bluuebunny/crossref_metadata_2025 huggingface dataset:
Increases in Neutral, Mg2+-Dependent and Acidic, Mg2+-Independent Sphingomyelinase Activities Precede Commitment to Apoptosis and Are Not a Consequence of Caspase 3–Like Activity in Molt-4 Cells in Response to Thymidylate Synthase Inhibition by GW1843
Authors: Laethem Ronald M. , Hannun Yusuf A. , Jayadev Supriya , Sexton Connie J. , Strum Jay C. , Sundseth Rebecca , Smith Gary K.
Published: October 2019

Thymidylate synthase (TS) inhibition causes cell death, and this enzyme is the target for the important chemotherapy regime 5-fluorouracil/leucovorin. GW1843 (1843U89) is a potent and specific folate analog TS inhibitor in clinical development. Because of the importance of TS as a chemotherapy target, we are studying the mechanism of TS inhibition-induced cell death by GW1843. Ceramide is a regulatory lipid generated by the action of sphingomyelinase and is believed to signal apoptosis. The role of the ceramide in apoptotic signaling was studied in Molt-4 human T-cell leukemia cells undergoing cell death after treatment with GW1843. In response to GW1843, Molt-4 cells undergo apoptosis with both acidic pH, Mg2+-independent sphingomyelinase (ASMase) and neutral pH, Mg2+-dependent sphingomyelinase (NSMase) activities elevated as early steps in the initiation of apoptosis before Molt-4 commitment to death. These activities lead to ceramide production with kinetics consistent with a role as an effector molecule signaling the initiation of apoptosis in Molt-4 cells. These changes were found to be independent of caspase 3–like (CPP32/apopain) activity and DNA degradation, but were not separable from membrane blebbing or cell lysis in this cell line. In this report, kinetic evidence is provided for a role of ceramide in initiating GW1843-induced cell death of Molt-4 T-cell leukemia cells.
Biliary Iron Excretion in Rats Following Treatment With Analogs of Pyridoxal Isonicotinoyl Hydrazone
Authors: Bláha Karel , Cikrt Miroslav , Nerudová Jana , Fornusková Helena , Ponka Premysl
Published: October 2019

Iron overload is a major life-threatening complication of thalassemia major and other iron-loading anemias treated by regular blood transfusions. Although the clinical manifestations of iron overload may be prevented by desferrioxamine, the only iron-chelating drug in routine clinical use, this treatment requires subcutaneous infusion of desferrioxamine for 12 hours each day. New orally effective iron chelators are urgently needed, and pyridoxal isonicotinoyl hydrazone (PIH), which was first recognized as an effective iron chelator in vitro and subsequently in vivo, shows promise for the treatment of iron overload. More recently, over 40 analogs of PIH were synthesized, and some of them proved to be very potent in mobilizing 59Fe in vitro from 59Fe-labeled cells. In this study, we show that PIH analogs such as pyridoxal benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone (PMBH), pyridoxal m-fluorobenzoyl hydrazone (PFBH), and pyridoxal-2-thiophenecarboxyl hydrazone, compounds previously shown to mobilize iron from cells in vitro, are also effective in vivo. All of these chelators significantly enhanced biliary excretion of iron (measured by atomic absorption spectrophotometry) following their intraperitoneal (IP) and/or oral administration to rats. The most effective was PFBH, which increased iron concentration in the bile about 150-fold, as compared with basal biliary iron concentration, within 1 hour following a single IP dose of 0.2 mmol/kg body weight. In contrast, desferrioxamine increased the biliary iron concentration only 20-fold to 30-fold under the same conditions. Moreover, while control rats excreted ≈ 0.8 μg Fe in 2 hours, treatment with PFBH, PMBH, and desferrioxamine resulted in cumulative excretions of 87, 59, and 22 μg Fe, respectively, in the same period of time. Interestingly, PMBH was also quite effective following gastric administration, resulting in a 6-hour cumulative value of 34 μg Fe. These compounds are nontoxic and are inexpensive and easy to make. Their further evaluation as candidate drugs for the treatment of iron overload is warranted.
Pgp and MRP Activities Using Calcein-AM Are Prognostic Factors in Adult Acute Myeloid Leukemia Patients
Authors: Legrand Ollivier , Simonin Ghislaine , Perrot Jean-Yves , Zittoun Robert , Marie Jean-Pierre
Published: October 2019

Thirteen cell lines with different levels of Pgp and MRP expression were used to assess the ability of calcein acetoxymethyl ester (calcein-AM) uptake and calcein efflux to measure Pgp and MRP functions, respectively. There was a good correlation between MRP expression and the modulatory effect of probenecid (a specific modulator of MRP) on the calcein efflux (r = .91, P= .0003) and between Pgp expression and the modulatory effect of CsA on calcein-AM uptake (r = .96, P < .0001). In light of the high correlations for both proteins, we tested calcein-AM uptake and efflux in fresh myeloid leukemic cells. In 53 acute myeloid leukemia (AML) patients, there was also a good correlation between MRP expression (measured by reverse transcription-polymerase chain reaction and by MRPm6 expression by flow cytometry) and the modulatory effect of probenecid on the calcein fluorescence (r = .92, P < .0001) and between Pgp expression as measured by UIC2 antibody binding on flow cytometry and the modulatory effect of cyclosporin A on calcein-AM uptake (r = .83,P < .0001). Pgp activity was higher in CD34+leukemia than in CD34− leukemia (2.26 ± 1.50 v1.46 ± 1.21, respectively; P = .003), and MRP activity was higher in CD34− leukemia than in CD34+leukemia (1.77 ± 0.40 v 1.4 ± 0.29, respectively; P= .004). Pgp expression and activity (P = .004 andP = .01, respectively) and MRP activity (P = .03) but not MRP expression were prognostic factors for achievement of complete remission. These results suggest that functional testing (with calcein-AM ± modulators) for the presence of both MRP and Pgp activities is of prognostic value and that MRP contributes to drug resistance in AML.
Rapid Activation of Protein C by Factor Xa and Thrombin in the Presence of Polyanionic Compounds
Authors: Rezaie Alireza R.
Published: October 2019

