bluuebunny/crossref_metadata_2025 huggingface dataset:
p16/INK4a and p15/INK4b Gene Methylation and Absence of p16/INK4a mRNA and Protein Expression in Burkitt's Lymphoma
Authors: Klangby Ulf , Okan Ismail , Magnusson Kristinn P. , Wendland Martin , Lind Peter , Wiman Klas G.
Published: October 2019

The fact that the p16/INK4a and p15/INK4b genes are frequently inactivated in human malignancies and that p16/INK4a null mice spontaneously develop B-cell lymphomas prompted us to examine the status of both genes in Burkitt's Lymphoma (BL). We found a low frequency of p16/INK4a and p15/INK4b deletions and mutations in BL cell lines and biopsies. However, p16/INK4a exon 1 was methylated in 17 out of 19 BL lines (89.5%) and in 8 out of 19 BL biopsies (42%) analyzed. p15/INK4b Exon 1 was also methylated, although at a lower frequency. p16/INK4a mRNA was readily detected in BL lines carrying unmethylated p16/INK4a, but not in those carrying methylated p16/INK4a. No p16/INK4a protein was detected in any of the BL lines and biopsies examined. In contrast, only one out of seven lymphoblastoid cell lines (LCLs) examined was methylated in p16/INK4a exon 1, and three out of the six LCLs with unmethylated p16/INK4a expressed detectable levels of p16/INK4a protein. Thus, the frequent p16/INK4a methylation in BL lines correlates with downregulation of p16/INK4a expression, suggesting that exon 1 methylation is responsible for silencing the p16/INK4a gene in BL.
Differential Induction of Apoptosis by Fludarabine Monophosphate in Leukemic B and Normal T Cells in Chronic Lymphocytic Leukemia
Authors: Consoli Ugo , El-Tounsi Iman , Sandoval Alex , Snell Virginia , Kleine Hans-Dieter , Brown Wendy , Robinson Johnnie R. , DiRaimondo Francesco , Plunkett William , Andreeff Michael
Published: October 2019

Fludarabine (F-ara-A), an adenine nucleoside analog with efficacy in B-cell chronic lymphocytic leukemia (B-CLL), has also been shown to have a long-lasting suppressive effect on T lymphocytes. In heterogeneous clinical samples, apoptosis cannot be detected by standard methods in small cellular subsets. We developed, therefore, a combined assay of in situ end-labeling of nicked DNA by terminal deoxynucleotide transferase, with measurements of cellular DNA content and surface antigens (CD3, CD4, CD8, and CD19) by multiparametric flow cytometry. This assay was used to determine F-ara-A–induced apoptosis in different lymphocyte subsets from CLL patients and normal controls treated with F-ara-A in vitro. Apoptosis was also correlated to bcl-2 protein levels. We observed a direct effect of F-ara-A on both B-CLL and T lymphocytes. The response to F-ara-A in B-CLL lymphocytes in vitro was Rai stage–dependent, the early-stages being more responsive (P = .01). Higher levels of spontaneous apoptosis were observed in B-CLL lymphocytes from early stage patients (P = .02). No difference was observed in spontaneous apoptosis of normal T cells in B-CLL, although T lymphocytes in late-stage disease were more sensitive to F-ara-A–induced apoptosis. Incubation with cyclosporin A did not affect B-CLL and T-lymphocyte survival compared with control cultures. Results suggested a direct apoptotic effect of F-ara-A on B-CLL lymphocytes that decreases with increasing clinical stage. No correlation was found between bcl-2 and spontaneous or F-ara-A–induced apoptosis. Apoptosis occurred at all cell-cycle stages and was not restricted to cells in S phase. The mechanisms of this stage-dependent apoptosis in CLL remain to be elucidated.
Molecular Cloning of Translocation t(1;14)(q21;q32) Defines a Novel Gene (BCL9) at Chromosome 1q21
Authors: Willis T.G. , Zalcberg I.R. , Coignet L.J.A. , Wlodarska I. , Stul M. , Jadayel D.M. , Bastard C. , Treleaven J.G. , Catovsky D. , Silva M.L.M. , Dyer M.J.S.
Published: October 2019

