bluuebunny/crossref_metadata_2025_split huggingface dataset:
GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation
Authors: Suwabe Naruyoshi , Takahashi Satoru , Nakano Toru , Yamamoto Masayuki 
Published: October 2019

AbstractAlthough the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.
Cooperative Effects of Growth Factors Involved in the Induction of Hematopoietic Mesoderm
Authors: Huber Tara L. , Zhou Yi , Mead Paul E. , Zon Leonard I. 
Published: October 2019

AbstractHematopoietic induction occurs on the ventral side ofXenopus gastrulae and is thought to be triggered by the growth factor bone morphogenetic protein 4 (BMP-4). To characterize this process, we developed a quantitative and sensitive assay for the induction of erythroid cells from totipotent ectoderm of the embryo. When high doses of BMP-4 were used in this explant assay, few erythroid cells were detected. In contrast, large numbers of differentiated erythroid cells were induced when ectoderm was treated with BMP-4 and the mesoderm inducers, activin, or fibroblast growth factor (FGF). Ectopic expression of GATA-1 also induced abundant erythroid cells in ectoderm treated with bFGF. This induction of erythroid cells by GATA-1 was blocked by coexpression with a dominant negative BMP-4 receptor, showing that GATA-1 requires the BMP signaling cascade to function. These results suggest that BMP-4 requires mesoderm induction to generate a program of gene expression, which regulates the specification of hematopoietic mesoderm by GATA factors.
Vascular Endothelial Growth Factor Inhibits the Development of Dendritic Cells and Dramatically Affects the Differentiation of Multiple Hematopoietic Lineages In Vivo
Authors: Gabrilovich Dmitry , Ishida Tadao , Oyama Tsunehiro , Ran Sophia , Kravtsov Vladimir , Nadaf Sorena , Carbone David P. 
Published: October 2019

AbstractDefective function of dendritic cells (DC) in cancer has been recently described and may represent one of the mechanisms of tumor evasion from immune system control. We have previously shown in vitro that vascular endothelial growth factor (VEGF), produced by almost all tumors, is one of the tumor-derived factors responsible for the defective function of these cells. In this study, we investigated whether in vivo infusion of recombinant VEGF could reproduce the observed DC dysfunction. Continuous VEGF infusion, at rates as low as 50 ng/h (resulting in serum VEGF concentrations of 120 to 160 pg/mL), resulted in a dramatic inhibition of dendritic cell development, associated with an increase in the production of B cells and immature Gr-1+ myeloid cells. Infusion of VEGF was associated with inhibition of the activity of the transcription factor NF-κB in bone marrow progenitor cells. Experiments in vitro showed that VEGF itself, and not factors released by VEGF-activated endothelial cells, affected polypotent stem cells resulting in the observed abnormal hematopoiesis. These data suggest that VEGF, at pathologically relevant concentrations in vivo, may exert effects on pluripotent stem cells that result in blocked DC development as well as affect many other hematopoietic lineages.
Proteasome Inhibitors Induce Apoptosis in Glucocorticoid-Resistant Chronic Lymphocytic Leukemic Lymphocytes
Authors: Chandra Joya , Niemer Irina , Gilbreath Joyce , Kliche Kay-Oliver , Andreeff Michael , Freireich Emil J. , Keating Michael , McConkey David J. 
Published: October 2019

AbstractOur previous work showed that the nuclear scaffold (NS) protease is required for apoptosis of both thymocytes and chronic lymphocytic leukemic (CLL) lymphocytes. Because partial sequencing of one of the subunits of the NS protease revealed homology to the proteasome, we tested the effects of classical proteasome inhibitors on apoptosis in CLL cells. Here we report that proteasome inhibition caused high levels of DNA fragmentation in all patients analyzed, including those resistant to glucocorticoids or nucleoside analogs, in vitro. Proteasome inhibitor-induced DNA fragmentation was associated with activation of caspase/ICE family cysteine protease(s) and was blocked by the caspase antagonist, zVADfmk. Analysis of the biochemical mechanisms involved showed that proteasome inhibition resulted in mitochondrial dysregulation leading to the release of cytochrome c and a drop in mitochondrial transmembrane potential (▵Ψ). These changes were associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in other systems. Together, our results suggest that drugs that target the proteasome might be capable of bypassing resistance to conventional chemotherapy in CLL.
Identification of New Nonrandom Translocations in Multiple Myeloma With Multicolor Spectral Karyotyping
Authors: Sawyer Jeffrey R. , Lukacs Janet L. , Munshi Nikhil , Desikan K. Raman , Singhal Seema , Mehta Jayesh , Siegel David , Shaughnessy John , Barlogie Bart 
Published: October 2019

