bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of an oncogenic form of the thrombopoietin receptor MPL using retrovirus-mediated gene transfer
Authors: Onishi M , Mui AL , Morikawa Y , Cho L , Kinoshita S , Nolan GP , Gorman DM , Miyajima A , Kitamura T
Published: October 2019

Thrombopoietin and its receptor (MPL) are important regulators of megakaryopoiesis. We have identified an activating mutation of MPL using a combination of a retrovirus-mediated gene transfer and polymerase chain reaction-driven random mutagenesis. This point mutation causes a single amino acid substitution from Ser498 to Asn498 in the transmembrane region and abrogates factor-dependency of all interleukin-3-dependent cell lines tested. Murine interleukin-3- dependent Ba/F3 cells expressing the mutated but not the normal form of MPL were tumorigenic when transduced into syngeneic mice. Analysis of intracellular signaling pathways indicated that the mutant MPL protein constitutively activated two distinct signaling pathways, SHC-Raf-MAPK and JAK2-STAT3/STAT5.
In vitro transcription and translation inhibition by anti-promyelocytic leukemia (PML)/retinoic acid receptor alpha and anti-PML peptide nucleic acid
Authors: Gambacorti-Passerini C , Mologni L , Bertazzoli C , le Coutre P , Marchesi E , Grignani F , Nielsen PE
Published: October 2019

Peptide nucleic acids (PNAs) complementary to the 15 bases around the fusion point of both genomic DNA and cDNA of the promyelocytic leukemia/retinoic acid receptor alpha (PML/ RAR alpha; P/R) hybrid gene present in acute promyelocytic leukemia cells were synthesized and shown by gel retardation experiments to specifically bind oligonucleotides corresponding to the fusion region of the P/R molecule. PNA was also able to successfully compete with anti-P/R DNA for duplex formation with P/R DNA and to displace the anti-P/R DNA from dsDNA. In vitro transcribed P/R RNA from two inserts of approximately 350 to approximately 700 bp were tested in gel acceleration experiments with fluorescein-conjugated PNA and showed stable binding (resistant to denaturing conditions) of PNA to the newly transcribed RNA. Control RNA or transcripts from the noncoding strand did not bind PNA. However, this PNA, although able to specifically clamp polymerase chain reaction, was incapable of inhibiting in vitro translation of the PML/RAR alpha mRNA, even when a bis-PNA was used. Therefore, a PNA was targeted against the start region of the P/R cDNA and against poly- purine regions of the gene. Specific inhibition of in vitro translation and transcription was shown, starting at concentrations as low as 100 nmol/L. When oligonucleotides presenting the same sequence were compared, PNA proved to be approximately 40 times more active. In conclusion, in vitro inhibition of translation and transcription of the P/R gene can be obtained with PNA; however, it is still necessary to target the ATG start region or poly-purine regions of the gene.
Localization by chromosome microdissection of a recurrent breakpoint region on chromosome 6 in human B-cell lymphoma
Authors: Guan XY , Horsman D , Zhang HE , Parsa NZ , Meltzer PS , Trent JM
Published: October 2019

Deletion of the long arm of chromosome 6 (6q) is one of the most common chromosomal alterations in human B-cell lymphomas. Conventional cytogenetic banding analysis and loss-of-heterozygosity (LOH) studies have detected several common regions of deletion ranging across the entire long arm (6q), with no defined recurrent breakpoint yet identified. We describe here a strategy combining chromosome microdissection and fluorescence in situ hybridization (Micro-FISH) to determine a minimal region of deletion along chromosome 6. Seven clinical cases and one cell line of follicular lymphoma containing a t(14;18) and one case of diffuse lymphoma, also with a t(14;18), were used for this study. All nine cases had previously defined abnormalities of chromosome 6 determined by cytogenetic analysis. The results of chromosome dissection were unexpected and in contrast to the suggestion of disparate breakpoints by conventional chromosome banding. Specifically, Micro-FISH analysis provided evidence for a common breakpoint at 6q11 in seven of nine cases. After Micro-FISH analysis, all of the presumed simple deletions of chromosome 6 were carefully reanalyzed and shown to actually represent either nonreciprocal translocations (three cases), interstitial deletions (five cases), or isochromosome (one case). The recurrent proximal breakpoint (6q11) was detected in seven of nine cases, with the minimal region of deletion encompassing 6q11 to 6q21. By analogy to other tumor systems, the identification of recurring breakpoints within 6q11 may suggest that a gene(s) important to the genesis or progression of follicular lymphoma can be localized to this band region.
LYSP100-associated nuclear domains (LANDs): description of a new class of subnuclear structures and their relationship to PML nuclear bodies
Authors: Dent AL , Yewdell J , Puvion-Dutilleul F , Koken MH , de The H , Staudt LM
Published: October 2019

