bluuebunny/crossref_metadata_2025 huggingface dataset:
Arachidonic Acid Mediates Interferon-γ–Induced Sphingomyelin Hydrolysis and Monocytic Marker Expression in HL-60 Cell Line
Authors: Višnjić Dora , Batinić Drago , Banfić Hrvoje
Published: October 2019

The biochemical signaling mechanisms involved in transducing the effects of interferon-γ (IFN-γ) on human leukemia-derived HL-60 cell differentiation are not completely understood. Recent studies established the existence of a sphyngomyelin (SM) cycle that operates in response to the action of IFN-γ on HL-60 cells, but the mechanisms by which IFN-γ induces the SM hydrolysis remain unexplored. In this study, biochemical events mediating IFN-γ effects on SM turnover and their specificity and role in HL-60 differentiation were investigated. The activation of the SM cycle by IFN-γ occurred rapidly, with a decrease of approximately 20% in the SM level observed after 60 minutes with a concomitant increase in ceramide level. Treatment of HL-60 cells with IFN-γ did not influence the 1,2-diacylglycerol concentration, intracellular Ca2+ concentration, or phospholipase D activity. IFN-γ stimulated a rapid release of arachidonic acid (AA) from HL-60 cells; the effect was abolished by the pretreatment of cells with pertussis toxin, suggesting a role for a pertussis-toxin–sensitive G protein in IFN-γ–mediated activation of phospholipase A2 (PLA2 ). At 4 to 120 hours after the stimulation of the cells with IFN-γ, a significant increase in the particulate and soluble PLA2 activity was observed, corresponding to an increase in the level of immunoreactive cPLA2 in both cytosol and membrane fractions. The treatment of cells with tyrosine kinase inhibitor herbimycin A completely abolished the effect of IFN-γ on PLA2 activity in membrane and cytosolic fractions, but had no effect on IFN-γ–mediated early AA release suggesting dual mechanism of PLA2 activation. Melittin, potent activator of PLA2 , and AA mimicked the effect of IFN-γ on SM hydrolysis. Pretreatment of HL-60 cells with the PLA2 inhibitor, bromophenacyl bromide (BPB), or pertussis toxin abolished the effect of IFN-γ on SM hydrolysis; exogenous addition of AA overcame the effects of BPB and pertussis toxin. Long-term exposure (5 days) of HL-60 cells to IFN-γ caused an increase in nitroblue tetrazolium (NBT)-reducing and nonspecific esterase (NSE) activity and induced expression of FcγRI (CD64) without significant effects on cell number, adherence, or fagocytic activity. The treatment of cells with AA or melittin induced NBT, NSE, and CD64 expression to the level similar to that observed with IFN-γ, and no further increase was observed with the combination of IFN-γ and AA or IFN-γ and melittin. Treatment of HL-60 cells with indomethacin, an inhibitor of cyclo-oxygenase, and nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, had no effects on IFN-γ–mediated induction of CD64 expression. These studies indicate a key role for the phospholipase A2/AA pathway, as an early biochemical signal elicited by the occupation of IFN-γ–receptor, in mediating IFN-γ induction of the SM cycle and phenotypic changes associated with differentiation of HL-60 along monocytic lineage.
Modulation of Calcium Channels in Human Erythroblasts by Erythropoietin
Authors: Cheung Joseph Y. , Zhang Xue-Qian , Bokvist Krister , Tillotson Douglas L. , Miller Barbara A.
Published: October 2019

Erythropoietin (Epo) induces a dose-dependent increase in intracellular free Ca2+ ([Ca2+]i ) in human erythroblasts, which is dependent on extracellular Ca2+ and blocked by high doses of nifedipine or Ni2+. In addition, pretreatment of human erythroblasts with mouse antihuman erythropoietin receptor antibody but not mouse immunopure IgG blocked the Epo-induced [Ca2+]i increase, indicating the specificity of the Ca2+ response to Epo stimulation. In this study, the erythropoietin-regulated calcium channel was identified by single channel recordings. Use of conventional whole cell patch-clamp failed to detect Epo-induced whole cell Ca2+ current. To minimize washout of cytosolic constituents, we next used nystatin perforated patch, but did not find any Epo-induced whole cell Ca2+ current. Using Ba2+ (30 mmol/L) as charge carrier in cell-attached patches, we detected single channels with unitary conductance of 3.2 pS, reversal potential of +72 mV, and whose unitary current (at +10 mV) increased monotonically with increasing Ba2+ concentrations. Channel open probability did not appreciably change over the voltage range (−50 to +30 mV) tested. Epo (2 U/mL) increased both mean open time (from 4.27 ± 0.75 to 11.15 ± 1.80 ms) and open probability (from 0.26 ± 0.06 to 2.56 ± 0.59%) of this Ba2+-permeable channel. Our data strongly support the conclusion that the Epo-induced [Ca2+]i increase in human erythroblasts is mediated via Ca2+ entry through a voltage-independent Ca2+ channel.
Implication of a New Molecule IK in CD34+ Hematopoietic Progenitor Cell Proliferation and Differentiation
Authors: Cao Liang-Xian , Le Bousse-Kerdilès Marie-Caroline , Clay Denis , Oshevski Sergey , Jasmin Claude , Krief Patricia
Published: October 2019