Abnormalities of chromosome 1q21 are common in B-cell malignancies and have been associated with a poor response to therapy. The nature of the involved gene(s) on chromosome 1q21 remains unknown. A cell line (CEMO-1) has recently been established from a patient with precursor-B–cell acute lymphoblastic leukemia (ALL), which exhibited a t(1;14)(q21;q32). To identify the gene involved in this translocation, we have cloned both rearranged IGHJ alleles using long-distance inverse polymerase chain reaction (LDI-PCR). TwoIGHJ fragments were amplified from CEMO-1 DNA and sequenced. One allele showed novel sequences upstream of JH5 with no homology to either IGH or any other sequences on the databases. Using a single-copy Xho I fragment immediately 5′ ofJH5, PAC clones were isolated and mapped to chromosome 1q21 on normal metaphases by fluorescence in situ hybridization (FISH), confirming that this allele represented the t(1;14)(q21;q32) breakpoint. Sequence analysis of the 1q21 XhoI fragment showed identity with an expressed sequence tag (EST), and this probe was therefore used to probe Northern blots. Two transcripts of 6.3 kb and 4.2 kb expressed at low level in mRNA from all tissues were detected: a third transcript of 1.6 kb was expressed only in thymus, spleen, and small intestine. Full-lengthBCL9 cDNA clones were obtained from a normal human fetal brain cDNA library supplemented by 5′ and 3′ RACE. Sequence analysis predicted a protein of 1394 amino acids containing 18% proline, 11% glycine, 11% serine, and 6% methionine, but no recognizable protein motifs or significant homologies to any other known proteins. The CEMO-1 1q21 breakpoint fell within the 3′ UTR of the BCL9 gene. Low-level expression of BCL9 was detected in Epstein-Barr virus-transformed normal B cells by Northern blot; in contrast, abundant BCL9 expression was observed in CEMO-1, indicating that deregulated expression of this gene was one pathological consequence of the translocation. Screening of a panel of 39 B-cell malignancies with 1q abnormalities by Southern blot showed one additional case with a breakpoint in the 3′ UTR ofBCL9, indicating that this was a recurrent breakpoint. FISH analysis using an 850-kb YAC spanning BCL9 identified a further case with t(1;22)(q21;q11) causing juxtaposition of BCL9 to theIGλ locus. Other breakpoints were heterogeneous, falling both centromeric (10 cases) and telomeric (10 cases) of the BCL9gene. These data suggest that BCL9 may be the target of translocation in some B-cell malignancies with abnormalities of 1q21 and that deregulated BCL9 expression may be important in their pathogenesis.
The Chemotactic Cytokine Eotaxin Acts as a Granulocyte-Macrophage Colony-Stimulating Factor During Lung Inflammation
Authors: Peled Amnon , Gonzalo Jose Angel , Lloyd Clare , Gutierrez-Ramos Jose-Carlos
Published: October 2019

During inflammatory processes, inflamed tissues signal the bone marrow (BM) to produce more mature leukocytes in ways that are not yet understood. We report here that, during the development of lung allergic inflammation, the administration of neutralizing antibodies to the chemotactic cytokine, Eotaxin, prevented the increase in the number of myeloid progenitors produced in the BM, therefore reducing the output of mature myeloid cells from BM. Conversely, the in vivo administration of Eotaxin increased the number of myeloid progenitors present in the BM. Furthermore, we found that, in vitro, Eotaxin is a colony-stimulating factor for granulocytes and macrophages. Eotaxin activity synergized with stem cell factor but not with interleukin-3 or granulocyte-macrophage colony-stimulating factor and was inhibited bypertussis toxin. We report also that CCR-3, the receptor for Eotaxin, was expressed by hematopoietic progenitors (HP). Thus, during inflammation, Eotaxin acts in a paracrine way to shift the differentiation of BM HP towards the myeloid lineage.
Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor
Authors: de Koning John P. , Soede-Bobok Amrita A. , Schelen Anita M. , Smith Louise , van Leeuwen Daphne , Santini Valeria , Burgering Boudewijn M.T. , Bos Johannes L. , Löwenberg Bob , Touw Ivo P.
Published: October 2019

The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF–mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF–containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF–mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF–driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.
Plasminogen Deficiency Differentially Affects Recruitment of Inflammatory Cell Populations in Mice
Authors: Ploplis Victoria A. , French Esther L. , Carmeliet Peter , Collen Desire , Plow Edward F.
Published: October 2019