The PML gene is fused to the retinoic acid receptor alpha (RAR alpha) gene in t(15;17) acute promyelocytic leukemia (APL), creating a PML-RAR alpha fusion oncoprotein. The PML gene product has been localized to subnuclear dot-like structures variously termed PODs, ND10s, Kr bodies, or PML nuclear bodies (PML NBs). The present study describes the cloning of a lymphoid-restricted gene, LYSP100, that is homologous to another protein that localizes to PML NBs, SP100. In addition to SP100 homology regions, one LYSP100 cDNA isoform contains a bromodomain and a PHD/TTC domain, which are present in a variety of transcriptional regulatory proteins. By immunofluorescence, LYSP100 was localized to nuclear dots that were surprisingly largely nonoverlapping with PML NBs. However, a minority of LYSP100 nuclear dots exactly colocalized with PML and SP100. We term the LYSP100 structures “LANDs,” for LYSP100- associated nuclear domains. Although LYSP100 is expressed only in lymphoid cells, LANDs could be visualized in HeLa cells by transfection of a LYSP100 cDNA. Immunoelectron microscopy revealed LANDs to be globular, electron-dense structures morphologically distinct from the annular structures characteristic of PML NBs. LANDs were most often found in the nucleoplasm, but were also found at the nuclear membrane and in the cytoplasm, suggesting that these structures may traffic between the cytoplasm and the nucleus. By double-immunogold labeling of PML and LYSP100, some LANDs were shown to contain both PML and LYSP100. Thus, PML is localized to a second subnuclear domain that is morphologically and biochemically distinct from PML NBs.
Detection of maternal DNA in placental/umbilical cord blood by locus- specific amplification of the noninherited maternal HLA gene
Authors: Scaradavou A , Carrier C , Mollen N , Stevens C , Rubinstein P
Published: October 2019

A critical issue regarding the broader utilization of placental/ umbilical cord blood (PCB) in unrelated bone marrow restoration is the possibility of contamination with maternal lymphocytes capable of immunological reactivity against the eventual recipient. On transplantation, such maternal cells might lead to graft-versus-host disease (GVHD) even if the intended donor's neonatal lymphocytes were unresponsive. We measured the proportion of PCB samples that were contaminated with maternal cells. Placental-maternal sample pairs were selected so that the mother was heterozygous for the DR53 haplotype, whereas the placental sample was DR53-negative. The PCB samples were investigated for the presence of the noninherited maternal gene DRB4, exclusive to the DR53 haplotypes. Locus-specific polymerase chain reaction amplification with DRB4 sequence-specific primers was followed by either gel electrophoresis or blotting and hybridization to an internal sequence DRB4 probe. Polymerase chain reaction products from DNA mixtures containing as low as 0.5 ng of a DRB4-positive DNA control in 1.0 microgram of a DRB4-negative DNA sample (1:2 x 10(3) dilution) showed a visible DRB4 band in agarose gels stained with ethidium bromide. Locus-specific hybridization increased the detection sensitivity to 1:10(5) (0.01 ng of the DRB4-positive DNA control). Control mixtures of known amounts of DRB4-positive and -negative DNA were included in all experiments. Comparison of the thickness of DRB4 bands after electrophoresis and the intensity of the DRB4-specific hybridization signals to the concentration controls allowed a rough estimation of the amount of maternal DNA in the placental blood specimens. A total of 213 PCB samples were tested. By gel electrophoresis, DRB4-specific bands were observed to be as strong or stronger in 23 (10.8%) samples as those in the 1:2 x 10(3) control, and 153 (17.8%) samples were negative in this test. The remaining 37 (17.3%) samples disclosed weaker DRB4 bands, suggesting the presence of maternal genetic material. By hybridization, 81 (38%) samples were positive and 132 were negative for the noninherited maternal gene. Review of the clinical characteristics of the mothers (demographics and labor and delivery information), the newborns (birth weight, sex, and gestational age), and PCB collections (placental weight, white blood cell count, and collected volume) failed to show any significant differences between the units testing positive or negative for the noninherited maternal gene. Thus, transplantable PCB units carry a high probability of having maternal DNA in detectable amounts. Whether this DNA comes from potentially graft-versus-host disease-inducing maternal lymphocytes or whether the putatively transplacentally-acquired maternal cells are immunologically dysfunctional, as in most infants with severe combined immunodeficiency disease, remains to be shown.
Megakaryocyte growth and development factor stimulates enhanced platelet recovery in mice after bone marrow transplantation
Authors: Molineux G , Hartley CA , McElroy P , McCrea C , McNiece IK
Published: October 2019

Megakaryocyte growth and development factor (MGDF) is a recently characterized ligand for the cell surface receptor mpl. We have evaluated the effects of polyethylene glycollated recombinant human MGDF (PEG-rHuMGDF) on recovery of hematopoietic cells in mice following bone marrow transplantation (BMT) to support lethal irradiation. Mice treated with PEG-rHuMGDF (50 micrograms/kg/d) had accelerated recovery of platelet numbers compared with BMT mice treated with carrier or recombinant human granulocyte colony-stimulating factor (rHuG-CSF, 72 or 200 micrograms/kg/d). In contrast, PEG-rHuMGDF had no effect on white blood cell (WBC) or red blood cell (RBC) recovery. As previously reported, animals treated with rHuG-CSF had an enhanced recovery of WBC but not platelet or RBC levels. Interestingly, BMT receipient mice treated with the combination of PEG-rHuMGDF and rHuG-CSF showed simultaneous enhanced recovery of both leukocytes and platelets. PEGylated rHuMGDF was found to be considerably more potent than non- PEGylated rHuMGDF in this setting. PEG-rHuMGDF is an effective growth factor for enhancing platelet recovery in mice following BMT either alone or in combination with rHuG-CSF. It will be of interest to evaluate in a clinical setting the ratios of PEG-rHuMGDF and rHuG-CSF for simultaneous administration of these factors and accelerated recovery of both leukocytes and platelets.
A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia
Authors: Maciejewski JP , Selleri C , Sato T , Anderson S , Young NS
Published: October 2019

