Characterization of the cells showed that HB-1 proliferated on hematopoietic supportive stromal cells (MS-10), but did not survive or proliferate on hematopoietic nonsupportive cells (MS-K). Direct contact between HB-1 and MS-10 appears to be necessary for HB-1 to proliferate on MS-10. We found that interleukin-1α (IL-1α) produced by MS-10 plays a major role in the survival and proliferation of HB-1. IL-11 did not support the proliferation of HB-1 cells by itself, but enhanced the proliferation of HB-1 cells in the presence of IL-1α. The expression of IL-1α and IL-11 was induced in MS-10 by the direct contact with HB-1 cells, and the expression of IL-1 receptor type I (IL-1RI) and interleukin-11 receptor (IL-11R) was induced in HB-1 cells by the attachment of the cells to MS-10. These findings show the existence of two-way interactions between HB-1 and MS-10.

© 1998 by The American Society of Hematology.
CD164, a Novel Sialomucin on CD34+ and Erythroid Subsets, Is Located on Human Chromosome 6q21
Authors: Watt Suzanne M. , Bühring Hans-Jörg , Rappold Irene , Chan James Yi-Hsin , Lee-Prudhoe Jane , Jones Tania , Zannettino Andrew C.W. , Simmons Paul J. , Doyonnas Regis , Sheer Denise , Butler Lisa H.
Published: October 2019

CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human CD34+ hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in hematopoiesis by facilitating the adhesion of CD34+ cells to bone marrow stroma and by negatively regulating CD34+hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes, defined by the monoclonal antibodies (MoAbs), 103B2/9E10 and 105A5. In this report, we show that these MoAbs, together with two other CD164 MoAbs, N6B6 and 67D2, show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical cord blood, and peripheral blood. Flow cytometric analyses revealed that, on average, 63% to 82% of human bone marrow and 55% to 93% of cord blood CD34+ cells are CD164+, with expression of the 105A5 epitope being more variable than that of the other identified epitopes. Extensive multiparameter flow cytometric analyses were performed on cells expressing the 103B2/9E10 functional epitope. These analyses showed that the majority (>90%) of CD34+ human bone marrow and cord blood cells that were CD38lo/− or that coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)+. This CD164 epitope was generally detected on a significant proportion of CD34+CD71lo/− or CD34+CD33lo/− cells. In accord with our previous in vitro progenitor assay data, these phenotypes suggest that the CD164(103B2/9E10) epitope is expressed by a very primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD34+CD164(103B2/9E10)lo/−cells in bone marrow are mainly CD19+ B-cell precursors, with the CD164(103B2/9E10) epitope subsequently appearing on CD34lo/−CD19+ and CD34lo/−CD20+ B cells in bone marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34lo/−CD164(103B2/9E10)+ subsets indicated that one of the most prominent populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/9E10) epitope is also found on developing myelomonocytic cells in bone marrow, being downregulated on mature neutrophils but maintained on monocytes in the peripheral blood. We have extended these studies further by identifying Pl artificial chromosome (PAC) clones containing the CD164 gene and have used these to localize the CD164 gene specifically to human chromosome 6q21.

© 1998 by The American Society of Hematology.
Negative Regulation by Interleukin-3 (IL-3) of Mouse Early B-Cell Progenitors and Stem Cells in Culture: Transduction of the Negative Signals by βc and βIL-3 Proteins of IL-3 Receptor and Absence of Negative Regulation by Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Matsunaga Takuya , Hirayama Fumiya , Yonemura Yuji , Murray Richard , Ogawa Makio
Published: October 2019

The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit βc. However, in the mouse, there is an additional IL-3 signaling protein, βIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from βc- and βIL-3–knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin−) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin−Sca-1–positive, c-kit–positive marrow cells from 5-FU–treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with “compromised” marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors.