A recent study indicated that negatively charged substances such as heparin and dextran sulfate accelerate thrombin activation of coagulation factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind heparin, it was reasonable to think that these compounds may also bind protein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this, PC activation by thrombin and factor Xa (fXa) was studied in the presence of these polysaccharides. With thrombin in the absence of thrombomodulin (TM), these polysaccharides markedly reduced the Km for PC and Gla-domainless PC (GDPC) activation in the presence of Ca2+. With TM containing chondroitin sulfate, heparin did not influence PC activation by thrombin, but with TM lacking chondroitin sulfate, the characteristic high-affinity PC interaction at low Ca2+ (∼50 to 100 μmol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the Km, but it inhibited PC activation by increasing the Km. PC activation in EDTA was insensitive to the presence of heparin if the exosite 2 mutant, R93,97,101A thrombin, was used for activation. These results suggest that, when the Gla-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this site prevents this interaction. Additional studies indicated that, in the presence of phospholipid vesicles, heparin and dextran sulfate dramatically accelerate PC activation by fXa by also reducing the Km. Interestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations (∼80 nmol/L) by fXa in the presence of dextran sulfate was nearly comparable to that observed by the thrombin-TM complex. The biochemical and potential therapeutical ramifications of these findings are discussed.
Interferon-γ–Dependent Inducible Expression of the Human Interleukin-12 p35 Gene in Monocytes Initiates From a TATA-Containing Promoter Distinct From the CpG-Rich Promoter Active in Epstein-Barr Virus-Transformed Lymphoblastoid Cells
Authors: Hayes Mark P. , Murphy Finbarr J. , Burd Parris R.
Published: October 2019

Interleukin-12 (IL-12) production by human monocytes is stringently regulated through the inducibility of both subunits, p35 and p40, and expression of p35 mRNA is the limiting factor for the secretion of the bioactive IL-12 p70 heterodimer. Optimal induction of p35 mRNA requires priming of the monocytes by interferon-γ (IFN-γ), followed by brief exposure to lipopolysaccharide or other bacterial products. To investigate control of p35 gene expression, we isolated genomic clones containing the human p35 gene and determined the 5′ end of the mRNA expressed in monocytes. We discovered that a unique p35 transcript is induced in monocytes that begins downstream of a consensus TATA box that lies within the 5′ end of the cDNA originally cloned from Epstein-Barr virus (EBV)-transformed B cells. Analysis of p35 mRNA by Northern blotting showed that the message from monocytes is approximately 200 bases shorter than message derived from the EBV-transformed B-cell line VDS. The initiation sites downstream from the TATA box were confirmed by RNase protection and 5′ RACE. The data indicate that p35 transcription can initiate from different sites depending on the cell type and that the shorter inducible transcript in monocytes is the one that accumulates after stimulation. Protein translation of these two forms may result in proteins of different sizes with potential implications for the regulation of IL-12 secretion and function.
The Role of Tumor Necrosis Factor α in Modulating the Quantity of Peripheral Blood-Derived, Cytokine-Driven Human Dendritic Cells and Its Role in Enhancing the Quality of Dendritic Cell Function in Presenting Soluble Antigens to CD4+ T Cells In Vitro
Authors: Chen Bing-guan , Shi Yijun , Smith Jeffrey D. , Choi David , Geiger James D. , Mulé James J.
Published: October 2019

Because dendritic cells (DC) are critically involved in both initiating primary and boosting secondary host immune responses, attention has focused on the use of DC in vaccine strategies to enhance reactivity to tumor-associated antigens. We have reported previously the induction of major histocompatibility complex class II-specific T-cell responses after stimulation with tumor antigen-pulsed DC in vitro. The identification of in vitro conditions that would generate large numbers of DC with more potent antigen-presenting cell (APC) capacity would be an important step in the further development of clinical cancer vaccine approaches in humans. We have focused attention on identifying certain exogenous cytokines added to DC cultures that would lead to augmented human DC number and function. DC progenitors from peripheral blood mononuclear cells (PBMC) were enriched by adherence to plastic, and the adherent cells were then cultured in serum-free XVIVO-15 medium (SFM) for 7 days with added granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). At day 7, cultures contained cells that displayed the typical phenotypic and morphologic characteristics of DC. Importantly, we have found that the further addition of tumor necrosis factor α (TNFα) at day 7 resulted in a twofold higher yield of DC compared with non–TNFα-containing DC cultures at day 14. Moreover, 14-day cultured DC generated in the presence of TNFα (when added at day 7) demonstrated marked enhancement in their capacity to stimulate a primary allogeneic mixed leukocyte reaction (8-fold increase in stimulation index [SI]) as well as to present soluble tetanus toxoid and candida albicans (10- to 100-fold increases in SI) to purified CD4+ T cells. These defined conditions allowed for significantly fewer DC and lower concentrations of soluble antigen to be used for the pulsing of DC to efficiently trigger specific T-cell proliferative responses in vitro. When compared with non–TNFα-supplemented cultures, these DC also displayed an increased surface expression of CD83 as well as the costimulatory molecules, CD80 and CD86. Removal of TNFα from the DC cultures after 2 or 4 days reduced its enhancing effect on DC yield, phenotype, and function. Thus, the continuous presence of TNFα over a 7-day period was necessary to achieve the maximum enhancing effect observed. Collectively, our findings point out the importance of exogenous TNFα added to cultures of cytokine-driven human DC under serum-free conditions, which resulted in an enhanced number and function of these APC. On the basis of these results, we plan to initiate clinical vaccine trials in patients that use tumor-pulsed DC generated under these defined conditions.
Homozygous Deletions at Chromosome 9p21 Involving p16 and p15 Are Associated With Histologic Progression in Follicle Center Lymphoma
Authors: Elenitoba-Johnson Kojo S.J. , Gascoyne Randy D. , Lim Megan S. , Chhanabai Mukesh , Jaffe Elaine S. , Raffeld Mark
Published: October 2019