We examined the stem cell compartment of patients with acquired aplastic anemia (AA) using the long-term culture-initiating cell assay (LTC-IC), in parallel with measurements of CD34+ cells and mature hematopoietic progenitors. Secondary colonies from cells surviving 5 weeks of long-term bone marrow culture (LTBMC) were determined for the peripheral blood (PB) of 68 AA patients and 13 normal controls and for BM of 49 AA patients and 14 controls; because of low cell numbers, formal limiting dilution analysis could only be performed in 10 patients. The relationship of cell input in LTBMC and the output of secondary colonies was linear, allowing quantification of LTC-IC number from bulk cultures. Secondary colony formation was markedly abnormal in severe AA. In contrast to 7.8 colony-forming cells (CFC)/10(5) mononuclear cells in normal BM and 0.14 CFC/10(5) normal PB mononuclear cells, patients with severe disease showed 0.024 CFC/10(5) in BM and 0.0068 CFC/10(5) in PB. Under limiting dilution conditions, patients' cells also showed markedly lower colony-forming ability. In contrast to 4.3 +/- 1 colonies/normal LTC-IC, we obtained only 1.27 +/- 0.09 and 2.0 +/- 0.35 colonies from BM of acute and recovered cases, respectively. These values were used to extrapolate LTC-IC numbers from secondary colony formation in suspension cultures. In PB, calculated LTC-IC were decreased 7.4-fold in new and relapsed severe AA and 2.8- fold in recovered AA. In BM, LTC-IC were decreased 10-fold in new and relapsed AA and sixfold in recovered cases. Compared with measurements obtained on presentation, LTC-IC were lower in post-treatment samples from patients who had failed to recover after intensive immunosuppression and relatively higher in cases at relapse. In recovered patients, LTC-IC number increased but remained below the normal range in 20 of 25. In patients studied serially for 3 to 12 months after treatment, LTC-IC numbers remained stable but low. LTC-IC number correlated with concurrently determined CD34+ cell number and primary hematopoietic colony formation. These results indicate that stem cell numbers, as quantitated by the LTC-IC assay, are markedly diminished in number in all severe AA. Additionally, the function of the stem cell or the stem cell compartment in AA is also abnormal, as inferred from the low clonogenic potential in secondary colony assays. Early hematologic improvement in some patients occurs without increasing numbers of LTC-IC, and a minority of recovered cases show apparent repopulation of the LTC-IC compartment years after treatment.
Canine CD34: cloning of the cDNA and evaluation of an antiserum to recombinant protein
Authors: McSweeney PA , Rouleau KA , Storb R , Bolles L , Wallace PM , Beauchamp M , Krizanac-Bengez L , Moore P , Sale G , Sandmaier B , de Revel T , Appelbaum FR , Nash RA
Published: October 2019

Increasingly, enriched populations of hematopoietic progenitors are used in experimental and clinical transplantation studies. The separation of progenitors is based on the expression of CD34, a marker preferentially expressed on progenitor cells. The dog model has been important for preclinical transplant studies, because it has proven predictive for outcomes in human hematopoietic stem cell transplantation. To identify and isolate canine hematopoietic progenitors, we have cloned a cDNA encoding a CD34 homologue from a canine myelomonocytic leukemia cell line, ML2. The CD34 homologue cDNA predicts an amino acid sequence that is highly conserved with human and murine CD34 in the cytoplasmic domain, transmembrane domain, and C- terminal end of the extracellular domain, but shows considerable divergence from these sequences at the amino-terminal end of the protein. In Western blotting studies, canine CD34 homologue (caCD34) appears to be a heavily and variably glycosylated protein with a molecular weight of approximately 100 kD and shows some tissue-specific differences in protein mass. To evaluate the expression of caCD34 protein, the extracellular domain of caCD34 was expressed as an Ig fusion protein and used as an immunogen to generate a rabbit polyclonal antiserum. The antiserum reacted against the fusion protein, against vascular endothelium, and with three leukemic cell lines. Approximately 1% of canine bone marrow cells stained brightly with antibodies to caCD34 and this population was 25- to 50-fold enriched for colony- forming units-granulocyte-macrophage as compared to unfractionated marrow mononuclear cells. These findings suggest that the canine CD34 homologue is expressed on bone marrow progenitor cells and, thus, that this molecule should be a valuable marker for identifying and isolating canine hematopoietic progenitors for experimental hematopoiesis and stem cell transplantation.
Thrombopoietin stimulates colony-forming unit-megakaryocyte proliferation and megakaryocyte maturation independently of cytokines that signal through the gp130 receptor subunit
Authors: Broudy VC , Lin NL , Fox N , Taga T , Saito M , Kaushansky K
Published: October 2019

Thrombopoietin (Tpo), the ligand for the c-Mpl receptor, is a major regulator of megakaryopoiesis. Treatment of mice with Tpo raises the platelet count fourfold within a few days. Conversely, c-mpl knock-out mice have platelet counts that are 15% that of normal. The subunit structure of the c-Mpl receptor is not fully understood. Some cytokines that stimulate megakaryopoiesis (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M) bind to receptors that use gp130 as a signal transduction subunit. For these reasons, we determined whether gp130 function was required for Tpo-induced signal transduction. Murine marrow cells were cultured in semi-solid media in the presence of Tpo or IL-3, with or without a neutralizing anti-gp130 monoclonal antibody (RX187) or a soluble form of c-Mpl receptor (soluble Mpl) that blocks Tpo bioactivity, and the numbers of colony-forming unit-megakaryocyte (CFU-Meg) colonies were counted on day 5. Murine marrow cells were also cultured in suspension under serum-free conditions for 5 days, and megakaryocyte DNA content was measured by flow cytometry, as an index of nuclear maturation. The addition of RX187 did not block Tpo-induced CFU-Meg colony growth nor CFU-Meg nuclear maturation in suspension culture. However, IL-3-induced CFU-Meg colony growth and megakaryocyte nuclear maturation decreased in the presence of RX187. Soluble Mpl completely ablated Tpo-induced CFU-Meg growth, and partially blocked IL- 3-stimulated CFU-Meg growth. Thus the effects of Tpo on megakaryopoiesis in vitro do not depend on cytokines that signal through gp130. Furthermore, it is unlikely that gp 130 serves as a beta chain for the c-Mpl receptor, as Tpo signalling is unimpaired in the presence of RX187. In contrast, the effects of IL-3 on CFU-Meg growth are mediated in part through Tpo and through gp130-signalling cytokines.
An Ets/Sp1 interaction in the 5'-flanking region of the megakaryocyte- specific alpha IIb gene appears to stabilize Sp1 binding and is essential for expression of this TATA-less gene
Authors: Block KL , Shou Y , Poncz M
Published: October 2019