HLA-DR is one of the markers associated with hematopoietic cell differentiation, since expression of this molecule is modulated throughout hematopoiesis. We have previously described and cloned the gene encoding factor IK, which inhibits both interferon gamma (IFN-γ)-induced and constitutive HLA-DR expression. The current study demonstrates that IK gene transcripts are present in CD34+ cells purified from human umbilical cord blood. IK expression increased and was therefore inversely correlated with the gradual loss of HLA-DR during growth factor–induced CD34+ cell proliferation and differentiation. To study the possible role of IK in hematopoiesis, antisense probes were used. IK expression was specifically inhibited by an antisense oligodeoxynucleotide containing two phosphorothioate internucleotide linkages at each of the 3′ and 5′ ends and corresponding to the initiation site of IK mRNA. A control oligonucleotide was also tested in parallel. A specific decrease of IK transcripts was correlated with an increase of HLA-DR antigen expression level. In colony-forming assays, IK antisense oligonucleotide inhibited colony formation by multilineage early erythroid and granulomonocytic CD34+ progenitors. The mean colony size was decreased 70% by IK antisense oligonucleotide in comparison to controls. These results provide evidence that the IK molecule participates in the regulation of HLA-DR expression on hematopoietic cells and plays a role in growth factor–dependent CD34+ cell proliferation and differentiation by modulating HLA-DR expression.
Transcription Factor GATA-2 Is Required for Proliferation/Survival of Early Hematopoietic Cells and Mast Cell Formation, But Not for Erythroid and Myeloid Terminal Differentiation
Authors: Tsai Fong-Ying , Orkin Stuart H.
Published: October 2019

The zinc-finger transcription factor GATA-2 plays a critical role in maintaining the pool of early hematopoietic cells. To define its specific functions in the proliferation, survival, and differentiation of hematopoietic cells, we analyzed the hematopoietic potential of GATA-2−/− cells in in vitro culture systems for proliferation and maintenance of uncommitted progenitors or differentiation of specific lineages. From a two-step in vitro differentiation assay of embryonic stem cells and in vitro culture of yolk sac cells, we demonstrate that GATA-2 is required for the expansion of multipotential hematopoietic progenitors and the formation of mast cells, but dispensable for the terminal differentiation of erythroid cells and macrophages. The rare GATA-2−/− multipotential progenitors that survive proliferate poorly and generate small colonies with extensive cell death, implying that GATA-2 may play a role in both the proliferation and survival of early hematopoietic cells. To explore possible mechanisms resulting in the hematopoietic defects of GATA-2−/− cells, we interbred mutant mouse strains to assess the effects of p53 loss on the behavior of GATA-2−/− hematopoietic cells. Analysis of GATA-2−/−/p53−/− compound-mutant embryos shows that the absence of p53 partially restores the number of total GATA-2−/− hematopoietic cells, and therefore suggests a potential link between GATA-2 and p53 pathways.
Pentoxifylline Promotes Replication of Human Cytomegalovirus In Vivo and In Vitro
Authors: Staak Kerstin , Prösch Susanna , Stein Joachim , Priemer Christina , Ewert Ralf , Döcke Wolf-Dietrich , Krüger Detlev H. , Volk Hans Dieter , Reinke Petra
Published: October 2019

OKT3 monoclonal antibody (MoAb) therapy is well established in the prevention and therapy of acute rejection in transplant patients. Unfortunately, this therapy is associated with several short-term (cytokine release syndrome) and long-term (infections, EBV-related lymphoma) side effects. Recently, we were able to demonstrate an association between the TNFα release following the first OKT3 MoAb infusions and the appearance of human cytomegalovirus (HCMV) reactivation several days later. In order to prevent this TNFα associated HCMV reactivation patients were additionally treated with pentoxifylline (PTX), a methylxanthine derivative that has been shown to suppress TNFα induction. Although the TNFα peak plasma level following OKT3 MoAb treatment was markedly reduced, the incidence of HCMV reactivation and HCMV disease was not influenced. In transient transfection experiments using HCMV immediate early enhancer/promoter CAT reporter gene constructs PTX enhanced the promoter activity independently from TNFα in premonocytic cells. Furthermore, PTX acted synergistically with TNFα. In virus-infected human embryonal lung fibroblasts HCMV replication was triggered in the presence of both PTX and TNFα, while either substance alone had only marginal effects. The stimulatory effect of PTX on the immediate early (IE) enhancer/promoter was mediated via CREB/ATF, a eukaryotic transcription factor that binds to the 19 bp sequence motif in the enhancer region, while TNFα stimulation was mediated by activation of the transcription factor NF-kB and its binding to the 18 bp sequence motif in the enhancer. These data suggest a potential side effect of cAMP-elevating drugs such as PTX.
Use of Partially Mismatched Related Donors Extends Access to Allogeneic Marrow Transplant
Authors: Henslee-Downey P. Jean , Abhyankar Sunil H. , Parrish Rudolph S. , Pati Asim R. , Godder Kamar T. , Neglia William J. , Goon-Johnson Karen S. , Geier Steven S. , Lee Carlos G. , Gee Adrian P.
Published: October 2019