Low-grade follicle center lymphoma (LGFCL) is characterized genetically by the t(14;18) translocation and an indolent clinical course. Histologic progression from LGFCL to an aggressive diffuse large B-cell lymphoma (DLCL) occurs in 60% to 80% of cases, and this transformation is associated with the accumulation of secondary genetic alterations. Using 10 polymorphic microsatellite markers spanning the chromosome 9p21 region harboring the p15 (p15INK4B/MTS-2/CDKN2B) and p16 (p16INK4A/MTS-1/CDKN2) tumor-suppressor gene loci, we analyzed 11 matched pairs of LGFCL and their corresponding progressed DLCL biopsies for loss of heterozygosity and homozygous deletions at 9p21. A comparative multiplex polymerase chain reaction assay was also used for the detection of homozygous deletions. Deletions were identified in 8 of the 11 cases studied (73%): 6 homozygous (54%) and 2 hemizygous (18%). The deletions were identified exclusively in the progressed DLCL biopsies. Immunohistochemical studies showed an excellent correlation with the results from the genetic analyses. Of the 9 matched pairs of LGFCL and progressed DLCL with interpretable immunohistochemical staining, 9 of 9 (100%) of the LGFCL showed diffuse reactivity for p16. Four of the 9 (44%) immunohistochemically evaluable cases of progressed DLCL showed loss of or, in 1 case, markedly diminished p16 expression. All 4 of these cases correspondingly showed homozygous deletions at 9p21. Five of the 9 progressed DLCL cases showed p16 expression and demonstrated retention of one or both 9p21 alleles by genetic analysis. This is the first longitudinal series examining sequential biopsy specimens of low-grade and progressed FCL for genetic loss at 9p21 encompassing the p16 and p15 loci. The high frequency and exclusive occurrence of deletions involving p16 in the progressed DLCLs suggests that genetic loss at 9p21 targeting p16 and/or p15 is an important secondary genetic event in the histologic progression of FCL.
Lymphocyte-Specific Protein 1 Expression in Eukaryotic Cells Reproduces the Morphologic and Motile Abnormality of NAD 47/89 Neutrophils
Authors: Howard Thomas H. , Hartwig John , Cunningham Casey
Published: October 2019

Despite its name, the actin-binding protein lymphocyte-specific protein1 (LSP1) is found in all hematopoetic cells, and yet its role in cell function remains unclear. Recently, LSP1 was identified as the 47-kD protein overexpressed in the polymorphonuclear neutrophils of patients with a rare neutrophil disorder, neutrophil actin dysfunction with abnormalities of 47-kD and 89-kD proteins (NAD 47/89). These neutrophils are immotile, defective in actin polymerization in response to agonists, and display distinctive, fine, “hairlike” F-actin-rich projections on their cell surfaces. We now show that overexpression of LSP1 produces F-actin bundles that are likely responsible for the morphologic and motile abnormalities characteristic of the NAD 47/89 phenotype. Coincident with LSP1 overexpression, cells from each of several different eukaryotic lines, including a highly motile human melanoma line, develop hairlike surface projections that branch distinctively and contain F-actin and LSP1. The hairlike projections are supported at their core by thick actin bundles, composed of actin filaments of mixed polarity, which periodically anastomose to generate a branching structure. The motility of the melanoma cells is inhibited even at low levels of LSP1 expression. Therefore, these studies show that overexpression of LSP1 alone can recreate the morphologic and motile defects seen in NAD 47/89 and suggest that LSP1 is distinct from other known actin binding proteins in its effect on F-actin network structure.
Retinoic Acid Inhibits Monocyte to Macrophage Survival and Differentiation
Authors: Kreutz Marina , Fritsche Jana , Ackermann Ute , Krause Stefan W. , Andreesen Reinhard
Published: October 2019