The megakaryocyte-specific integrin, alpha IIb, is the alpha-subunit of the alpha IIb/beta 3 complex found on the surface of platelets. This complex is a receptor for fibrinogen and other ligands when platelets are activated. Because the alpha IIb gene is specifically expressed in megakaryocytes, the 5′-flanking region was studied as a potential model for megakaryocyte-specific gene expression. Previous studies have defined some of the important regulatory elements in the 5′-flanking region of this gene. The present studies focus on the issue of the molecular basis by which this TATA-less gene is properly transcribed. A GA-rich region centered 14 bp upstream from the transcriptional start site appears to be a nonconsensus Sp1-binding site. Binding to this site is of low affinity, but is markedly improved by interaction with protein(s) binding at an Ets-consensus site approximately 20 bp further upstream. Mutation of the Ets site greatly reduces the ability of Sp1 to bind to its site. Trans-acting nuclear factors binding to and interaction of the proteins at these two sites have direct effects on the observed promoter activity in primary megakaryocyte transient expression studies. These studies provide further evidence of the role of interactions between Ets-like proteins and Sp1 in transcriptional activation when a TATA box is not present in the promoter region of a gene. Based on the presented studies and previous results, a model is proposed for the regulation of expression of the alpha IIb gene.
Insulin-like growth factor I is a growth and survival factor in human multiple myeloma cell lines
Authors: Georgii-Hemming P , Wiklund HJ , Ljunggren O , Nilsson K
Published: October 2019

Human multiple myeloma (MM) represents a highly aneuploid tumor as shown by cytogenetic studies. This may partly explain the heterogeneity with regard to growth factor requirements demonstrated among MM cells. We have previously reported the expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNA in some MM cell lines. In this study we investigated the role of IGF-I as a growth and/or survival factor in three MM cell lines: LP-1, EJM, and Karpas 707. We report that all cell lines expressed IGF-I and IGF-IR mRNA and protein. LP-1 and Karpas 707, but not EJM, were stimulated to proliferation in a dose-dependent manner by exogenous IGF-I. An IGF-IR blocking antibody inhibited both the IGF-I-induced and spontaneous growth of LP-1, and Karpas 707, while the EJM cell line was unaffected by the addition of the antibody. In conclusion, our results show that IGF-I can act as a growth factor in human MM, and they suggest that an autocrine IGF-I loop may contribute to the growth and survival in some MM cell lines.
Inhibition of tartrate-resistant acid phosphatase gene expression by hemin and protoporphyrin IX. Identification of a hemin-responsive inhibitor of transcription
Authors: Reddy SV , Alcantara O , Roodman GD , Boldt DH
Published: October 2019

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein encoded by the same gene that codes for uteroferrin, a placental iron transport protein. In human peripheral mononuclear cells, TRAP expression is inhibited by both hemin (ferric protoporphyrin IX) and protoporphyrin IX. Nuclear run-on assays confirmed that this inhibition occurs at the level of gene transcription. Previous studies with mTRAP deletion mutants showed that the hemin effect was dependent on repressor activity in the mTRAP 5′- flanking region at -1846 bp to -1240 bp relative to ATG (Reddy et al, J Bone Mineral Res 10:601, 1995). We now report that gel shift assays showed a DNA binding protein in nuclear extracts of hemin-treated cells termed hemin response element binding protein (HREBP). Additional studies have localized the HREBP binding region in the mTRAP 5′- flanking DNA to a 27-bp sequence at -1815 to -1789 bp relative to ATG. A tandem repeat sequence, GAGGC;GAGGC, contained within this DNA segment, was shown to be involved in binding of HREBP. Highly homologous sequences are present in the 5′-flanking region of the hTRAP gene. Binding of HREBP to the mTRAP DNA sequence was inhibited by anti- HAP1 antibodies, indicating homology between the hemin-responsive factor and the yeast heme-dependent transcription factor, HAP1. A 607- bp segment of the mTRAP 5′-flanking region containing the candidate hemin response element and surrounding sequences conferred hemin regulation on the viral SV40 promoter. Southwestern blotting experiments probing nuclear extracts of hemin-treated U937 cells with the 27-bp binding sequence showed two protein bands at 37 and 133 kD representing candidate HREBPs. A GENINFO search showed several other mammalian genes with tandem GAGGC motifs in noncoding regions, providing the possibility that additional genes may also be regulated by hemin at the level of transcription. These studies provide the first description of a novel iron/hemin-responsive transcriptional regulatory mechanism in mammalian cells.
The Fanconi anemia complementation group C protein corrects DNA interstrand cross-link-specific apoptosis in HSC536N cells
Authors: Marathi UK , Howell SR , Ashmun RA , Brent TP
Published: October 2019