Abstract
Multicolor spectral karyotyping (SKY) was performed on bone marrow samples from 50 patients with multiple myeloma (MM) in anticipation of discovering new previously unidentified translocations. All samples showed complex karyotypes with chromosome aberrations which, in most cases, were not fully characterized by G-banding. Patients of special interest were those who showed add(14)(q32), add(8)(q24) and those whose G-banding karyotypes showed poor chromosome morphology. Three new recurring chromosome translocations not previously reported in MM were identified. Two of the translocations involve recurring aberrations at band 14q32.3, the site of the IgH locus, with different exchange partners. The most frequently recurring rearrangement was a subtle translocation at 14q32.3 designated as a t(14;16)(q32;q22∼23), which was identified in six patients. A second and larger translocation at 14q32, identified in two patients, was designated as a t(9;14)(p13;q32), previously associated with Waldenstrom’s macroglobulinemia and lymphoplasmacytoid lymphoma. A third translocation, identified in two patients, involved a whole-arm t(6;8)(p10;q10) translocation. The SKY technique was able to refine the designations of over 156 aberrations not fully characterized by G-banding in this study and resolved additional chromosome aberrations in every patient studied except two. The t(14;16)(q32;q22∼23) identified by SKY in this study suggests this may be a frequent translocation in MM associated with complex karyotypes and disease progression. Therefore, the SKY technique provides a useful adjunct to routine G-banding and fluorescence in situ hybridization studies in the cytogenetic analysis of MM.
Most Acute Myeloid Leukemia Progenitor Cells With Long-Term Proliferative Ability In Vitro and In Vivo Have the Phenotype CD34+/CD71−/HLA-DR−
Authors: Blair A. , Hogge D.E. , Sutherland H.J. 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) occurs as the result of malignant transformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML cells are capable of proliferation in vitro, suggesting that AML cells may be organized in a hierarchy, with only the most primitive of these cells capable of maintaining the leukemic clone. To further investigate this hypothesis, we have evaluated a strategy for purifying these primitive cells based on surface antigen expression. As an in vitro endpoint, we have determined the phenotype of AML progenitor cells which are capable of producing AML colony-forming cells (CFU) for up to 8 weeks in suspension culture (SC) and compared the phenotype with that of cells which reproduce AML in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activated cell sorted (FACS) for coexpression of CD34 and CD71, CD38, and/or HLA-DR and the subfractions were assayed in vitro and in vivo at various cell doses to estimate purification. While the majority of primary AML CFU lacked expression of CD34, most cells capable of producing CFU after 2 to 8 weeks in SC were CD34+/CD71−. HLA-DR expression was heterogeneous on cells producing CFU after 2 to 4 weeks. However, after 6 to 8 weeks in SC, the majority of CFU were derived from CD34+/HLA-DR− cells. Similarly, the majority of cells capable of long-term CFU production from SC were CD34+/CD38−. Most cells that were capable of engrafting NOD/SCID mice were also CD34+/CD71− and CD34+/HLA-DR−. Engraftment was not achieved with CD34+/CD71+ or HLA-DR+subfractions, however, in two patients, both the CD34+and CD34− subfractions were capable of engrafting the NOD/SCID mice. A three-color sorting strategy combining these antigens allowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cells in one experiment. Phenotyping studies suggest even higher purification could be achieved by combining lack of CD38 expression with the CD34+/CD71− or CD34+/HLA DR− phenotype. These results suggest that most AML cells capable of long-term proliferation in vitro and in vivo share the CD34+/CD71−/HLA-DR− phenotype with normal stem cells. Our data suggests that in this group of patients the leukemic transformation has occurred in a primitive progenitor, as defined by phenotype, with some degree of subsequent differentiation as defined by functional assays.
CBFβ-SMMHC, Expressed in M4eo Acute Myeloid Leukemia, Reduces p53 Induction and Slows Apoptosis in Hematopoietic Cells Exposed to DNA-Damaging Agents
Authors: Britos-Bray Martin , Ramirez Manuel , Cao Wangsen , Wang Xinping , Liu P. Paul , Civin Curt I. , Friedman Alan D. 
Published: October 2019