Vitamin A metabolites are potent differentiation-inducing agents for myelomonocytic cell lines in vitro and are successfully used for the treatment of patients with acute promyelocytic leukemia. However, little is known about the effects of vitamin A on normal hematopoietic cells. Therefore, we investigated the effect of vitamin A on differentiation and activation of human blood monocytes (MO). Culturing MO for up to 4 days with 9-cis retinoic acid (RA) and all-trans RA but not retinol reduced MO survival, with the remaining cells being morphologically comparable to control cells. Because macrophage colony-stimulating factor (M-CSF) is a well-known survival factor for MO, we measured the M-CSF content of MO culture supernatants using enzyme-linked immunosorbent assay and found that RA suppressed the constitutive secretion of M-CSF. Northern analysis showed that the M-CSF mRNA expression was only slightly reduced by RA treatment, suggesting regulation on the posttranscriptional level. In contrast to MO, M-CSF secretion by MO-derived macrophages (MAC) was not altered by RA, suggesting a differentiation-dependent switch in the responsiveness of MO/MAC to RA. Because M-CSF is not only a survival-promoting but also a differentiation-promoting factor for myeloid cells, we analyzed the effect of RA on MO to MAC maturation. RA suppressed the expression of the maturation-associated antigen carboxypeptidase M (CPM)/MAX.1 at both the protein and mRNA levels and modulated the lipopolysaccharide-stimulated cytokine secretion of MO/MAC. The addition of exogenous M-CSF to RA-containing MO cultures fails to overcome the RA-induced inhibition of MO differentiation. However, the survival rate was improved by exogenous M-CSF. We conclude that RA acts via two different mechanisms on monocyte survival and differentiation: posttranscriptionally by controlling M-CSF secretion, which decreases MO survival, and transcriptionally regulating the expression of differentiation-associated genes. The regulation of M-CSF production may contribute to the antileukemic effect of RA in vivo by reducing autocrine M-CSF production by leukemic cells.
Adenosine Diphosphate (ADP) and ADP Receptor Play a Major Role in Platelet Activation/Aggregation Induced by Sera From Heparin-Induced Thrombocytopenia Patients
Authors: Polgár János , Eichler Petra , Greinacher Andreas , Clemetson Kenneth J.
Published: October 2019

The molecular basis for heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, is not yet fully understood. We found that pretreatment of platelets with AR-C66096 (formerly FPL 66096), a specific platelet adenosine diphosphate (ADP) receptor antagonist, at a concentration of 100 to 200 nmol/L that blocked ADP-dependent platelet aggregation, resulted in complete loss of platelet aggregation responses to HIT sera. AR-C66096 also totally inhibited HIT serum-induced dense granule release, as judged by measurement of adenosine triphosphate (ATP) release. Apyrase, added to platelets at a concentration that had only minor effects on thrombin- or arachidonic acid-induced aggregation, also blocked completely HIT serum-induced platelet aggregation. Furthermore, AR-C66096 inhibited platelet aggregation and ATP release induced by cross-linking FcγRIIA with specific antibodies. These data show that released ADP and the platelet ADP receptor play a pivotal role in HIT serum-induced platelet activation/aggregation. The thromboxane receptor inhibitor, Daltroban, had no effect on HIT serum-induced platelet activation whereas GPIIb-IIIa antagonists blocked platelet aggregation but had only a moderate effect on HIT serum-induced dense granule release. Pretreatment of platelets with chondroitinases but not with heparinases resulted in concentration dependent inhibition of HIT serum-induced platelet aggregation. These novel data relating to the mechanism of platelet activation induced by HIT sera suggest that the possibility should be examined that ADP receptor antagonists or compounds that inhibit ADP release may be effective as therapeutic agents for the prevention or treatment of complications associated with heparin therapy.
The Upregulation of p27Kip1 by Rapamycin Results in G1 Arrest in Exponentially Growing T-Cell Lines
Authors: Kawamata Shin , Sakaida Hitoshi , Hori Toshiyuki , Maeda Michiyuki , Uchiyama Takashi
Published: October 2019

An immunosuppressant Rapamycin (Rap) has been reported to cause G1 arrest by inhibiting p70 S6 kinase and G1 cyclin/cdks kinase activities when added to quiescent cells with mitogens. However, antiproliferative effects of Rap on exponentially growing cells have been poorly investigated. We examined the intracellular events after the treatment of Rap in exponentially growing T cells and found that Rap upregulated a cdks inhibitor, p27Kip1 at both mRNA and protein levels in Rap-sensitive cells. Antiproliferative effect of Rap was mainly ascribed to the inhibition of cyclin E/cdk2 kinase activity through the formation of cyclin E/cdk2-p27Kip1 complex rather than inhibition of p70 S6 kinase activity. Furthermore, we showed that Rap-sensitive cells with elevated p27Kip1 expression lost sensitivity to Rap when antisense p27Kip1 was introduced, which indicates that the basal level of p27Kip1 is one of the limiting factors that determine the sensitivity to Rap in already cycling cells. These data suggest the presence of a putative threshold level of p27Kip1 at late G1 phase in already cycling cells. Rap may cause G1 arrest by upregulating the amount of p27Kip1 beyond the threshold in some Rap-sensitive cells that are exponentially growing.
Lipopolysaccharide Activates Caspase-1 (Interleukin-1–Converting Enzyme) in Cultured Monocytic and Endothelial Cells
Authors: Schumann Ralf R. , Belka Claus , Reuter Dirk , Lamping Norbert , Kirschning Carsten J. , Weber Joerg R. , Pfeil Dagmar
Published: October 2019