Fanconi anemia (FA) cells are hypersensitive to cytotoxicity, cell cycle arrest, and chromosomal aberrations induced by DNA cross-linking agents, such as mitomycin C (MMC) and nitrogen mustard (HN2). Although MMC hypersensitivity is complemented in a subset of FA cells (complementation group C [FA-C]) by wild-type FAC cDNA, the cytoprotective mechanism is unknown. In the current study, we tested the hypothesis that FAC protein functions in the suppression of DNA interstand cross-link (ISC)-induced cell cycle arrest and apoptosis. Comparison of HN2-induced cell cycle arrest and apoptosis with those of its non-cross-linking analogs, diethylaminoethyl chloride and 2- dimethylaminoethyl chloride, delineated the DNA ISC specificity of FAC- mediated cytoprotection. Overexpression of wild-type FAC cDNA in FA-C lymphoblasts (HSC536N cell line) prevented HN2-induced growth inhibition, G2 arrest, and DNA fragmentation that is characteristic of apoptosis. In contrast cytoprotection was not conferred against the effects of the non-cross-linking mustards. Our data show that DNA ISCs induce apoptosis more potently than do DNA monoadducts and suggest that FAC suppresses specifically DNA ISC-induced apoptosis in the G2 phase of the cell cycle.
P-selectin glycoprotein ligand-1 (PSGL-1) is a ligand for L-selectin in neutrophil aggregation
Authors: Guyer DA , Moore KL , Lynam EB , Schammel CM , Rogelj S , McEver RP , Sklar LA
Published: October 2019

In inflammation, activated neutrophils adhere to endothelial cells and aggregate with one another. While beta 2-integrin and L-selectin are essential for aggregation, their ligands remain to be identified. We have previously shown that L-selectin mediates a carbohydrate-dependent interaction in aggregation (Simon et al: J Immunol 149:2765, 1992; Rochon et al: J Immunol 152:1385, 1994). We have suggested that the L-selectin counter-structure is a mucinlike protein and proposed that aggregation occurs through a two-step process involving L-selectin, beta 2-integrin, and their distinct counter-structures (Bennett et al: J Leuk Biol 58:510, 1995). A candidate ligand for L-selectin is P-selectin glycoprotein ligand-1 (PSGL-1), a mucinlike protein on neutrophils that binds P-and E-selectin. Using flow cytometry we show that the number and size of neutrophil aggregates is reduced with Fab fragments of PL1, an anti-PSGL-1 monoclonal antibody that blocks the interaction between P-selectin and PSGL-1 (Moore et al: J Cell Biol 128:661, 1995). In addition, monoclonal antibodies to L-selectin and PSGL-1 were used simultaneously to modulate the availability of these adhesion molecules on individual cell populations. The inhibition of aggregation by these antibodies is consistent with L-selectin and PSGL-1 being counter-structures. We suggest that L-selectin and PSGL-1 support a collisional cell-cell interaction that represents the first step in neutrophil aggregation.
Dominant type 1 von Willebrand disease caused by mutated cysteine residues in the D3 domain of von Willebrand factor
Authors: Eikenboom JC , Matsushita T , Reitsma PH , Tuley EA , Castaman G , Briet E , Sadler JE
Published: October 2019

No defects have been reported in moderately severe type 1 von Willebrand disease (vWD) with a clear autosomal dominant inheritance pattern, and the mechanism underlying this form of vWD remains obscure. We have studied a type 1 vWD family with such a dominant phenotype. The entire coding sequence of the von Willebrand factor (vWF) gene was analyzed by direct sequencing of DNA fragments amplified by polymerase chain reaction. Only one candidate mutation T(3445)-->C in exon 26 was detected that predicts a replacement of cysteine (C) at position 386 of the mature vWF subunit by arginine (R). Both mutant and normal vWF alleles were expressed as shown by analysis of platelet mRNA. This substitution segregates with vWD in the family and was not found in 100 unrelated individuals. The recombinant mutant vWF(C386R) was characterized by expression in 293T cells. The secretion of vWF(C386R) was greatly impaired due to retention in the endoplasmic reticulum. In cotransfections of normal and mutant vWF constructs, the vWF(C386R) subunits caused a dose-dependent decrease in the secretion of vWF. The multimer pattern remained nearly normal and consistent with a dominant vWD type 1 phenotype. The importance of the cysteine residues in the D3 domain of vWF in the pathogenesis of dominant type 1 vWD was further shown by the detection of another cysteine mutation, Cys367-->Phe, in two additional unrelated patients with a similar dominant type 1 vWD phenotype. We conclude that the loss of cysteine pairing in the D3 domain, leaving one free cysteine, can induce a purely quantitative deficiency of vWF by dominantly suppressing the secretion of normal vWF.
Macrophage colony-stimulating factor induces substantial osteoclast generation and bone resorption in human bone marrow cultures
Authors: Sarma U , Flanagan AM
Published: October 2019