Most patients requiring allogeneic bone marrow transplant (allo-BMT) do not have an HLA-matched sibling donor. A phenotypically matched unrelated donor graft has been made available for approximately 50% of Caucasians and less than 10% of ethnic and racial minorities in need. However, almost all patients have a readily available partially mismatched related donor (PMRD). We summarize our experience with 72 patients who ranged from 1 to 50 years of age (median, 16 years) and who were recipients of a PMRD allo-BMT from haploidentical family members following conditioning therapy using total body irradiation (TBI) and multiagent, high-dose chemotherapy. T-cell depletion and post-BMT immunosuppression were combined for graft-versus-host disease (GVHD) prophylaxis. The probability of engraftment was 0.88 at 32 days. Six of 10 patients who failed to engraft achieved engraftment after secondary transplant. Grade II to IV acute GVHD was seen in 9 of 58 (16%) evaluable patients; extensive chronic GVHD was seen in 4 of 48 (8%) evaluable patients. There was a statistically significant difference in 2-year survival probability between low-risk and high-risk patients (0.55 v 0.27, P = .048). Prognostic factors that affected outcomes in multivariate analysis were (1) a lower TBI dose and 3-antigen rejection mismatch decreased stable engraftment (P = .005 and P = .002, respectively); (2) a higher T-cell dose increased acute GVHD (P = .058); (3) a higher TBI dose increased chronic GVHD (P = .016); and (4) a high-risk disease category increased treatment failure from relapse or death (P = .037). A PMRD transplant can be performed with acceptable rates of graft failure and GVHD. Using sequential immunomodulation, the disease status at the time of transplant is the only prognostic factor significantly associated with long-term successful outcome after PMRD allo-BMT. When allogeneic rather than autologous BMT is indicated, progression in disease status before transplant can be avoided using a PMRD with equal inclusion of all ethnic or racial groups.
Lytic Replication of Epstein-Barr Virus in the Peripheral Blood: Analysis of Viral Gene Expression in B Lymphocytes During Infectious Mononucleosis and in the Normal Carrier State
Authors: Prang Nadja S. , Hornef Mathias W. , Jäger Michael , Wagner Hans J. , Wolf Hans , Schwarzmann Fritz M.
Published: October 2019

Epstein-Barr virus (EBV) has been shown to establish latency in resting B lymphocytes of the peripheral blood. This creates a virus reservoir in contrast to lytic virus replication, which is thought to be restricted to differentiated epithelial cells in vivo. So far, the route of transmission between B cells and the production of progeny virus in the epithelial tissue has remained unclear. Reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry analysis of 16 patients with acute infectious mononucleosis (IM) and 25 healthy seropositive donors was performed to detect lytic replication gene products in B lymphocytes of the peripheral blood. Transcriptional activity was found in peripheral blood B lymphocytes (PBLs) for BZLF1 in 88%, BALF2 in 50%, and BcLF1 in 25% of the tested IM patients. All positive results were further confirmed in enriched B-cell populations by antigen determination using immunostaining with the APAAP technique. Furthermore, we detected transcripts for BZLF1 in 72% and for BALF2 in 16% of peripheral B lymphocytes of healthy seropositive donors. In contrast to patients with IM, no signals for BcLF1 were ever found in healthy seropositive donors. In these individuals, lytic replication of EBV is probably restricted by immunologic and gene regulatory mechanisms, whereas in the absence of immunologic control, reflected here by IM patients, the production of infectious virus becomes visible in PBLs.
Epstein-Barr Virus Strains With Latent Membrane Protein-1 Deletions: Prevalence in the Italian Population and High Association With Human Immunodeficiency Virus–Related Hodgkin's Disease
Authors: Dolcetti Riccardo , Zancai Paola , De Re Valli , Gloghini Annunziata , Bigoni Beatrice , Pivetta Barbara , De Vita Salvatore , Carbone Antonino , Boiocchi Mauro
Published: October 2019

Six Epstein-Barr virus (EBV)-related lymphoproliferative disorders were investigated to verify whether the EBV strain harbored by neoplastic cells had the same EBNA-2 and latent membrane protein-1 (LMP-1) DNA sequences of the virus carried by normal lymphocytes of the same patients. Within each case, the analysis of neoplastic lymph nodes, reactive lymphadenopathies, and/or EBV+ spontaneous lymphoblastoid cell lines gave concordant results with respect to type-specific EBNA-2 region and LMP-1 gene. In particular, five cases showed the same deletion in the 3′ end of the LMP-1 gene in both normal and neoplastic cells. We also determined the prevalence of LMP-1 deletions in a large series of normal peripheral blood mononucleated cells (PBMCs) from Italian individuals. The analysis showed that 50% (9 of 18) of PBMCs from human immunodeficiency virus (HIV)-seronegative donors carried a 30-bp deletion in the C-terminal portion of the LMP-1 gene, whereas a nondeleted fragment was amplified in about 44% (8 of 18) of the cases. Only one sample (5.6%) showed the amplification of a full-length LMP-1 band together with a deleted fragment. Similarly, PBMCs from HIV-infected patients showed an almost equivalent prevalence of full-length (17 of 37, 46%) and deleted (16 of 37, 43.2%) LMP-1 fragments, whereas about 11% of samples (4 of 37) showed evidence of double infections. Of note, deletions in the LMP-1 gene were detected with similar prevalence values in EBV+ Hodgkin's disease (HD) (13 of 30, 43.3%) and non-Hodgkin's lymphoma (NHL) (2 of 5, 40%) cases from HIV-seronegative patients and in HIV-related, EBV+ NHLs (4 of 7, 57.1%). Conversely, a 30-bp LMP-1 deletion was found in 10 of 12 HIV-associated HD cases (83%), a prevalence significantly higher than that detected in HIV-unrelated HD (P = .01). These findings indicate that: (1) the same EBV strain carrying LMP-1 deletions is harbored by normal and neoplastic cells of patients with EBV+ disorders, ruling out that these mutations might result from immunoselection phenomena; (2) in the Italian population, the prevalence of LMP-1 deletion mutants is comparable to that of EBV strains with full-length LMP-1; (3) HIV-induced immunosuppression is not associated with an increased prevalence of LMP-1 deletions in PBMCs; and (4) HIV-related HD cases, but not those of HIV-seronegative Italian patients, are closely correlated with the presence of LMP-1 deletions, suggesting that infection with these strains may increase the risk of developing HD in the HIV setting.
Involvement of Reactive Oxygen Intermediates in Spontaneous and CD95(Fas/APO-1)–Mediated Apoptosis of Neutrophils
Authors: Kasahara Yoshihito , Iwai Kazuyuki , Yachie Akihiro , Ohta Kunio , Konno Akihiro , Seki Hidetoshi , Miyawaki Toshio , Taniguchi Noboru
Published: October 2019