Interleukin-1β (IL-1β) is a pleiotropic proinflammatory cytokine. Mechanisms leading to its secretion include not only release of newly synthesized protein, but also cleavage of a preformed immature precursor protein into an active secretory form by the intracellular protease caspase-1 (formerly termed IL-1–converting enzyme [ICE]). Caspase-1 belongs to a rapidly growing family of cysteine proteases with substrate specificity for aspartate involved in cellular apoptosis. We have used an assay determining the caspase-1 activity based on cleavage of a fluorogenic peptide substrate to elucidate its role in lipopolysaccharide (LPS)-induced secretion of IL-1β. We show that LPS induces moderate caspase-1 activity in the monocytic cell line THP-1, in freshly isolated peripheral blood monocytes, and in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent fashion. Caspase-1 activation by LPS was associated with cleavage of the IL-1β precursor protein that was followed by release of the mature IL-1β protein in monocytic cells. In contrast, subsequent release of IL-1β by HUVECs was not significant. LPS-induced caspase-1 activation appeared not to result from modulation of caspase-1 transcript accumulation and inhibition of caspase-1 activity was accomplished by two specific inhibitors, YVAD-CHO and YVAD-CMK, capable of alleviating the release of mature IL-1β. Taken together, these results show that LPS moderately activates caspase-1 and that caspase-1 activation contributes to LPS induction of IL-1β secretion.
Primary Diffuse Large B-Cell Lymphoma of the Mediastinum: Outcome Following High-Dose Chemotherapy and Autologous Hematopoietic Cell Transplantation
Authors: Sehn Laurie H. , Antin Joseph H. , Shulman Lawrence N. , Mauch Peter , Elias Anthony , Kadin Marshall E. , Wheeler Catherine
Published: October 2019

We performed a retrospective analysis of 35 patients with primary diffuse large B-cell lymphoma of the mediastinum treated with high-dose cyclophosphamide, carmustine, and etoposide (CBV) plus autologous hematopoietic cell transplantation to determine outcome and prognostic features for progression-free survival (PFS). Thirty-five patients with primary diffuse large B-cell lymphoma of the mediastinum in first response (complete remission [CR] or partial remission [PR]) with poor prognostic features, with primarily refractory disease, or with relapsed disease following conventional chemotherapy, were treated with CBV and autologous hematopoietic cell transplantation. PFS and overall survival were assessed by the Kaplan-Meier method. Patient characteristics before transplantation were examined by univariate analysis using the log-rank test and by Cox's proportional hazards regression analysis to determine predictors of PFS. Estimated 5-year PFS varied significantly with patient disease status at transplantation. Patients transplanted in first response had an estimated 5-year PFS rate of 83%, compared with 58% and 27% for primarily refractory and relapsed patients, respectively (P = .02). The strongest predictor of PFS was chemotherapy responsiveness immediately before transplantation. Patients with chemotherapy-responsive disease had a significantly greater PFS rate than patients with chemotherapy-nonresponsive disease (risk ratio, 3.60; 95% confidence interval [CI], 1.14 to 11.4). No other factors were found to be significant on univariate or multivariate analysis. Patients with primary diffuse large B-cell lymphoma of the mediastinum can achieve prolonged PFS following high-dose chemotherapy and autologous hematopoietic cell transplantation. Outcomes are strongly correlated with disease status (first response v refractoryv relapsed) at transplantation and chemotherapy responsiveness immediately before transplantation.
Cyclosporin A Induces Apoptosis in Childhood Acute Lymphoblastic Leukemia Cells
Authors: Ito Chikako , Ribeiro Raul C. , Behm Frederick G. , Raimondi Susana C. , Pui Ching-Hon , Campana Dario
Published: October 2019

In an effort to identify novel antileukemic agents that can bypass the mechanisms of multidrug resistance, we found that cyclosporin A ([CyA] 5 μmol/L) produced a median cell kill of 69% (range, 47% to 85%) in seven B-lineage acute lymphoblastic leukemia (ALL) cell lines (OP-1, SUP-B15, KOPN-55bi, RS4;11, NALM6, REH, and 380) and three T-lineage ALL cell lines (MOLT4, CCRF-CEM, and CEM-C7) after 4 days of culture. At 10 μmol/L, median CyA toxicity was 99% (range, 88% to >99%). CyA was equally toxic to both a multidrug-resistant cell line, CEM-VLB100, which overexpresses gp-170 P-glycoprotein, and one resistant to topoisomerase II inhibitors, CEM-VM1-5, which has a mutation in the topoisomerase II gene. CyA was also toxic to primary leukemic cells maintained in stroma-based culture, a system that substantially prolongs in vitro cell survival. Against lymphoblasts from 21 patients with B-lineage ALL, the compound (at 5 μmol/L) reduced the leukemic cell number by a median of 87% (range, 27% to >99%) compared with results for parallel control cultures lacking CyA. Seven of these samples were from cases with unfavorable genetic features (eg, Philadelphia-chromosome orMLL gene rearrangements); three were obtained at relapse. Against T lymphoblasts (from six patients), the median reduction in cell number was 79% (range, 30% to >99%). At 10 μmol/L, the cell kill exceeded 97% in all cases studied. The mechanism of CyA cytotoxicity was found to be the activation of apoptosis, which was suppressed by phorbol myristate acetate but not by inhibitors of ceramide-mediated apoptosis, phosphatidyl inositol-3 kinase activity, or tyrosine kinase activity. These findings demonstrate high levels of CyA-induced toxicity against ALL cells at concentrations achievable in vivo, thus providing a strong rationale for clinical testing of this agent in patients with ALL.
Genomic Alterations of the p19ARF Encoding Exons in T-Cell Acute Lymphoblastic Leukemia
Authors: Gardie Betty , Cayuela Jean-Michel , Martini Sophie , Sigaux François
Published: October 2019