It is widely held that the plasminogen (Plg) system plays a role in inflammation through plasmin-mediated directional cell migration. However, substantial evidence for its involvement in the inflammatory response has been obtained from indirect studies and lacks firm biological confirmation. To directly characterize plasminogen's involvement in the inflammatory response, we used thioglycollate to induce a peritoneal inflammatory reaction in Plg(+/+),Plg(+/−), and Plg(−/−) mice. At 6 hours poststimulation, neutrophil recruitment into the peritoneum was maximal and similar between Plg(+/+), Plg(+/−), andPlg(−/−) mice. In contrast, monocyte recruitment was significantly diminished after 24 hours poststimulation inPlg(−/−) mice relative to Plg(+/+) mice. Lymphocyte recruitment also was blunted. Blood monocyte levels in these mice indicated that diminished recruitment into the peritoneum was not the result of a diminished source of cells in the circulation. Macrophage phagocytic function was similar between Plg(+/+) and Plg(−/−) mice. This study establishes a direct involvement of plasminogen in monocyte recruitment during a representative inflammatory response.
Stromelysin-1 (MMP-3)–Independent Gelatinase Expression and Activation in Mice
Authors: Lijnen H.R. , Silence J. , Van Hoef B. , Collen D.
Published: October 2019

A potential physiological role of stromelysin-1 (MMP-3) in the expression or activation of gelatinase A (MMP-2) or gelatinase B (MMP-9) in the wall of injured arteries was studied with the use of homozygous MMP-3–deficient (MMP-3−/−) mice. One week after perivascular electric injury of the carotid or femoral artery in wild-type (MMP-3+/+) or MMP-3−/− mice, 70 kD and 65 kD proMMP-2 levels were enhanced by twofold to fourfold, with corresponding increases of 20- to 40-fold for active 61 kD and 58 kD MMP-2, and of 10- to 80-fold for 94 kD proMMP-9. Active MMP-2 species represented approximately one third of the total MMP-2 concentration for both MMP-3+/+ and MMP-3−/− mice. Active 83 kD MMP-9 was not detected in noninjured carotid or femoral arteries, whereas one week after injury its contribution to the total MMP-9 level was 11% to 18% for MMP-3+/+ and MMP-3−/− mice. Immunostaining of arterial sections confirmed enhanced expression of both MMP-2 and MMP-9 after vascular injury. Double immunostaining showed colocalization of MMP-9 with macrophages in the adventitia, whereas MMP-2 was also detected mainly in the adventitia but failed to colocalize with smooth muscle cells. Cell culture experiments confirmed comparable ratios of active versus latent MMP-2 in skin fibroblasts and smooth muscle cells derived from MMP-3+/+ and MMP-3−/− mice. Addition of plasmin(ogen) did not significantly affect activation of proMMP-2. In MMP-3+/+ and MMP-3−/− macrophages, comparable levels of 94 kD proMMP-9 were detected, and plasmin(ogen)-mediated conversion to 83 kD MMP-9 was obtained in both genotypes. These data thus indicate that proMMP-2 activation may occur via a plasmin- and MMP-3–independent mechanism, whereas plasmin can directly activate proMMP-9 via a MMP-3–independent mechanism.
The Role of Transcription Factor PU.I in the Activity of the Intronic Enhancer of the Eosinophil-Derived Neurotoxin (RNS2) Gene
Authors: van Dijk Thamar B. , Caldenhoven Eric , Raaijmakers Jan A.M. , Lammers Jan-Willem J. , Koenderman Leo , de Groot Rolf P.
Published: October 2019

Eosinophil-derived neurotoxin (EDN) found in the granules of human eosinophils is a cationic ribonuclease toxin. Expression of the EDN gene (RNS2) in eosinophils is dependent on proximal promoter sequences in combination with an enhancer located in the first intron. We further define here the active region of the intron using transfections in differentiated eosinophilic HL60 cells. We show that a region containing a tandem PU.I binding site is important for intronic enhancer activity. This region binds multiple forms of transcription factor PU.I as judged by gel-shift analysis and DNA affinity precipitation. Importantly, introducing point mutations in the PU.I site drastically reduces the intronic enhancer activity, showing the importance of PU.I for expression of EDN in cells of the eosinophilic lineage.
Three Molecular Structures Cause Rhesus D Category VI Phenotypes With Distinct Immunohematologic Features
Authors: Wagner Franz F. , Gassner Christoph , Müller Thomas H. , Schönitzer Diether , Schunter Friedrich , Flegel Willy A.
Published: October 2019