Apoptosis is well known to be mediated by oxidative stress. To evaluate the functional role of reactive oxygen intermediates (ROI) produced by neutrophils, we compared the rates of apoptosis in neutrophils isolated from normal donors and from patients with chronic granulomatous disease (CGD), a hereditary defect in ROI production. Spontaneous cell death in CGD neutrophils in vitro was significantly inhibited relative to normal neutrophils. The acceleration of apoptosis induced by anti-Fas monoclonal antibody (MoAb) in CGD neutrophils was much slower than that seen in normal neutrophils. These findings suggest that the apoptosis of neutrophils may be mediated by endogenous oxidative products. This suggestion was confirmed by observation that apoptosis of normal neutrophils was markedly inhibited by reduction of intracellular levels of hydrogen peroxide (H2O2 ). The inhibition of apoptosis in normal neutrophils by adding catalase occurred regardless of the presence of anti-Fas MoAb. H2O2 increased both spontaneous apoptosis and Fas-mediated apoptosis of the CGD neutrophils in proportion to that seen in normal neutrophils. Although several factors that mediate the apoptosis of neutrophils remain to be determined, these results suggest that ROI are major mediators of the apoptosis in neutrophils and may be involved in Fas-mediated signal transduction pathway.
Molecular Analysis of 29 Pyruvate Kinase–Deficient Patients From Central Europe With Hereditary Hemolytic Anemia
Authors: Lenzner Claudia , Nürnberg Peter , Jacobasch Gisela , Gerth Christa , Thiele Bernd-Joachim
Published: October 2019

We investigated the DNA of 29 unrelated pyruvate kinase (PK) deficiency (PKD) patients from Central Europe with hereditary nonspherocytic hemolytic anemia for mutations in the PK-L/R gene. Among 58 potentially affected alleles, 53 mutations were identified, of which 17 were different from each other. Of these 17 mutations, 13 were single-nucleotide (nt) substitutions resulting in amino acid exchanges, G787A (Gly263-Arg), G994A (Gly332-Ser), G1006T (Ala336-Ser), G1010A (Arg337-Gln), A1081G (Asn361-Asp), G1127T (Ser376-Ile), G1174A (Ala392-Thr), G1281T (Glu427-Asp), C1454T (Ser485-Phe), C1456T (Arg486-Trp), G1493A (Arg498-His), G1529A (Arg510-Gln), and C1594T (Arg532-Trp); 1 in-frame triplet deletion, 1060delAAG (delLys354); 1 in-frame triplet insertion, 1203insAGC (insSer after Cys401); 1 splicesite mutation, 101-1G-A; and 1 frameshift deletion, 628delGT. Six mutations, 628delGT, G787A, G1010A, G1127T, G1281T, and C1454T, are described for the first time. To test the hypothesis of a single origin of the most common PK mutation in the European population, G1529A, we investigated all patients at four polymorphic sites in the PK-L/R gene: C/A at nt 1705, C/T at nt 1992, the (ATT)n microsatellite in intron J, and a polymorphism (T)10/(T)19 in intron I. Nine patients homozygous for mutation G1529A were consistent in all four markers. In the group of patients homozygous for mutation G1529A, the hematologic parameters and clinical manifestations have been studied in detail. Although having an identical mutation in the PK-L/R gene, the patients are affected differently. Their appearance ranges from a very mild compensated hemolysis to a severe anemia. Possible molecular explanations are discussed.
Association of HLA-C Disparity With Graft Failure After Marrow Transplantation From Unrelated Donors
Authors: Petersdorf Effie W. , Longton Gary M. , Anasetti Claudio , Mickelson Eric M. , McKinney Susan K. , Smith Anajane G. , Martin Paul J. , Hansen John A.
Published: October 2019

Disparity for HLA-A or HLA-B antigens increases the risk of marrow graft rejection, but the relevance of HLA-C is unknown because typing methods have not been sufficiently accurate for clinical use. We designed a matched case-control study and employed DNA sequencing methods to evaluate the role of HLA-C disparity in 21 patients who experienced graft failure (cases) following transplantation with unmanipulated marrow from either HLA-A, B serologically matched, DRB1 matched (n = 14) or single locus mismatched (n = 7) unrelated donors. For each case, two patients who successfully engrafted were selected as controls based on similarity for factors known or suspected to influence engraftment. The estimated odds ratio (OR) of graft failure for an HLA-C mismatch relative to match (univariable model) was 5.2 (95% CI, 1.4, 19; P = .01). Serologically undetectable HLA-A or HLA-B allele disparity was also associated with graft failure. The association between HLA-C disparity and graft failure remained significant even after accounting for the contribution of HLA-A and/or HLA-B allele disparity (OR 4.0; 95% CI, 1.1, 15; likelihood ratio test P = .03). These results show that HLA-C functions as a transplantation antigen and that HLA-A and HLA-B allele mismatches are biologically important. Molecular-based methods for pretransplant assessment of class I compatibility should be implemented for the selection of unrelated marrow donors.
Reversible Alterations in Platelet Morphology Produced by Anticoagulants and by Cold
Authors: ZUCKER MARJORIE B. , BORRELLI JENNIE
Published: October 2019