Macrophage colony-stimulating factor (M-CSF) is essential for murine osteoclast formation and its role in human hematopoiesis in vitro is not fully defined. Therefore, we have investigated the effect of M-CSF on the formation of human osteoclasts in vitro. M-CSF was found to induce substantial bone resorption and osteoclast formation in a dose-responsive and time-dependent manner above that induced by 1,25 dihydroxyvitamin D3 (1,25 vitamin D3) in cultures of human bone marrow (BM) stromal cells sedimented onto devitalized bone. By day 14 there was a mean of approximately 50% of the surfaces of the bone slices resorbed compared with only 6% in cultures treated with 1,25 vitamin D3 alone. Osteoclasts were identified as 23c6+ cells (an antibody that recognizes the vitronectin receptor), 87.5% of which coexpressed the calcitonin receptor. The number of 23c6+ cells correlated strongly with bone resorption spatially, and in a dose-responsive and time-dependent manner; the correlation coefficient in cultures treated with 1,25 vitamin D3 alone was 0.856 and those treated with both M-CSF and 1,25 vitamin D3 was 0.880. Granulocyte-macrophage colony-stimulating factor, IL-1 beta, IL-3, IL-6, tumor necrosis factor-alpha, transforming growth factor-beta, leukemia inhibitory factor, and IL-11 did not increase bone resorption above that in 1,25 vitamin D3-treated cultures. We also found that 1,25 vitamin D3 increased, to a minor but significant degree, both bone resorption and the concentration of M-CSF in the culture supernatants above that in vehicle-treated cultures, indicating that M-CSF is present in our BM cultures, but that there is insufficient to induce substantial osteoclast formation. These results define a critical role for M-CSF in the formation of human osteoclasts.
Interleukin-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells
Authors: Weber-Nordt RM , Henschler R , Schott E , Wehinger J , Behringer D , Mertelsmann R , Finke J
Published: October 2019

Bcl-2 expression has been shown in hematopoietic progenitor cells. Through the use of Bcl-2 specific antisense oligonucleotides we herein report the biologic importance of Bcl-2 expression in primary human CD34+ hematopoietic progenitor cells committed to the myeloid lineage. In bone marrow or peripheral blood derived CD34+ cells Bcl-2 specific antisense decreased cell survival and inhibited the outgrowth of mixed myeloid colonies. A short-term overnight pretreatment of CD34+ cells with 25 mumol/L of Bcl-2 antisense in liquid culture completely ablated the growth of granulocyte-macrophage colony-forming cells (GM-CFC) in a subsequent 14 days methylcellulose colony assay. Control experiments using corresponding Bcl-2 sense or nonsense oligonucleotides did not significantly impair cell survival or growth of GM-colony-forming unit. Western blot analyses revealed the Bcl-2 antisense dependent inhibition of expression of the Bcl-2 protein in CD34+ progenitor cells. Furthermore, regulation of Bcl-2 expression by various cytokines including interleukin-10 (IL-10) was studied. IL-10′s effects on the formation of mixed myeloid colonies were examined in the absence or presence of Bcl-2 specific antisense. In the absence of Bcl-2 antisense IL-10 significantly extended the colony forming potential of mixed myeloid colonies to 14 days. In the presence of Bcl-2 antisense rhIL-10 completely restored GM-CSF driven colony growth. Fluorescent microscopy, Western blot analysis, and reverse transcriptase-polymerase chain reaction revealed the IL-10 dependent increase in cellular expression of Bcl-2 protein and Bcl-2 mRNA transcripts in CD34+ cells. Thus these results show that Bcl-2 expression is necessary for the formation of GM-CSF-dependent colony growth in vitro and that rhIL-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells that are committed to the myeloid lineage.
Genetic analysis and functional characterization of prothrombins Corpus Christi (Arg382-Cys), Dhahran (Arg271-His), and hypoprothrombinemia
Authors: O'Marcaigh AS , Nichols WL , Hassinger NL , Mullins JD , Mallouh AA , Gilchrist GS , Owen WG
Published: October 2019

The structural abnormalities and functional characteristics of dysfunctional prothrombin variants in two new kindreds have been determined. Prothrombin Corpus Christi (family 1) was purified and found to have markedly reduced fibrinogen clotting activity, yet normal amidolytic and near-normal platelet aggregating activity. A transition (C to T) at nucleotide position 8885, present in the heterozygous form in affected family members, resulted in the substitution of Cys for Arg 382. This substitution results in the loss of a positive charge within the fibrinogen-binding exosite of thrombin, thus accounting for the observed functional defect. A heterozygous C to T transition was also present at position 19994 in other family members with a hypoprothrombinemic phenotype. This mutation results in the replacement of Gln 541 (CAA) by a premature stop codon (TAA). Prothrombin Dhahran (family 2) was found to have markedly reduced fibrinogen clotting activity, but normal amidolytic activity. Affected family members were found to have a G to A transition at nucleotide position 7312 resulting in the substitution of His for Arg 271. This substitution results in the abolition of a factor Xa cleavage site, yielding meizothrombin rather than thrombin, on activation of prothrombin Dhahran by factor Xa. All but one of the above mutations occur at CpG dinucleotides, thus further supporting the observation of a high incidence of CpG transitions in hereditary dysprothrombinemia. The significant bleeding tendencies of individuals homozygous for prothrombin Dhahran (prothrombin clotting activity 5% to 7%) contrast sharply with the absence of significant chronic bleeding in the proband expressing prothrombin Corpus Christi (prothrombin clotting activity 2%). Our findings underscore the capacity of thrombin to contribute to clinical hemostasis by mechanisms other than its fibrinogen clotting activity.
Markedly disturbed glutathione redox status in CD45RA+CD4+ lymphocytes in human immunodeficiency virus type 1 infection is associated with selective depletion of this lymphocyte subset
Authors: Aukrust P , Svardal AM , Muller F , Lunden B , Nordoy I , Froland SS
Published: October 2019