Rhesus D category VI (DVI) is the clinically most important partial D. DVI red blood cells were assumed to possess very low RhD antigen density and to be caused by twoRHD-CE-D hybrid alleles. Because there was no population-based work-up, we screened three populations in central Europe for DVI. Twenty-six DVI samples were detected and examined by exon-specific RHD polymerase chain reaction with sequence-specific primers (PCR-SSP). A new genotype, hereby designated D category VI type III, was characterized as a RHD-Ce(3-6)-D hybrid allele by sequencing of the cDNA, parts of intron 1, and by PCR-restriction fragment length polymorphism (PCR-RFLP) of intron 2. Rhesus introns 5 and 6 were sequenced and the 3′ breakpoints of all knownDVItypes shown to be distinct. We differentiated the 5′ breakpoints of DVItypeI andDVItype II by a newly devised RHD-PCR. Thus, the DVI phenotype originated in at least three independent molecular events. Each DVI type showed distinct immunohematologic features in flow cytometry. The number of RhD proteins accessible on the red blood cells' surface ofDVItype III was normal (about 12,000 antigens/cell; DVItypeI, 500;DVItype II, 2,400) based on the determination of an RhD epitope density profile. DVItype II and DVItype III occurred as CDe haplotypes, and DVItype I as a cDE haplotype.The distribution of the DVItypes varied significantly in three German-speaking populations. Genotyping strategies should take account of allelic variations in partial RhD. The reconsideration of previous serologic and clinical data for partial D in view of the underlying molecular structures may be worthwhile.
Systemic Overexpression of BCL-2 in the Hematopoietic System Protects Transgenic Mice From the Consequences of Lethal Irradiation
Authors: Domen Jos , Gandy Kimberly L. , Weissman Irving L.
Published: October 2019

A new transgenic mouse has been generated in which the proto-oncogene BCL-2 is ubiquitously overexpressed. H2K-BCL-2 transgenic mice overexpress BCL-2 in all cells of the hematolymphoid system and have been used to assess the role of BCL-2 in protecting cells of the hematolymphoid system from the consequences of ionizing radiation. We have expanded on previous studies that have demonstrated protection for specific (lymphoid) cell populations and show that systemic overexpression of BCL-2 can protect the hematopoietic system as a whole, including hematopoietic stem cells (HSC), thus increasing the radioresistance of the animal. The increase in radioresistance in H2K-BCL-2 transgenic mice has two components: an increase in the radioresistance of individual cells and, to a lesser extent, an increase in the size of certain critically important cell populations, such as HSC. Bone marrow transplantation experiments show that the increased radioresistance of the transgenic animals is provided by cells of the hematopoietic system. Protection against the consequences of irradiation is not limited to the increased expression levels of BCL-2 in transgenic mice; levels of endogenous BCL-2 are higher in lymphocyte populations that survive irradiation in wild-type mice. We show that ubiquitous overexpression of BCL-2 in the hematopoietic system can be used to increase the resistance of animals to lethal challenges such as irradiation.
Human T-Cell Leukemia Virus Type II Directly Acts on CD34+ Hematopoietic Precursors by Increasing Their Survival Potential. Envelope-Associated HLA Class II Molecules Reverse This Effect
Authors: Casoli Claudio , Re Maria Carla , Monari Paola , Furlini Giuliano , Tosi Giovanna , Gradozzi Chiara , Bertazzoni Pier Paolo Dall'Aglio, Umberto , Accolla Roberto S.
Published: October 2019

The role of human T-cell leukemia virus type II (HTLV-II) in human lymphoproliferative and hematopoietic abnormalities in which the retrovirus can be isolated is still elusive. Here we show that the C344 T-cell–derived lymphotropic HTLV-II type IIa Mo strain acts directly on CD34+ hematopoietic precursors by rescuing them from apoptosis induced by interleukin-3 (IL-3) deprivation. This effect is viral strain-specific, as it is not observed with the B-lymphotropic HTLV-II type IIb Gu strain, it does not require infection of the hematopoietic precursors, and, interestingly, it is strongly dependent on the infected cellular host from which the virus was derived. Indeed, growth adaptation of the Mo strain to the permissive B-cell line, BJAB, renders the virus no longer capable of mediating the antiapoptotic effect. However, pretreatment of the BJAB-adapted Mo strain with antibodies specific for HLA class II, but not class I, histocompatibility antigens restores the antiapoptotic potential of the virus. These results constitute the first evidence that HTLV-II retrovirus can directly influence the homeostasis of human progenitors, without infecting them, and that this crucial activity is strongly inhibited by the presence of host-derived envelope-associated HLA class II antigens.
Multivesicular Bodies Are an Intermediate Stage in the Formation of Platelet α-Granules
Authors: Heijnen Harry F.G. , Debili Najet , Vainchencker William , Breton-Gorius Janine , Geuze Hans J. , Sixma Jan J.
Published: October 2019