The morphology of the blood platelets has been studied under various conditions by fixing the platelets, observing them under the phase microscope, and classifying them according to shape. It has been found that most platelets maintain their native disc form in citrated or oxalated blood kept at 37 C. In EDTA blood they are converted to spheres. The platelets in citrated and oxalated blood are sphered by 10 minutes' exposure to 0 C., but many of them resume the disc form when body temperature is restored. Washed platelets prepared from EDTA blood are spherical even when resuspended in saline or in gelatin solution and incubated at 37 C. for 45 minutes. When resuspended in oxalated plasma or in albumin or gum acacia solutions, however, many of them revert to the disc shape. A smaller proportion of unwashed platelets, separated from EDTA blood by centrifugation and resuspended in oxalated plasma also resume their disc form after incubation.
Differential Incorporation of Ara-C, Gemcitabine, and Fludarabine Into Replicating and Repairing DNA in Proliferating Human Leukemia Cells
Authors: Iwasaki Hiromichi , Huang Peng , Keating Michael J. , Plunkett William
Published: October 2019

The major actions of nucleoside analogs such as arabinosylcytosine (ara-C) and fludarabine occurs after their incorporation into DNA, during either replication or repair synthesis. The metabolic salvage and DNA incorporation of the normal nucleoside, deoxycytidine, is functionally compartmentalized toward repair synthesis in a process regulated by ribonucleotide reductase. The aim of this study was to investigate the metabolic pathways by which nucleoside analogs that do (fludarabine, gemcitabine) or do not (ara-C) affect ribonucleotide reductase are incorporated into DNA in proliferating human leukemia cells. Using alkaline density-gradient centrifugation to separate repaired DNA from replicating DNA and unreplicated parental DNA strands, approximately 60% of ara-C nucleotide in DNA was incorporated by repair synthesis in CCRF-CEM cells; the remainder was incorporated by replication. In contrast, fludarabine and gemcitabine, nucleosides that inhibit ribonucleotide reductase and decreased deoxynucleotide pools, were incorporated mainly within replicating DNA. Hydroxyurea also depleted deoxynucleotide pools and increased the incorporation of ara-C into DNA by replicative synthesis. Stimulation of DNA repair activity by UV irradiation selectively enhanced the incorporation of all nucleosides tested through repair synthesis. These findings suggest that the pathways by which therapeutically useful nucleoside analogs are incorporated into DNA are affected by cellular dNTP pools from de novo synthesis and by the relative activities of DNA repair and replication. The antitumor activity of these drugs may be enhanced by combination with either ribonucleotide reductase inhibitors to increase their incorporation into replicating DNA or with agents that induce DNA damage and evoke the DNA repair process.
Calreticulin Biosynthesis and Processing in Human Myeloid Cells: Demonstration of Signal Peptide Cleavage and N-Glycosylation
Authors: Denning Gerene M. , Leidal Kevin G. , Holst Valerie A. , Iyer Shankar S. , Pearson Doran W. , Clark Joshua R. , Nauseef William M. , Clark Robert A.
Published: October 2019

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.
Relationship Between the Phenotypes of Circulating Erythrocytes and Cultured Erythroblasts in Paroxysmal Nocturnal Hemoglobinuria
Authors: Shichishima Tsutomu , Saitoh Yurie , Terasawa Takashi , Ogawa Kazuei , Maruyama Yukio
Published: October 2019

To investigate erythropoiesis in paroxysmal nocturnal hemoglobinuria (PNH), we studied the expression of glycosylphosphatidylinositol (GPI)-anchored membrane proteins on circulating erythrocytes and erythroblasts obtained by erythropoietic cell culture in nine patients with this disease. One-color and two-color flow cytometric analyses were performed using monoclonal antibodies for decay-accelerating factor (DAF ) and/or CD59/membrane attack complex-inhibitory factor (MACIF). In addition, terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL) analysis was performed to assess apoptosis of erythroblasts from six patients. On flow cytometric analysis, cases 1 to 6 had positive and negative erythrocyte populations, case 7 intermediate and negative populations, case 8 positive, intermediate, and negative populations, and case 9 a single double-negative population. In addition, cases 1 to 6 and 8 had positive, intermediate, and negative erythroblast populations, while cases 7 and 9 had intermediate and negative populations. The percentage of double-negative erythrocytes showed a significant correlation with that of double-negative erythroblasts (r = .741, P < .05). In seven of nine patients, more erythroblasts than erythrocytes were negative for the two membrane proteins. Also, some patients with an intermediate population of erythrocytes did not necessarily show an increase of PNH II erythroblasts. Apoptosis of PNH erythroblasts was also detected, but the percentage of apoptotic cells in PNH patients showed no difference from that in healthy volunteers. These findings suggest that the final phenotype of mature erythrocytes in PNH is determined during maturation from erythroblasts to erythrocytes by the disappearance or persistence of PNH II erythroblasts. In addition, PNH erythroblasts in vitro may be partly lost by apoptosis, but apoptosis does not play an important role in determining GPI-linked protein expression.
The Novel Cyclin-Dependent Kinase Inhibitor Flavopiridol Downregulates Bcl-2 and Induces Growth Arrest and Apoptosis in Chronic B-Cell Leukemia Lines
Authors: König Andrea , Schwartz Gary K. , Mohammad Ramzi M. , Al-Katib Ayad , Gabrilove Janice L.
Published: October 2019