Abstract
CBFβ-SMMHC is expressed in M4Eo acute myeloid leukemia (AML) as a result of inv(16), but how it contributes to leukemogenesis is unknown. p53 mutations are rare in de novo AML, but they are common in many malignancies. Expression of CBFβ-SMMHC in Ba/F3 cells reduced p53 induction in response to ionizing radiation or etoposide 3- to 4-fold. However, p53 induction was normal in Ba/F3 cells expressing a CBFβ-SMMHC variant that does not interfere with DNA binding by CBF, indicating that a CBF genetic target regulates p53 induction. The p53 gene may be regulated by CBF, because p53 mRNA levels were reduced by CBFβ-SMMHC. Reduced p53 induction was not caused by slowed cell proliferation, a consequence of CBFβ-SMMHC expression, because p53 was induced similarly in control cultures and in cultures propagated in 10-fold less interleukin-3 (IL-3). CBFβ-SMMHC did not slow apoptosis resulting from IL-3 withdrawal, where p53 induction is minimal, but slowed apoptosis in Ba/F3 cells exposed to 10 Gy of ionizing radiation or 3 to 8 μg/mL etoposide, providing 2-fold protection at 6 or 18 hours. Inhibition of apoptosis was temporary, because all the cells exposed to these doses ultimately died, and clonal survival assays performed using 0.04 μg/mL etoposide did not show protection by CBFβ-SMMHC. p21 levels were increased in cells subjected to DNA damage, regardless of CBFβ-SMMHC expression and attenuated p53 induction. Bcl-2, bcl-xL, bcl-xS, and bax levels were unaffected by CBFβ-SMMHC. Attenuated p53 induction may contribute to leukemogenesis by CBFβ-SMMHC by slowing apoptosis via a p21-independent mechanism.
Characterization of Multiple Isoforms of Protein 4.1R Expressed During Erythroid Terminal Differentiation
Authors: Gascard P. , Lee G. , Coulombel L. , Auffray I. , Lum M. , Parra M. , Conboy J.G. , Mohandas N. , Chasis J.A. 
Published: October 2019

AbstractIn erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4.1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5′ end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2′ and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2′ and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.
Recombination Breakpoints in the Human β-Globin Gene Cluster
Authors: Smith Rachelle A. , Ho P. Joy , Clegg John B. , Kidd Judith R. , Thein Swee Lay 
Published: October 2019

AbstractThe human β-globin gene complex spans a region of 70 kb and contains numerous sequence variants. These variant sites form a 5′ cluster (5′ β-haplotype) and a 3′ cluster (3′ β-haplotype) with strong linkage disequilibrium among the sites within each cluster, but not between the two clusters. The 9-kb region between the 5′ and 3′ clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a ‘hotspot’ of recombination. We describe three families with evidence of meiotic recombination within this ‘hotspot’ of the β-globin gene cluster and in which the cross-over breakpoints have been defined at the sequence level. In one family, the recombination has occurred in the maternal chromosome within a region of 361 bp between positions −911 and −550 5′ to the β-globin gene. In the other two families, the recombination has occurred in the paternal chromosome within a region of approximately 1,100 bp between positions −542 and +568 relative to the β-globin gene cap site. Both regions occur within the 2-kb region of replication initiation (IR) in the β-globin gene domain with no overlap. The IR region contains a consensus sequence for a protein (Pur), which binds preferentially to single-stranded DNA, a role implicated in recombination events.
Correction of the PNH Defect by GPI-Anchored Protein Transfer
Authors: Sloand Elaine M. , Maciejewski Jaroslaw P. , Dunn Daniel , Moss Joel , Brewer Bryan , Kirby Martha , Young Neal S. 
Published: October 2019