We have used ultrathin cryosectioning and immunogold cytochemistry to study the position of α-granules in the endocytic and biosynthetic pathways in megakaryocytes and platelets. Morphologically, we distinguished three types of granules; so-called multivesicular bodies type I (MVB I) with internal vesicles only, granules with internal vesicles and an electron dense matrix (MVB II), and the α-granules with mainly a dense content and often internal membrane vesicles at their periphery. The MVBs were prominent in cultured megakaryocytes and the megakaryoblastic cell line CHRF-288, but were less numerous in bone marrow megakaryocytes and platelets, whereas α-granules were most prominent in mature bone marrow megakaryocytes and in platelets. The internalization kinetics of bovine serum albumin-gold particles and of fibrinogen positioned the MVB subtypes and α-granules sequentially in the endocytic pathway. MVBs contained the secretory proteins von Willebrand factor (vWF) and β-thromboglobulin (β-TG), the platelet-specific membrane protein P-selectin, and the lysosomal membrane protein CD63. Within the MVBs, endocytosed fibrinogen and endogenous β-TG were restricted to the matrix, while vWF was predominantly associated with internal vesicles. CD63 was also observed in association with internal membrane vesicles in the α-granules. These observations, and the gradual morphologic transition from granules containing vesicles to granules containing predominantly dense material, suggest that MVBs represent a developmental stage in α-granule maturation.
Expression of Functional CD32 Molecules on Human NK Cells Is Determined by an Allelic Polymorphism of the FcγRIIC Gene
Authors: Metes Diana , Ernst Linda K. , Chambers William H. , Sulica Andrei , Herberman Ronald B. , Morel Penelope A.
Published: October 2019

Human natural killer (NK) cells were thought to express only FcγRIIIA (CD16), but recent reports have indicated that NK cells also express a second type of FcγR, ie, FcγRII (CD32). We have isolated, cloned, and sequenced full-length cDNAs of FcγRII from NK cells derived from several normal individuals that may represent four different products of the FcγRIIC gene. One transcript (IIc1) is identical with the already described FcγRIIc form. The other three (IIc2-IIc4) appear to represent unique, alternatively spliced products of the same gene, and include a possible soluble form. Analyses of the full-length clones have revealed an allelic polymorphism in the first extracellular exon, resulting in either a functional open reading frame isoform or a null allele. Stable transfection experiments enabled us to determine a unique binding pattern of anti-CD32 monoclonal antibodies to FcγRIIc. Further analyses of NK-cell preparations revealed heterogeneity in CD32 expression, ranging from donors lacking CD32 expression to donors expressing high levels of CD32 that were capable of triggering cytotoxicity. Differences in expression were correlated with the presence or absence of null alleles. These data show that certain individuals express high levels of functional FcγRIIc isoforms on their NK cells.
c-kit Is Expressed in Soft Tissue Sarcoma of Neuroectodermic Origin and Its Ligand Prevents Apoptosis of Neoplastic Cells
Authors: Ricotti Emanuela , Fagioli Franca , Garelli Emanuela , Linari Claudia , Crescenzio Nicoletta , Horenstein Alberto L. , Pistamiglio Paola , Vai Sergio , Berger Massimo , diMontezemolo Luca Cordero , Madon Enrico , Basso Giuseppe
Published: October 2019