Flavopiridol is a novel, potent inhibitor of cyclin-dependent kinases (CDK). This synthetic flavone has been reported to exhibit antitumor activity in murine and human tumor cell lines in vitro and in vivo and is currently undergoing clinical phase I evaluation. In the present study, 1 Epstein-Barr virus (EBV)-transformed B-prolymphocytic cell line (JVM-2), 1 EBV-transformed B-CLL cell line (I83CLL), and 1 non-EBV transformed B-CLL cell line (WSU-CLL) were used as targets. Treatment of the cells with flavopiridol (100 nmol/L to 400 nmol/L) led to a marked dose- and time-dependent inhibition of cell growth and survival as determined using trypan blue exclusion. Morphologic analysis showed characteristic apoptotic changes such as chromatin condensation and fragmentation, membrane blebbing, and formation of apoptotic bodies. Furthermore, quantitative assessment of apoptosis-associated DNA strand breaks by in situ TdT labeling showed that a significant number of flavopiridol-treated cells underwent apoptosis. These cellular effects were associated with a significant decrease in bcl-2 expression as observed by Northern and Western blotting. The results showed that flavopiridol downregulates bcl-2 mRNA and bcl-2 protein expression within 24 hours. Genistein and quercetin, two flavonoids that do not inhibit CDKs, did not affect bcl-2 expression. These data suggest an additional mechanism of action of this new flavone which might be useful as an agent in the treatment of chronic lymphoid malignancies.
Predominant Expression of Murine Bmx Tyrosine Kinase in the Granulo-Monocytic Lineage
Authors: Weil Dominique , Power Mary-Anne , Smith Shirley I. , Leung Li Chung
Published: October 2019

In the course of systematic cloning of protein tyrosine kinases (PTKs) expressed in hematopoietic stem and progenitor cells, we have identified the murine homologue of human Bmx. It encodes a protein containing the five domains characteristic of the Tec family of cytoplasmic src-related PTKs: pleckstrin homology (PH), Tec homology (TH), src homology 3 and 2 (SH3 and SH2), and tyrosine kinase (TK). In adults, Bmx expression was found primarily in bone marrow and at a lower level in lung and heart. During fetal development it was also found in the spleen at late stage of gestation and in neonates. Analysis of bone marrow subpopulations showed that Bmx was expressed in the progenitor cell population and maturing hematopoietic cells of the granulo/monocytic lineage where expression increased with maturation and differentiation. At the periphery, a high level of Bmx expression was also found in neutrophils and monocytes/macrophages. Bmx expression was not detected in the primitive hematopoietic stem cell population, and cells of the B-, T-, and erythroid-lineages. It was also not detected in most of the cell lines examined. Our results indicate that Bmx is another member of the Btk/Itk/Tec PTK family, which is predominantly expressed in the granulo-monocytic lineage within the hematopoietic system.
Thrombopoietin Upregulates the Promoter Conformation of p53 in a Proliferation-Independent Manner Coincident With a Decreased Expression of Bax: Potential Mechanisms for Survival Enhancing Effects
Authors: Ritchie Alec , Gotoh Akihiko , Gaddy Jay , Braun Stephen E. , Broxmeyer Hal E.
Published: October 2019

Thrombopoietin (Tpo) has proliferative and maturational effects on immature and more committed cells, respectively. We previously reported a role for Tpo as a survival factor in the factor-dependent human cell line M07e by demonstrating that Tpo suppresses apoptosis in the absence of induced proliferation. Wild-type p53 is a tumor suppressor gene that can play a vital role in mediating growth factor withdrawal-induced apoptosis in factor-dependent hematopoietic cells. Wild-type p53 can switch from a suppressor conformation, with an antiproliferative, pro-apoptotic phenotype, to a promoter conformation that has a diminished ability to mediate cell cycle arrest and apoptosis. In an effort to elucidate the mechanisms through which Tpo suppresses apoptosis, we investigated the effects of Tpo treatment on p53-mediated apoptosis in M07e cells. Tpo upregulated the expression of the promoter conformation of p53 in M07e cells coincident with a downregulation of Bax and Mdm2 protein levels. Protein levels of Bcl-2 and Bcl-xL did not significantly vary as a function of growth-factor stimulation. Conversely, the levels of suppressor conformation p53 were maximal when M07e was in a growth arrested state and decreased during factor stimulation. Furthermore, Tpo treatment induced an extranuclear buildup and greatly weakened the DNA binding capacity of p53. p53-specific antisense oligonucleotide treatment recapitulated the effects of Tpo treatment on the levels of Bax, Mdm-2, and Bcl-2. These results suggest that Tpo is suppressing growth factor withdrawal induced-apoptosis, at least in part, by downregulating the expression of pro-apoptotic Bax protein levels, through modulating the conformation of p53, which results in a functional inactivation of its pro-apoptotic abilities.
The c-kit Ligand Stem Cell Factor and Anti-IgE Promote Expression of Monocyte Chemoattractant Protein-1 in Human Lung Mast Cells
Authors: Baghestanian Mehrdad , Hofbauer Roland , Kiener Hans P. , Bankl Hans C. , Wimazal Friedrich , Willheim Martin , Scheiner Otto , Füreder Wolfgang , Müller Michael R. , Bevec Dorian , Lechner Klaus , Valent Peter
Published: October 2019