We have previously shown that the disruption/deletion of the MTS(MTS1-MTS2) locus due to illegitimate V(D)J recombinase activity is a genetic event characteristic of T-cell acute lymphoblastic leukemia (T-ALL). Inactivation of the p16INK4a tumor suppressor protein, encoded by MTS1, is thought to be the major functional consequence of these chromosomal rearrangements. The two other cell cycle inhibitors encoded by genes identified in the locus (p19ARF by MTS1 and p15INK4b by MTS2), also represent possible candidates for inactivating events. By analyzing p16INK4aexpression in three cases in which an identical 36-kb deletion had deleted MTS2 and disrupted the p19ARF, but spared the p16INK4aMTS1 encoding exons, we have excluded p16INK4a and pinpointed p19ARF and/or p15INK4b as the functional target(s) of this rearrangement. Moreover, by the study of the MTS genomic configuration of 149 rearranged alleles from a large series of T-ALL cases, we have shown that p19ARF encoding exons were always disrupted or deleted, whereas p16INK4a and p15INK4b encoding exons were spared in four and 21 cases, respectively. These results suggest that p19ARF may be targeted by the genetic events that occur in the MTS locus in the majority of T-ALLs.
Nitric Oxide–Mediated Induction of Ferritin Synthesis in J774 Macrophages by Inflammatory Cytokines: Role of Selective Iron Regulatory Protein-2 Downregulation
Authors: Recalcati Stefania , Taramelli Donatella , Conte Dario , Cairo Gaetano
Published: October 2019

Cytokine-treated macrophages represent a useful model to unravel the molecular basis of reticuloendothelial (RE) iron retention in inflammatory conditions. In the present study, we showed that stimulation of murine macrophage J774 cells with interferon (IFN)-γ/lipopolysaccharide (LPS) resulted in a nitric oxide-dependent modulation of the activity of iron regulatory proteins (IRP)-1 and 2, cytoplasmic proteins which, binding to RNA motifs called iron responsive elements (IRE), control ferritin translation. Stimulation with cytokines caused a small increase of IRP-1 activity and a strong reduction of IRP-2 activity accompanied by increased ferritin synthesis and accumulation. Cytokines induced only a minor increase of H chain ferritin mRNA, thus indicating that IRP-2–mediated posttranscriptional regulation plays a major role in the control of ferritin expression. This was confirmed by direct demonstration that the translational repression function of IRP was impaired in stimulated cells. In fact, translation in cell-free extracts of a reporter transcript under the control of an IRE sequence was repressed less efficiently by IRP-containing lysates from cytokine-treated cells than by lysates from control cells. Our findings throw light on the role of IRP-2 showing that: (1) this protein responds to a stimulus in opposite fashion to IRP-1; (2) when abundantly expressed, as in J774 cells, IRP-2 is sufficient to regulate intracellular iron metabolism in living cells; and (3) by allowing increased ferritin synthesis, IRP-2 may play a role in the regulation of iron homeostasis in RE cells during inflammation.
Expression of the Wilms' Tumor Suppressor Gene, WT1, Is Upregulated by Leukemia Inhibitory Factor and Induces Monocytic Differentiation in M1 Leukemic Cells
Authors: Smith Shirley I. , Weil Dominique , Johnson Gregory R. , Boyd Andrew W. , Li Chung L.
Published: October 2019

The Wilms' tumor gene, WT1, encodes a transcription factor of the Cys2-His2 zinc finger type. The functional significance of WT1 expression in leukemias, in addition to tissues and cell lines of hematopoietic origin, has not been determined. Using the murine myeloblastic leukemia cell line M1 as a model for macrophage differentiation, expression of WT1 is shown to be activated in M1 cells 24 hours after differentiation induction by leukemia inhibitory factor (LIF). Upregulation ofWT1 in these cells is associated with cellular differentiation, coinciding with expression of the monocyte/macrophage marker c-fms, and the appearance of mature cells. WT1 isoforms lacking the KTS insert are unable to be ectopically expressed in M1 cells. Stable expression of the WT1 isoforms containing the KTS insert leads to spontaneous differentiation of the M1 myeloblasts through the monocytic differentiation pathway. These cells express c-fms,in addition to the myeloid-specific cell surface marker Mac-1. Exposure of these cells to LIF results in the rapid onset of terminal macrophage differentiation, accompanied by apoptotic cell death. These results show that the WT1 gene is an important regulator of M1 cell monocytic differentiation in vitro, and suggests a potential role for this gene in the molecular control of hematopoiesis.
Stimulating Cell Proliferation Through the Pharmacologic Activation of c-kit
Authors: Jin Liqing , Asano Haruhiko , Anthony Blau C.
Published: October 2019