During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Consequently, accumulating evidence suggests that the c-kit/SCF system plays a crucial role in all of these processes and in tumors which derive from them. Especially in neuroblastoma (infant tumors of neuroectoderm crest derivation such as melanocytes) it would appear that an autocrine loop exists between c-kit and SCF, and that the functional block of the c-kit receptors with monoclonal antibodies (MoAbs) results in a significant decrease in cellular proliferation. We studied the expression and role of c-kit and SCF in cell lines of soft tissue sarcoma of neuroectodermic origin, such as Ewing's sarcoma (ES) and peripheral neuro-ectodermal tumors (PNET). Using flow cytometry with MoAb CD117 PE, c-kit expression was highlighted in all six of the cell lines examined. This receptor was specifically and functionally activated by SCF, as shown by the binding experiments and the intracellular phosphotyrosine and immunoprecipitation studies that were performed. Using reverse transcriptase polymerase chain reaction analysis, five of the six cellular lines expressed the mRNA of SCF. In the medium measured by using an enzyme- linked immunosorbent assay, low concentrations of SCF were found: only the TC32 cellular line produced significantly higher levels (32 pg) than control. In serum-free culture the addition of SCF reduced the percentage of apoptotic cells from 25% to 90% in five out of the six cellular lines. This observation was confirmed by (1) the functional block of c-kit with MoAb: after 7 days of culture more than 30% of the cells were apoptotic (range 31.5% to 100%) in five out of six cell lines and there was also a decrease in the percentage of cells in phase S, and (2) c-kitantisense oligonucleotides: in the cellular lines treated with oligonucleotides (in relation to the untreated lines) there was a notable reduction (P < .001) both in the absolute number of cells and the 3H-thymidine uptake. These results indicate that ES and PNET express c-kit and its ligand SCF and that SCF is capable of protecting the tumor cells against apoptosis. Furthermore, the reverse transcriptase–polymerase chain reaction performed on the biopsies revealed the presence of mRNA both of SCF and c-kit in practically all of the samples studied. Our in vitro data lead us to assume that SCF may also inhibit tumor cell apoptosis in vivo.
Evidence for the Involvement of Both Retinoic Acid Receptor- and Retinoic X Receptor-Dependent Signaling Pathways in the Induction of Tissue Transglutaminase and Apoptosis in the Human Myeloma Cell Line RPMI 8226
Authors: Joseph Bertrand , Lefebvre Olga , Méreau-Richard Claude , Danzé Pierre-Marie , Belin-Plancot Marie-Thérèse , Formstecher Pierre
Published: October 2019

In this study, we show that both all-trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA) are potent inducers of tissue transglutaminase (TGase II), an enzyme involved in apoptosis, at the level of both enzyme activity and mRNA in the human myeloma cell line RPMI 8226. RPMI 8226 cells were shown to express mRNAs for all the retinoid receptors subtypes, ie, RARα, RARβ, RARγ, RXRα, RXRβ, and RXRγ. To identify which of these receptors are involved in regulating TGase II expression, several receptor-selective synthetic retinoids were used. Neither CD367, a very potent retinoid that selectively binds and activates receptors of the RAR family, nor CD2425, an RXR-selective agonist, induced TGase II when used alone. However, combination of CD367 and CD2425 resulted in nearly full induction of the enzyme. Moreover, when used in combination with atRA, CD367 partially inhibited the atRA-dependent induction of TGase II, whereas CD2425 enhanced it. The effects of Am 580, CD417, and CD437, three synthetic retinoids selective for the RARs subtypes RARα, RARβ, and RARγ, respectively, were also investigated. None of these compounds was able to induce TGase II when used alone; however, the combination of each of them with CD2425 resulted in strong induction of the enzyme activity, reaching 30% to 50% of the values obtained in the presence of retinoic acid and suggesting functional redundancy between the RAR subtypes. Finally, treatment with atRA or the combination of CD367 and CD2425, but not with CD367 or CD2425 alone, was also shown to trigger apoptosis in RPMI 8226 cells, with prominent accumulation of TGase II immunoreactivity in apoptotic cells. Taken together these data suggest that the induction of TGase II expression and apoptosis in the RPMI 8226 myeloma cell line required ligand-dependent activation of both the RAR and RXR receptors.
Enhanced MDR1 Gene Expression in Human T-Cell Leukemia Virus-I–Infected Patients Offers New Prospects for Therapy
Authors: Lau Alan , Nightingale Simon , Taylor Graham P. , Gant Timothy W. , Cann Alan J.
Published: October 2019

Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. This phenotype is reminiscent of human T-cell leukemia virus (HTLV)-transformed leukemic cells, for which no consistently effective chemotherapeutic regime has been found. The presence of an active multiple drug resistance (MDR) phenotype in freshly isolated peripheral blood mononuclear cells (PBMC) from HTLV-I–infected subjects was investigated. Significant P-gp–mediated efflux activity and enhanced MDR1 mRNA expression was observed in nine of 10 HTLV-infected subjects. The development of MDR phenotypes was found to be independent of disease type or status with significant MDR activities being observed in adult T-cell leukemia (ATL), HTLV-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and asymptomatic HTLV-infected individuals. P-gp–mediated drug efflux was also found to be restricted to CD3+ T-cell populations. Furthermore, we show the novel finding that theMDR1 gene promoter is transcriptionally activated by the HTLV-I tax protein, suggesting a molecular basis for the development of drug resistance in HTLV-I infections. These observations open up the possibility of new chemotherapeutic approaches to HTLV-associated diseases through the use of P-gp inhibitors.
Mechanisms of Growth Control of Kaposi's Sarcoma–Associated Herpes Virus–Associated Primary Effusion Lymphoma Cells
Authors: Asou Hiroya , Said Jonathan W. , Yang Rong , Munker Reinhold , Park Dorothy J. , Kamada Nanao , Koeffler H. Phillip
Published: October 2019