Recent data suggest that mast cells (MC) are involved in the regulation of leukocyte accumulation in inflammatory reactions. In this study, expression of leukocyte-chemotactic peptides (chemokines) in purified human lung MC (n = 16) and a human mast cell line, HMC-1, was analyzed. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) showed baseline expression of monocyte chemoattractant protein (MCP)-1 mRNA in unstimulated MC. Exposure of MC to recombinant stem cell factor (rhSCF, 100 ng/mL) or anti-IgE (10 μg/mL) was followed by a substantial increase in expression of MCP-1 mRNA. Neither unstimulated nor stem cell factor (SCF )-stimulated lung MC expressed transcripts for interleukin-8 (IL-8), macrophage inflammatory protein-1α (MIP-1α), MIP-1β, or RANTES by Northern blotting. The mast cell line HMC-1, which contains a mutated and intrinsically activated SCF-receptor, was found to express high levels of MCP-1 mRNA in a constitutive manner. Exposure of HMC-1 cells to rhSCF resulted in upregulation of MCP-1 mRNA expression, and de novo expression of MIP-1β mRNA. The SCF-induced upregulation of MCP-1 mRNA in lung MC and HMC-1 was accompanied by an increase in immunologically detectable MCP-1 in cell supernatants (sup) (lung MC [<98%], control medium, 1 hour: 159 ± 27 v SCF, 100 ng/mL, 1 hour: 398 ± 46 pg/mL/106 cells; HMC-1: control, 1 hour: 894 ± 116 v SCF, 1 hour: 1,536 ± 265 pg/mL/106). IgE-dependent activation was also followed by MCP-1 release from MC. MC-sup and HMC-1–sup induced chemotaxis in blood monocytes (Mo) (control: 100% ± 12% v 2-hour–MC-sup: 463% ± 38% v HMC-1–sup: 532% ± 12%), and a monoclonal antibody (MoAb) to MCP-1 (but not MoAb to IL-8) inhibited Mo-chemotaxis induced by MC-sup or HMC-1–sup (39% to 55% inhibition, P < .05). In summary, our study identifies MCP-1 as the predominant CC-chemokine produced and released in human lung MC. MCP-1 may be a crucial mediator in inflammatory reactions associated with MC activation and accumulation of MCP-1–responsive leukocytes.
The Role of Interleukin-10 (IL-10) in IL-15–Mediated T-Cell Responses
Authors: Körholz Dieter , Banning Ursula , Bönig Halvard , Grewe Markus , Schneider Marion , Mauz-Körholz Christine , Klein-Vehne Anne , Krutmann Jean , Burdach Stephan
Published: October 2019

Interleukin-15 (IL-15) is a potent T-cell stimulating factor, which has recently been used for pre-clinical in vivo immunotherapy. Here, the IL-15 effect on CD3-stimulated peripheral human T cells was investigated. IL-15 induced a significant T-cell proliferation and upregulated CD25 expression. IL-15 significantly enhanced T-cell production of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-10. Between 10- and 100-fold greater concentrations of IL-15 were necessary to reach a biological effect equivalent to that of IL-2. Blockade of IL-2 binding to the high-affinity IL-2 receptor did not affect the IL-15 effects, suggesting that IL-15 did not act by inducing endogenous IL-2. Exogenously administered IL-10 significantly reduced the IL-15 and IL-2–mediated IFN-γ and TNF-α production, whereas T-cell proliferation and CD25 expression were not affected. The inhibitory effects of exogenously administered IL-10 on T-cell cytokine production appeared indirect, and are likely secondary to decreased IL-12 production by accessory cells. Inhibition of endogenous IL-10 binding to the IL-10 receptor significantly increased IFN-γ and TNF-α release from T cells. These data suggest that endogenous IL-10 can regulate activated T-cell production of IFN-γ and TNF-α via a paracrine negative feedback loop. The observations of this study could be of relevance for the therapeutic use of IL-15 in vivo.
Adult Patients With De Novo Acute Myeloid Leukemia and t(9; 11)(p22; q23) Have a Superior Outcome to Patients With Other Translocations Involving Band 11q23: A Cancer and Leukemia Group B Study
Authors: Mrózek Krzysztof , Heinonen Kristiina , Lawrence David , Carroll Andrew J. , Koduru Prasad R.K. , Rao Kathleen W. , Strout Matthew P. , Hutchison Robert E. , Moore Joseph O. , Mayer Robert J. , Schiffer Charles A. , Bloomfield Clara D.
Published: October 2019

Following reports of childhood acute myeloid leukemia (AML) showing that patients with t(9; 11)(p22; q23) have a better prognosis than those with translocations between 11q23 and other chromosomes, we compared response to therapy and survival of 24 adult de novo AML patients with t(9; 11) with those of 23 patients with other 11q23 translocations [t(11q23)]. Apart from a higher proportion of French-American-British (FAB) M5 subtype in the t(9; 11) group (83% v 43%, P = .006), the patients with t(9; 11) did not differ significantly from patients with t(11q23) in terms of their presenting clinical or hematologic features. Patients with t(9; 11) more frequently had an extra chromosome(s) 8 or 8q as secondary abnormalities (46% v 9%, P = .008). All patients received standard cytarabine and daunorubicin induction therapy, and most of them also received cytarabine-based intensification treatment. Two patients, both with t(9; 11), underwent bone marrow transplantation (BMT) in first complete remission (CR). Nineteen patients (79%) with t(9; 11) and 13 (57%) with t(11q23) achieved a CR (P = .13). The clinical outcome of patients with t(9; 11) was significantly better: the median CR duration was 10.7 versus 8.9 months (P = .02), median event-free survival was 6.2 versus 2.2 months (P = .009), and median survival was 13.2 versus 7.7 months (P = .009). All patients with t(11q23) have died, whereas seven (29%) patients with t(9; 11) remain alive in first CR. Seven of eight patients with t(9; 11) who received postremission regimens with cytarabine at a dose of 100 (four patients) or 400 mg/m2 (2 patients) or who did not receive postremission therapy (2 patients) have relapsed. In contrast, 7 (64%) of 11 patients who received intensive postremission chemotherapy with high-dose cytarabine (at a dose 3 g/m2) (5 patients), or underwent BMT (2 patients) remain in continuous CR. We conclude that the outcome of adults with de novo AML and t(9; 11) is more favorable than that of adults with other 11q23 translocations; this is especially true for t(9; 11) patients who receive intensive postremission therapy.
Mitochondrial Antisense RNA for Cytochrome C Oxidase (MARCO) Can Induce Morphologic Changes and Cell Death in Human Hematopoietic Cell Lines
Authors: Shirafuji Naoki , Takahashi Satoshi , Matsuda Satoru , Asano Shigetaka
Published: October 2019