We investigated the percentage of CD45RA+ and CD45RO+ T cells in peripheral blood and the intracellular glutathione redox balance in these lymphocyte subsets in patients with human immunodeficiency virus type 1 (HIV-1) infection and healthy controls. In HIV-1-infected patients there was a preferential depletion of CD45RA+CD4+ cells, which was most pronounced in symptomatic patients. In CD4+ lymphocytes from HIV-1-infected patients the glutathione abnormalities were clearly most pronounced in the CD45RA+ subset with a marked increase in level of oxidized glutathione and decreased ratio of reduced to total glutathione as the major characteristics. These abnormalities were shown in CD45RA+ CD4+ lymphocytes from both symptomatic and asymptomatic patients, whereas similar abnormalities in CD45RO+CD4+ cells were found only in symptomatic patients. The glutathione abnormalities in CD45RA+CD4+ lymphocytes were significantly correlated with low numbers of total CD4+ lymphocytes, decreased proportion of CD45RA+CD4+ lymphocytes, and raised serum levels of tumor necrosis factor-alpha. In the CD8+ lymphocytes a decrease in both proportion and absolute numbers of CD45RA+ cells was found, with markedly increased level of oxidized glutathione and decreased ratio of reduced to total glutathione in this subset. These findings suggest that glutathione redox disturbances in CD45RA+ T cells may be of pathogenic importance for the preferential depletion of this subset considered to represent naive T cells, during HIV-1 infection.
Establishment and characterization of a primary effusion (body cavity- based) lymphoma cell line (BC-3) harboring kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus
Authors: Arvanitakis L , Mesri EA , Nador RG , Said JW , Asch AS , Knowles DM , Cesarman E
Published: October 2019

The recently identified Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), has been found to be consistently associated with an unusual subset of acquired immunodeficiency syndrome-related lymphomas, the so-called body cavity-based lymphomas (BCBL) or primary effusion lymphomas (PEL). These lymphomas are characterized by a unique spectrum of morphologic and molecular characteristics, and grow as lymphomatous effusions without an identifiable contiguous tumor mass. Until now, efforts to delineate the role of KSHV in the pathogenesis of PELs have been hampered by the lack of appropriate model systems and the concomitant presence of Epstein-Barr virus (EBV) in nearly all cases examined, and in all previously established cell lines. We now report the establishment and characterization of a novel PEL cell line, BC-3, which is KSHV+ by polymerase chain reaction (PCR) but EBV- as assessed by a variety of methods including PCR for EBER, EBNA-2, and EBNA-3C. This cell line was established from a lymphomatous effusion obtained from an HIV- patient, and has immunophenotypic and molecular features consistent with the diagnosis of PEL, including an indeterminate immunophenotype with a B-cell immunogenotype and lack of c-myc proto-oncogene rearrangements. Pulsed-field gel electrophoresis shows an intact KSHV genome of about 170 kb both in the cell line and in the viral isolate, whereas herpesvirus-like capsids are visible by electron microscopy. Consequently, the BC-3 cell line represents an invaluable tool as a source of KSHV, for both the evaluation of the pathogenic potential of this virus and the mechanistic characterization of its role in the development of Kaposi's sarcoma and malignant lymphoma.
Polymerase chain reaction-based diagnosis of del (5q) in acute myeloid leukemia and myelodysplastic syndrome identifies a minimal deletion interval
Authors: Horrigan SK , Westbrook CA , Kim AH , Banerjee M , Stock W , Larson RA
Published: October 2019

Loss of all or part of the long arm of human chromosome 5 is a recurrent abnormality in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), especially after chemotherapy for a prior malignancy. It is one of the worst prognostic indicators in AML, associated with chemotherapy resistance and short survival. These deletions center at band 5q31, which has thus been proposed as the location of a tumor suppressor gene; this site is to be distinguished from that observed in 5q- syndrome, centering at 5q32. To define the molecular extent and the clinical prevalence of 5q31 deletions, we collected a series of AML and MDS cases of mixed karyotype, taking care to exclude MDS cases with 5q- syndrome. The samples were analyzed for loss of heterozygosity (LOH) using a panel polymerase chain reaction (PCR)-based microsatellite markers from 5q, comparing malignant cells with normal tissue derived from lymphoblastoid cell lines or buccal mucosa scrapings. Losses were detected in seven of 29 matched samples, including four of 17 with MDS, and three of 12 with AML; six of these seven also had a cytogenetically-visible del(5q) or -5. The one case without a cytogenetic deletion showed molecular loss of three contiguous markers, with retention of flanking markers interleukin-9 (IL-9) and D5S414, and thus contained a small deletion that is below cytogenetic resolution. PCR failed to detect 5q loss in two cases with large cytogenetic deletions, but both had been treated and contained low percentages of malignant cells in the samples. This study thus led to the identification of a case with a minimal deletion for the 5q31 tumor suppressor gene, specified by IL-9-D5S414, that is approximately 1 Mb (2 cM) in extent. Additionally, we demonstrate that PCR-based allelotyping is a reliable method for the detection of chromosomal deletion in myeloid malignancy, providing the specimens contain a high proportion of malignant cells. These studies will help to identify the tumor-suppressor gene at 5q31, and will help to develop molecular methods for diagnosis and monitoring of these disorders.
Interstitial deletion constitutes the major mechanism for loss of heterozygosity on chromosome 20q in polycythemia vera
Authors: Asimakopoulos FA , Gilbert JG , Aldred MA , Pearson TC , Green AR
Published: October 2019