Primary effusion lymphoma (PEL) is a distinct clinicopathologic entity associated with Kaposi's sarcoma-associated herpes virus (KSHV). Several cytokines, including interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) may be important for survival of KS cells. However, little is known about the interaction of cytokines with KSHV-infected lymphocytes from PEL. Therefore, we investigated what cytokines were produced by KSHV-infected PEL cell lines (KS-1, BC-1, BC-2), what cytokine receptors were expressed by these cells, what response these cells had to selected cytokines, and what was the effect of IL-6 antisense phosphorothioated oligonucleotides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and protein studies showed that these three cell lines produced IL-10, IL-6, and the receptors for IL-6. The granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-8, IL-12, bFGF, PDGF, and c-kit transcripts were not detected in the cell lines. High levels (0.7 to 5 ng/mL/106cells/48 hours) of IL-6 protein were consistently detected in supernatants of the cell lines by enzyme-linked immunosorbent assay (ELISA) tests. In clonogenic assays, interferon-α (IFN-α) and IFN-γ suppressed the clonal growth of the PEL cells, but GM-CSF, IL-4, IL-6, IL-8, IL-10, and oncostatin M did not change it. We examined for several autocrine loops that have been suggested to occur in KS. Experiments using antisense oligonucleotides showed that the clonal growth of KS-1 and BC-1 was nearly 100% inhibited by IL-6 antisense oligonucleotides (10 μmol/L), but not at all by either oligonucleotides (≤10 μmol/L) to IL-6 sense, IL-6 scrambled, viral IL-6 (vIL-6) antisense, or IL-10 antisense. Furthermore, the IL-6 antisense oligonucleotides had no effect on two B-cell lymphoma cell lines, which were not infected with KSHV. Addition of IL-6 antibody did not inhibit clonal growth of any of the cell lines. Taken together, we have defined the cytokines and their receptors expressed on PEL cells and have found that these cells synthesized IL-6 and IL-6 receptors; interruption of this pathway by IL-6 antisense oligonucleotides specifically prevented the growth of these cells. These findings will offer potential new therapeutic strategies for PEL.
The Fcγ Receptor–Mediated Respiratory Burst of Rolling Neutrophils to Cytokine-Activated, Immune Complex-Bearing Endothelial Cells Depends on L-Selectin But Not on E-Selectin
Authors: Fröhlich Dieter , Spertini Olivier , Moser René
Published: October 2019

Intracellular H2O2 generation, as a measure of the respiratory burst, was determined after stimulation of neutrophils by immune complex (IC)-bearing human umbilical vein endothelial cells. Under static conditions, neutrophils basically responded to the immune deposits on resting endothelial cells. The rotating shear forces of ≈0.7 dynes/cm2, corresponding to the physiological flow in postcapillary venules, completely abolished this basal H2O2 generation. After activation of the IC-bearing endothelial layers with interleukin-1 (IL-1) or tumor necrosis factor (TNF), or both, for 4 hours, rolling adhesion of the neutrophils was induced, accompanied by considerable H2O2 production. The neutrophil respiratory burst was prominently inhibited by anti-FcγRIII MoAb 3G8 (72.4%), and partially by MoAb 2E1 against FcγRII (38.5%). Both MoAbs together inhibited the Fc-mediated H2O2generation by 93.4%. The respiratory burst and rolling adhesion were markedly blocked by MoAb LAM1-3 against L-selectin (91.3%), whereas the nonfunctional anti-L-selectin MoAb LAM1-14 was ineffective. F(ab)2′ fragments of MoAb 7A9 against E-selectin inhibited neutrophil rolling by 98.6%, but not the respiratory burst. Moreover, rolling adhesion of neutrophils and the related oxidative burst were CD11b/CD18- independent. In summary, L-selectin has a unique auxiliary function in triggering the FcγR-mediated respiratory burst of rolling neutrophils to IC-bearing endothelial cells, thereby substituting CD11b/CD18 under conditions of flow.
Response of Monocyte Iron Regulatory Protein Activity to Inflammation: Abnormal Behavior in Genetic Hemochromatosis
Authors: Recalcati Stefania , Pometta Roberta , Levi Sonia , Conte Dario , Cairo Gaetano
Published: October 2019