Previous studies have shown that expression of a membrane targeted chimeric protein containing the erythropoietin receptor (EpoR) cytoplasmic domain fused to the FK506-binding peptide FKBP12 allowed Ba/F3 cells to be rescued from interleukin-3 (IL-3) deprivation using a dimeric form of FK506, called FK1012. In this report, a similar approach is applied to the c-kit receptor. Expression of a membrane targeted fusion protein containing the c-kit receptor linked to one or more copies of FKBP12 allowed Ba/F3 cells to be switched from IL-3 dependence to FK1012-dependence. Similar results were obtained using an alternative dimerizer of FKBP12 domains called AP1510. Pharmacologic dimerization of chimeric proteins containing only a single FKBP12 domain confirmed that receptor dimerization is sufficient for proliferative signaling. Interestingly, while the proliferative effects of both FK1012 and AP1510 were reversible, FK1012-driven proliferation persisted for several days after drug withdrawal. Furthermore, much higher concentrations of FK506 were required to inhibit FK1012-mediated proliferation than were required to inhibit AP1510-mediated proliferation. The persistence of FK1012's effect appeared to be specific to clones expressing c-kit–containing fusion proteins. These results suggest that pharmacologically-responsive fusion proteins containing c-kitmay be useful for specifically and reversibly expanding genetically modified hematopoietic cell populations.
Dendritic Cells Stimulate the Expansion of bcr-abl Specific CD8+ T Cells With Cytotoxic Activity Against Leukemic Cells From Patients With Chronic Myeloid Leukemia
Authors: Nieda Mie , Nicol Andrew , Kikuchi Akiko , Kashiwase Koichi , Taylor Kerry , Suzuki Kenji , Tadokoro Kenji , Juji Takeo
Published: October 2019

The role of T lymphocytes in the control of chronic myeloid leukemia (CML) after bone marrow transplantations has been clearly shown. This effect closely correlates with graft-versus-host disease (GVHD). A specific graft-versus-leukemia (GVL) effect separate from GVHD has been postulated but has been difficult to show. One possible target for specific GVL activity is the bcr-abl fusion protein characteristic of CML. We have investigated the use of normal peptide-pulsed dendritic cells for the generation of cytotoxic, bcr-abl–specific T cells from normal donors. T cells (CD3+, CD8+, TCRαβ+, and NK receptor-negative) generated from a normal donor (HLA A24, B52, B59, Cw1) after stimulation with autologous dendritic cells, primed with a 16 mer peptide spanning the b3a2 breakpoint of bcr-abl, lysed CML cells from the peripheral blood of seven patients with CML with the b3a2 breakpoint. CML cells from four patients with only the b2a2 breakpoint were not lysed. Phytohemagglutinin (PHA) blasts derived from peripheral blood of patients with CML were not lysed, suggesting that cytotoxicity was not due to alloreactivity. Blocking experiments with anti–HLA-A,B,C indicated that cytotoxicity was dependent on recognition of major histocompatibility complex (MHC) class I molecules, although cytotoxicity was not MHC-restricted because not all patients shared HLA types with the T-cell donor. Specificity for bcr-abl and absence of alloreactivity was confirmed by the presence of lytic activity against autologous and allogeneic class I HLA-A matched monocytes pulsed with the 16 mer bcr-abl fusion peptide, but not against unpulsed monocytes or monocytes pulsed with other peptides. These results show that bcr-abl–specific T cells with marked cytotoxic activity against CML cells can be generated and amplified from normal donor peripheral blood. Recognition of HLA molecules is essential for cytotoxicity but strict HLA identity is not required.
Elevated Expression of the Apoptotic Regulator Mcl-1 at the Time of Leukemic Relapse
Authors: Kaufmann Scott H. , Karp Judith E. , Svingen Phyllis A. , Krajewski Stan , Burke Philip J. , Gore Steven D. , Reed John C.
Published: October 2019

Bcl-2, Bcl-xL, and Mcl-1 are three related intracellular polypeptides that have been implicated as negative regulators of apoptosis. In contrast, the partner protein Bax acts as a positive regulator of apoptosis. Based on the observation that all four of these polypeptides are expressed in a variety of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) cell lines, cellular levels of these polypeptides were examined by immunoblotting in bone marrow samples harvested from 123 adult AML patients and 36 adult ALL patients before initial antileukemic therapy. Levels of Bcl-2, Mcl-1, Bcl-xL, and Bax each varied over a more than 10-fold range in different pretreatment leukemia specimens. When the 54 AML and 23 ALL samples that contained greater than 80% malignant cells were examined in greater detail, it was observed that pretreatment levels of Bcl-2 and Mcl-1 correlated with each other (R = .44,P < .001 for AML and R = .79,P < .0001 for ALL). In addition, a weak negative correlation between Bax expression and age was observed in AML samples (R = −0.35, P < .02) but not ALL samples. There was no relationship between pretreatment levels of these polypeptides and response to initial therapy. However, examination of 19 paired samples (the first harvested before chemotherapy and the second harvested 23 to 290 days later at the time of leukemic recurrence) revealed a greater than or equal to twofold increase in Mcl-1 levels in 10 of 19 pairs (7 of 15 AML and 3 of 4 ALL) at recurrence. In contrast, 2 of 19 pairs contained twofold less Mcl-1 at the time of recurrence. Approximately equal numbers of samples showed twofold increases and decreases in Bcl-2 (5 increases, 3 decreases) and Bcl-xL (1 increase, 4 decreases) at recurrence. Bax levels did not show a twofold decrease in any patient. These results, coupled with recent observations that cells overexpressing Mcl-1 are resistant to a variety of chemotherapeutic agents, raise the possibility that some chemotherapeutic regimens might select for leukemia cells with elevated levels of this particular apoptosis inhibitor.
Tec and Jak2 Kinases Cooperate to Mediate Cytokine-Driven Activation of c-fos Transcription
Authors: Yamashita Yoshihiro , Watanabe Sumiko , Miyazato Akira , Ohya Ken-ichi , Ikeda Uichi , Shimada Kazuyuki , Komatsu Norio , Hatake Kiyohiko , Miura Yasusada , Ozawa Keiya , Mano Hiroyuki
Published: October 2019