An acquired deletion of the long arm of chromosome 20 is a recurrent abnormality in myeloproliferative disorders, particularly polycythemia vera and myelodysplastic syndromes. The association of 20q deletions with myeloid “stem cell” disorders suggests that the deletions mark the site of one or more genes, loss or inactivation of which plays a role in the regulation of normal hematopoietic progenitors. We have recently performed a detailed molecular analysis of 20q deletions in peripheral blood (PB) granulocytes and defined a commonly deleted region of 16 to 21 centimorgan (cM). To further reduce the size of the common deleted region we have searched for small deletions or mitotic recombination events, neither of which would be detected by conventional cytogenetics. We have studied 48 patients with polycythemia vera and four patients with idiopathic myelofibrosis. In each case, cytogenetic analysis had either failed or had shown no abnormalities of chromosome 20. Seventeen microsatellite markers that span the common deleted region were used to search for loss of heterozygosity in granulocyte DNA. No instance of microsatellite instability was observed in a total of 880 comparisons of granulocyte and T-cell DNA. Granulocyte DNA from four patients exhibited allele loss on 20q. In each case the allele loss was caused by an interstitial deletion because heterozygosity at distal markers was retained and because quantitative Southern blotting demonstrated hemizygosity. Loss of heterozygosity in PB granulocytes would be masked by the presence of significant numbers of normal granulocytes not derived from the malignant clone. Therefore, the human androgen receptor assay (HUMARA) was used to determine granulocyte clonality. In 21 of 27 informative female patients the majority of the granulocytes were clonally derived. In 5 patients the granulocytes appeared polyclonal and in 1 patient unilateral X inactivation was observed in both granulocytes and T cells. These results show that, in the vast majority of patients presented here, the failure to detect loss of heterozygosity cannot be attributed to the presence of normal polyclonal granulocytes. Our results therefore show that allele loss on chromosome 20q in polycythemia vera does not commonly involve mitotic recombination or chromosome loss and that microsatellite instability is a rare event in this disorder.
Activating mutations of N- and K-ras in multiple myeloma show different clinical associations: analysis of the Eastern Cooperative Oncology Group Phase III Trial
Authors: Liu P , Leong T , Quam L , Billadeau D , Kay NE , Greipp P , Kyle RA , Oken MM , Van Ness B
Published: October 2019

Mutations of members of the ras family are among the most common oncogene mutations found in multiple myeloma (MM). We have examined the mutational status of the N- and K-ras genes at codons 12, 13, and 61 in 160 newly diagnosed MM patients enrolled on the Eastern Cooperative Oncology Group (ECOG) phase III clinical trial E9486. The total incidence of ras mutations was found to be 39% of the samples analyzed. Five patients showed evidence of more than one mutation. We obtained 22 marrow samples from patients at the time of disease progression or relapse, for whom a ras mutation was identified at diagnosis. In all cases, the ras mutation of the disease progression sample was identical to that found at diagnosis. In contrast, three of 25 patients who did not show any ras mutation at diagnosis acquired a ras mutation at the time of disease progression. No significant association was observed between any ras mutation and stage of disease, beta 2-microglobulin levels, labelling index, or protein type. The mean tumor burden and median survival for patients with mutations of N-ras was indistinguishable from patients with no ras mutations. However, patients with K-ras mutations had a significantly higher mean bone marrow tumor burden at diagnosis than patients with no ras mutations (57% v 36%, P ‼ .02); and the median survival of patients with a K-ras mutation was significantly shorter (2.0 v 3.7 years, P ‼ .02). To determine if the status of ras mutations could affect treatment response, we examined patient survival on the three treatment arms of E9486. Although the presence of a ras mutation in the multidrug treatment, VBMCP alone, showed a marginal significance, neither the VBMCP, nor the addition of interferon-alpha showed statistically significant survival differences between mutant and wildtype ras status. Interestingly, there appeared to be a statistically significant difference in survival of patients treated with VBMCP and alternating high doses of cyclophosphamide + prednisone. Patients with ras mutations had a median survival of 2.1 years; patients with wild-type ras had a median survival of 4.0 years (P ‼ .01).
Targeted disruption of guanosine diphosphate-dissociation inhibitor for Rho-related proteins, GDID4: normal hematopoietic differentiation but subtle defect in superoxide production by macrophages derived from in vitro embryonal stem cell differentiation
Authors: Guillemot JC , Kruskal BA , Adra CN , Zhu S , Ko JL , Burch P , Nocka K , Seetoo K , Simons E , Lim B
Published: October 2019

The Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, through their role in cytoskeletal organization, is involved in diverse cellular functions, including cell motility and morphologic changes during differentiation. Rac also has a special role in the production of superoxide, a key component in phagocytic antimicrobial function. Guanosine diphosphate (GDP)-dissociation inhibitors (GDIs) belong to one of three classes of proteins that regulate the critical cycling of GTP-binding proteins between the inactive and active states. Two homologous GDIs for the Rho subfamily have been identified. GDID4 is preferentially expressed in hematopoietic cells, while RhoGDI is ubiquitously expressed. Whether different physiologic functions are subserved by the two GDIs is unknown. We have derived embryonal stem (ES) cells with targeted disruption of both alleles of the GDID4 gene and examined hematopoiesis and phagocytic functions of macrophages derived from in vitro ES-cell differentiation. GDID4-/- ES cells develop like wild-type cells into colonies that contain heterogeneous populations of progenitor cells and differentiated erythromyeloid cells. GDID4-/- cells express no GDID4 protein, but have normal levels of RhoGDI. GDID4-/- macrophages phagocytose yeasts and antibody-opsonized erythrocytes as effectively as wild-type macrophages. However, a slight but consistent reduction in their capacity to generate superoxide was observed, which suggests new insight into the cellular role of GDID4. The minimal phenotypic effect of a loss of function of GDID4 also indicates a significant redundancy of function between GDID4 and RhoGDI. Their functional repertoire may be better revealed by a disruption of both genes. The use of hematopoietic cells derived in vitro from genotypically altered ES cells avoids the difficulties inherent in generating knockout animals and is a useful complementary approach for evaluating the gene function.