© 1998 by The American Society of Hematology.
MDM2 Protein Overexpression Inhibits Apoptosis of TF-1 Granulocyte-Macrophage Colony-Stimulating Factor–Dependent Acute Myeloblastic Leukemia Cells
Authors: Urashima Mitsuyoshi , Teoh Gerrard , Chauhan Dharminder , Ogata Atsushi , Shirahama Shuya , Kaihara Chiharu , Matsuzaki Masaharu , Matsushima Hiroshi , Akiyama Masaharu , Yuza Youki , Maekawa Kihei , Anderson Kenneth C.
Published: October 2019

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.

© 1998 by The American Society of Hematology.
A Single Genetic Origin for the Common Prothrombotic G20210A Polymorphism in the Prothrombin Gene
Authors: Zivelin Ariella , Rosenberg Nurit , Faier Shlomit , Kornbrot Nurit , Peretz Hava , Mannhalter Christine , Horellou Marie Helene , Seligsohn Uri
Published: October 2019

The polymorphism G20210A in the 3′ untranslated region of the prothrombin gene is associated with an increased level of factor II activity and confers a twofold to fivefold increase in the risk for venous thromboembolism. Among Caucasian populations, the prevalence of factor II G20210A heterozygotes is 1% to 6%, whereas in non-Caucasian populations it is very rare or absent. The aim of the present study was to discern whether factor II G20210A originated from a single or recurrent mutational events. Allele frequencies of four dimorphisms spanning 16 of 21 kb of the factor II gene were determined in 133 unrelated Caucasian subjects of Jewish, Austrian, and French origins who bore factor II G20210A (10 homozygotes and 123 heterozygotes) and 110 Caucasian controls. Remarkable differences in the allele frequencies for each dimorphism were observed between the study groups (P = .0007 or less), indicating strong linkage disequilibrium and suggesting a founder effect. Indeed, a founder haplotype was present in 68% of 20210A mutant alleles and only in 34% of 20210G normal alleles (P < .0001). These data strongly support a single origin for factor II G20210A that probably occurred after the divergence of Africans from non-Africans and of Caucasoid from Mongoloid subpopulations.

© 1998 by The American Society of Hematology.
ABI-1, a Human Homolog to Mouse Abl-Interactor 1, Fuses theMLL Gene in Acute Myeloid Leukemia With t(10;11)(p11.2;q23)
Authors: Taki Tomohiko , Shibuya Noriko , Taniwaki Masafumi , Hanada Ryoji , Morishita Kazuhiro , Bessho Fumio , Yanagisawa Masayoshi , Hayashi Yasuhide
Published: October 2019

Recurrent translocation t(10;11) has been reported to be associated with acute myeloid leukemia (AML). Recently, two types of chimeric transcripts, MLL-AF10 in t(10;11)(p12;q23) andCALM-AF10 in t(10;11)(p13;q14), were isolated. t(10;11) is strongly associated with complex translocations, including invins(10;11) and inv(11)t(10;11), because the direction of transcription of AF10 is telomere to centromere. We analyzed a patient of AML with t(10;11)(p11.2;q23) and identified ABI-1 on chromosome 10p11.2, a human homolog to mouse Abl-interactor 1 (Abi-1), fused with MLL. Whereas the ABI-1 gene bears no homology with the partner genes of MLL previously described, the ABI-1 protein exhibits sequence similarity to protein of homeotic genes, contains several polyproline stretches, and includes asrc homology 3 (SH3) domain at the C-terminus that is required for binding to Abl proteins in mouse Abi-1 protein. Recently, e3B1, an eps8 SH3 binding protein 1, was also isolated as a human homolog to mouse Abi-1. Three types of transcripts of ABI-1 gene were expressed in normal peripheral blood. Although e3B1 was considered to be a full-length ABI-1, the MLL-ABI-1fusion transcript in this patient was formed by an alternatively spliced ABI-1. Others have shown that mouse Abi-1 suppresses v-ABL transforming activity and that e3B1, full-length ABI-1, regulates cell growth. In-frame MLL-ABI-1 fusion transcripts combine the MLL AT-hook motifs and DNA methyltransferase homology region with the homeodomain homologous region, polyproline stretches, and SH3 domain of alternatively spliced transcript of ABI-1. Our results suggest that the ABI-1 gene plays a role in leukemogenesis by translocating to MLL.

© 1998 by The American Society of Hematology.