To identify essential molecules capable of inducing terminal morphologic maturation and cell death of myeloid progenitor cells, we isolated cDNA clones by functional expression cloning using a library constructed from all-trans retinoic acid (ATRA)-treated human promyelocytic HL-60 cells. Clones which induced morphologic changes in HL-60 cells from blastic cells to mature neutrophilic granulocytes were selected. The isolated positive cDNA clone was demonstrated to encode an antisense RNA for cytochrome c oxidase/serine tRNA derived from a mitochondrial gene (MARCO). When MARCO was expressed in HL-60 cells with the lac switch system, blastic cell morphology became neutrophilic after 48-hour incubation with IPTG, and cell death was observed after 3 days. Also, high molecular weight DNA fragmentation was observed after 36 hours in culture. Similar results were observed using transformants from human K562 cells and CMK cells. RT-PCR analysis revealed that MARCO was transcribed in both ATRA and TNF-α systems, and also in human blood neutrophilic granulocytes. Following transfection with cytochrome c oxidase expression plasmids, TNF-α–induced high molecular weight DNA fragmentation in U937 cells and HL-60 cells was inhibited in these transformants. These results indicate that maturational changes in hematopoietic cells and the process of cell death may be induced by mitochondrial respiratory insufficiency, and also that the mitochondrial gene MARCO may be used as one of the candidates for gene supplementation therapy for the acute leukemias.
Modulation of Idarubicin-Induced Apoptosis in Human Acute Myeloid Leukemia Blasts by All-Trans Retinoic Acid, 1,25(OH)2 Vitamin D3, and Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Ketley Nicolas J. , Allen Paul D. , Kelsey Stephen M. , Newland Adrian C.
Published: October 2019

The relationship between differentiation of human myeloid cells and apoptosis remains unclear. Recent studies have shown that terminal differentiation need not necessarily lead to the apoptotic demise of myeloid cells, while other studies have shown that induction of differentiation is associated with increased resistance to apoptosis-inducing agents, such as chemotherapy and γ-irradiation. Such results are pertinent to the treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome, where differentiating agents and hemopoietic growth factors are being combined with chemotherapy to enhance response and limit toxicity. To elucidate the factors governing apoptosis in human AML blasts, we have studied the cytotoxic effect of idarubicin on HL60, U937 and KG1 cells, after incubation with all-trans retinoic acid (ATRA), 1,25(OH)2 D3, and granulocyte-macrophage colony-stimulating factor (GM-CSF ). We show that prior incubation of human myeloid leukemic cells with ATRA or 1,25(OH)2 D3 induced resistance to idarubicin-induced apoptosis, which was modulated by coincubation with GM-CSF. The altered chemosensitivity of cells depended on the degree of G0/G1 cell-cycle arrest induced by incubation with ATRA, 1,25(OH)2 D3, and GM-CSF and was independent of differentiation status or Bcl-2 oncoprotein expression. These findings suggest that cell-cycle arrest in human leukemic cells can be induced by exogenous agents and may promote drug resistance. Determining the mechanisms by which cell-cycle arrest is induced may permit understanding of the processes by which the cells escape cytotoxic drug-mediated apoptosis.
Implications of Potential Cure in Acute Myelogenous Leukemia: Development of Subsequent Cancer and Return to Work
Authors: de Lima Marcos , Strom Sara S. , Keating Michael , Kantarjian Hagop , Pierce Sherry , O'Brien Susan , Freireich Emil , Estey Elihu
Published: October 2019

Chemotherapy produces extended remissions, and potential cures, in a small minority of patients with acute myeloid leukemia (AML). We explored whether potentially cured patients were at increased risk of subsequent invasive cancer and were able to return to work. Potentially cured patients were defined as those in first or second complete remission (CR) for at least 3 years based on hazard rates for recurrence or death in CR, which declined sharply after this time. Patients who received allogeneic marrow transplant were excluded. We used questionnaires, phone contact, and chart review to obtain information about subsequent cancer and work status. The number of patients who developed invasive cancer was compared with the number expected based on age, gender, and years of follow-up using the Connecticut Tumor Registry. A total of 215 patients met our criteria for potential cure: 203 in first CR and 12 in second CR (of 1,663 treated between 1965 and December, 1992). At a median of 9.2 years from first or second CR, 163 (76%) remain alive in CR. Fifteen patients developed 18 invasive cancers (expected number of patients, 8.8; observed/expected, 1.70; 95% CI, 0.96 to 2.84; P = .06). Patients initially treated between 1973 to 1979, patients above the potentially cured cohort's median age of 40 years, and those who presented with abnormalities of chromosomes 5 and/or 7 were more likely to develop subsequent cancer, whereas the observed/expected ratio for younger patients was 1.05 (95% CI, 0.13 to 3.80; P = .56). Seventy-four percent of the patients who were working full-time and who were under age 50 at time of treatment for AML have been working full-time in the last 6 months. Only 17 of 56 patients who are currently not working cited physical limitation as the reason. Patients with potentially cured AML are likely to be able to return to work, and at least if younger do not, on average, have an increased risk of invasive cancer.