Although transcriptional activation of the c-fosproto-oncogene plays an intrinsic role in the mechanism of blood cell growth, it is still obscure how protein-tyrosine kinases (PTKs) regulate the cytokine-driven c-fos activation pathway. We present here that Tec PTK is tyrosine-phosphorylated and activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in a human GM-CSF–dependent cell line. Moreover, we could show that introduction of Tec into mouse BA/F3-hGMRαβ cells can profoundly activate the c-fos promoter in response to GM-CSF or to interleukin-3 (IL-3). In contrast, introduction of a kinase-deleted Tec could suppress cytokine-driven c-fos activation, indicating that Tec is directly involved in the regulation of c-fos transcription. Interestingly, strong activation by Tec of the c-fos promoter was blocked by the co-expression of dominant negative Jak2. The molecular interaction between Tec and Jak2 was then investigated both in mammalian and insect cell systems, revealing that they can not only bind to each other, but either of the two can phosphorylate the other. Thus, Tec and Jak2 can “cross-talk” in a complexed way to mediate cytokine-driven c-fos activation.
Glycoprotein IIb Leu214Pro Mutation Produces Glanzmann Thrombasthenia With Both Quantitative and Qualitative Abnormalities in GPIIb/IIIa
Authors: Grimaldi Christine M. , Chen Fangping , Wu Changhong , Weiss Harvey J. , Coller Barry S. , French Deborah L.
Published: October 2019

Glanzmann thrombasthenia is an inherited bleeding disorder due to a functional reduction or absence of platelet GPIIb/IIIa (αIIbβ3) integrin receptors. Based on a prolonged bleeding time and absence of platelet aggregation in response to physiologic agonists, a 55-year-old white man was diagnosed as having Glanzmann thrombasthenia. The patient's platelet fibrinogen level was ≈5% of normal. As judged by complex-dependent monoclonal antibody (MoAb) binding, surface expression of platelet GPIIb/IIIa receptors was less than 5.5% of normal, whereas the binding of an anti-GPIIIa specific MoAb (7H2) was ≈12% of normal. Immunoblot analysis of the patient's platelet lysates showed ≈35% of normal levels of GPIIIa, ≈30% of normal levels of GPIIb, and an abnormally migrating fragment of GPIIb. Biotinylation of the surface proteins on the patient's platelets followed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed only GPIIb and GPIIIa subunits of normal size. Surface expression of platelet αvβ3 receptors was 192% of normal, suggesting that the patient's' defect was in GPIIb. Sequence analysis of the patient's GPIIb cDNA identified a T to C transition at nucleotide 643, predicting a Leu214Pro substitution. Direct sequencing of GPIIb exon 6 indicated that the patient is homozygous for the mutation. The nature of the Leu214Pro mutation was analyzed by expression in Chinese hamster ovary (CHO) cells. As judged by subunit-specific MoAb binding, surface expression of mutant receptors was ≈60% of normal, but these receptors were not recognized by the complex-dependent monoclonal antibodies, 10E5 and 7E3. In addition, mutant receptors pretreated with the ligand-induced binding site MoAb AP5 were not recognized by the activation-dependent MoAb PAC-1 and mutant expressing CHO cells did not adhere to immobilized fibrinogen. These data suggest that the Leu214Pro mutation in GPIIb disrupts the structural conformation, and either directly or indirectly, the ligand binding properties of the heterodimeric complex. This is in accord with studies from other integrins that have implicated a β-turn in a homologous region as important in ligand binding. Thus, the Leu214Pro mutation appears to produce the Glanzmann thrombasthenia phenotype by both qualitative and quantitative abnormalities. In addition, the mutation appears to confer susceptibility of the GPIIb subunit to proteolysis.
Prolonged Bleeding Time With Defective Platelet Filopodia Formation in the Wistar Furth Rat
Authors: Stenberg Paula E. , Barrie Rosemary J. , Pestina Tamara I. , Steward Shirley A. , Arnold Julie T. , Murti Aparna K. , Hutson Nancy K. , Jackson Carl W.
Published: October 2019

Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine–coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF rats.
Apoptosis of Malignant Human B Cells by Ligation of CD20 With Monoclonal Antibodies
Authors: Shan Daming , Ledbetter Jeffrey A. , Press Oliver W.
Published: October 2019

CD20 is a nonglycosylated 33 to 37 kD phosphoprotein involved in B-cell signaling that subserves important functions in the regulation of B-cell proliferation and differentiation. In addition, this B-cell surface antigen has been shown recently to be an effective target for immunotherapy of B-cell malignancies using chimeric (mouse/human) or radiolabeled murine monoclonal anti-CD20 antibodies. In this report we show that extensive crosslinking of CD20 with murine anti-CD20 monoclonal antibodies (MoAbs) in the presence of either goat anti-mouse IgG or Fc receptor (FcR)-expressing cells directly inhibits B-cell proliferation, induces nuclear DNA fragmentation, and leads to cell death by apoptosis. The apoptotic effects of these MoAbs can be inhibited by chelation of extracellular or intracellular Ca2+ by EGTA or Bapta AM, indicating that anti-CD20–mediated apoptosis may be related to changes in Ca2+ concentration. These findings suggest that ligation of CD20 in vivo by anti-CD20 antibodies in the presence of FcR-expressing cells may initiate signal transduction events that induce elevation of [Ca2+]i and lead to apoptosis of malignant B cells, thereby contributing to the impressive tumor regressions observed in mouse models and clinical trials using anti-CD20 MoAbs.