AbstractHemolytic anemia is a major feature of paroxysmal nocturnal hemoglobinuria (PNH). Intravascular red blood cell (RBC) destruction is caused by increased sensitivity of the abnormal erythrocyte to complement-mediated lysis, due to the GPI absence of a membrane-bound glycosylphosphatidylinositol (GPI)-linked protein, which functions as an inhibitor of reactive lysis (CD59). Both in vivo and in vitro models have suggested the feasibility of cell-to-cell transfer of GPI proteins, and patients with hemolysis could potentially benefit from transfer of CD59 to their deficient erythrocytes. We studied the ability of RBC components prepared from outdated packed RBC collections, as well as high-density lipoprotein (HDL) preparations, rich in CD55 and CD59, to promote protein transfer, as assessed by flow cytometry, immunoblotting, and susceptibility to complement-mediated lysis. By flow cytometry, CD55 and CD59 were present on RBC-derived microvesicles that stained with an antiglycophorin antibody Ab; in addition, soluble CD59 and CD55 were detected by immunoblot in soluble fractions eluated from RBC units stored for more than 35 days, but not in fresh blood. Both commercial HDL preparations and those prepared in our laboratory contained CD55 and CD59, as assayed by immunoblot. When RBC that were deficient (GPI)-anchored protein, obtained from five patients, with PNH were incubated with HDL preparations for 2 to 4 hours, there was significant transfer of both proteins to the cell surface, as demonstrated by flow cytometry. Washed RBC microvesicles, prepared by ultrasonification, also mediated transfer of GPI-linked proteins to deficient RBC. Pretreatment of microvesicles, RBC eluate preparations, and HDL with phosphatidylinositol-specific, phospholipase C, abrogated protein transfer to deficient cells, indicating that increased cell-associated CD55 and CD59 levels were related to insertion of the intact GPI moiety, rather than to simple adhesion. PNH RBC that were exposed to HDL, RBC eluate preparations, or microvesicles demonstrated decreased in vitro complement-mediated hemolysis in the Ham test. Transfer of GPI-linked proteins from soluble preparations containing CD55 and CD59 to PNH erythrocytes is feasible and may have clinical utility.
Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells
Authors: Georas Steve N. , Cumberland John E. , Burke Thomas F. , Chen Rongbing , Schindler Ulrike , Casolaro Vincenzo 
Published: October 2019

AbstractThe differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4–secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4–activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.
Adenovirus Vector-Based Purging of Multiple Myeloma Cells
Authors: Teoh Gerrard , Chen Ling , Urashima Mitsuyoshi , Tai Yu-Tzu , Celi Leo A. , Chen Dongshu , Chauhan Dharminder , Ogata Atsushi , Finberg Robert W. , Webb Iain J. , Kufe Donald W. , Anderson Kenneth C. 
Published: October 2019

AbstractAdenoviruses are efficient gene delivery agents for a variety of neoplasms. In the present study, we have investigated the use of adenoviruses for the delivery of the thymidine kinase(tk) gene into multiple myeloma (MM) cells. We first demonstrated that MM cell lines and MM patient cells express both adenovirus receptors as well as the DF3/MUC1 protein, thus providing a rationale for using adenoviruses to selectively deliver genes under the control of the DF3 promoter. By using an adenoviral construct containing β-galactosidase (β-gal) gene driven by the DF3 promoter (Ad.DF3-βgal), we demonstrate greater than 80% transduction efficiency in OCI-My5 and RPMI 8226 MM cell lines at a multiplicity of infection of 1 to 100. Importantly, transduction with the tk gene driven by the DF3 promoter (Ad.DF3-tk)followed by treatment with 50 μmol/L ganciclovir (GCV) purged ≥6 log of contaminating OCI-My5 and RPMI 8226 MM cells within bone marrow mononuclear cells. In contrast, normal human hematopoietic progenitor cell number was unaffected under these conditions. Selectivity of DF3/MUC1 promoter was further confirmed, because Ad.DF3-βgalor Ad.DF3-tk did not transduce MUC1-negative HeLa cervical carcinoma cells. In addition, GCV treatment of Ad.DF3-tk–transduced RPMI 8226 MM cells did not induce a significant bystander effect. These findings demonstrate that transduction with Ad vectors using a tumor-selective promoter provides a highly efficient and selective approach for the ex vivo purging of MM cells.
Expression Trapping: Identification of Novel Genes Expressed in Hematopoietic and Endothelial Lineages by Gene Trapping in ES Cells
Authors: Stanford William L. , Caruana Georgina , Vallis Katherine A. , Inamdar Maneesha , Hidaka Michihiro , Bautch Victoria L. , Bernstein Alan 
Published: October 2019

AbstractWe have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZreporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressedlacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.