In genetic hemochromatosis (GH), iron overload affects mainly parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells. We previously found that RE cells from GH patients had an inappropriately high activity of iron regulatory protein (IRP), the key regulator of intracellular iron homeostasis. Elevated IRP should reflect a reduction of the iron pool, possibly because of a failure to retain iron. A defect in iron handling by RE cells that results in a lack of feedback regulation of intestinal absorption might be the basic abnormality in GH. To further investigate the capacity of iron retention in RE cells of GH patients, we used inflammation as a model system as it is characterized by a block of iron release from macrophages. We analyzed the iron status of RE cells by assaying IRP activity and ferritin content after 4, 8, and 24 hours of incubation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). RNA-bandshift assays showed that in monocytes and macrophages from 16 control subjects, IRP activity was transiently elevated 4 hours after treatment with LPS and IFN-γ but remarkably downregulated thereafter. Treatment with NO donors produced the same effects whereas an inducible Nitric Oxide Synthase (iNOS) inhibitor prevented them, which suggests that the NO pathway was involved. Decreased IRP activity was also found in monocytes from eight patients with inflammation. Interestingly, no late decrease of IRP activity was detected in cytokine-treated RE cells from 12 GH patients. Ferritin content was increased 24 hours after treatment in monocytes from normal subjects but not in monocytes from GH patients. The lack of downregulation of IRP activity under inflammatory conditions seems to confirm that the control of iron release from RE cells is defective in GH.
Lymphoid Reconstitution After Autologous PBSC Transplantation With FACS-Sorted CD34+ Hematopoietic Progenitors
Authors: Bomberger Catherine , Singh-Jairam Meeta , Rodey Glenn , Guerriero Anastasia , Yeager Andrew M. , Fleming William H. , Holland H. Kent , Waller Edmund K.
Published: October 2019

T-cell and B-cell reconstitution was studied in nine patients who received fluorescence activated cell sorter (FACS)-sorted autologous CD34+ hematopoietic progenitor cells (HPC). The mean numbers of T cells (CD3+), B cells (CD19+) and CD34+ HPC administered to each patient were .004, .002, and 1.8 × 106 cells/kg, respectively. After high-dose myeloablative chemotherapy (busulfan, cyclophosphamide, etoposide) CD34+ HPC were infused and lymphoid reconstitution was monitored using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VDJ T-cell receptor (TcR) sequences. Restoration of normal numbers of peripheral blood T cells and B cells among recipients of FACS-sorted CD34+ HPC was delayed compared to recipients of non-T-cell–depleted PBSC autografts. In both patient groups, the circulating T cells were primarily CD4−, CD8+, αβ TcR+, and CD45RO+, CD45RA− during the first 2 months after transplant. Subsequent increases in the frequency of CD45RA+ CD45RO− T cells occurred at 2 to 3 months after transplant, suggesting maturation of CD34+hematopoietic progenitors to “naive” T cells. Analysis of the TcR repertoire after hematopoietic reconstitution demonstrated decreased diversity of Vβ TcR expression associated with global decreases in the absolute number of total peripheral blood T cells and most Vβ TcR+ subsets. Three of nine recipients of FACS-sorted CD34+ HPC demonstrated significant increases in the percentage of γδ+ peripheral T cells and CD5+ B cells at 3 to 9 weeks after transplantation, and all patients had transient oligoclonal expansions of T cells expressing specific Vβ TcR. Transplantation with highly purified CD34+ HPC results in reduced diversity of the peripheral T-cell repertoire during the early post-transplant period compared with patients receiving unmanipulated or MoAb-depleted transplants.
Differential Deactivation of Human Dendritic Cells by Endotoxin Desensitization: Role of Tumor Necrosis Factor-α and Prostaglandin E2
Authors: Rieser Claudia , Papesh Christine , Herold Manfred , Böck Günther , Ramoner Reinhold , Klocker Helmut , Bartsch Georg , Thurnher Martin
Published: October 2019

The endotoxin (lipopolysaccharide)-induced cytokine response is followed by a state of unresponsiveness to lipopolysaccharide (LPS) referred to as LPS tolerance or endotoxin desensitization. LPS tolerance, which can be experimentally induced in vitro and in vivo, is also known to occur in septic disease. Here, we evaluated whether dendritic cells (DC), the most potent antigen-presenting cells, are also subject to this phenomenon. Single doses of LPS added at the initiation of DC culture inhibited in a dose-dependent fashion the production of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and IL-12, but not the production of IL-8, in response to a second LPS challenge in day-5 DC. In addition, the LPS-induced expression of the CD83 maturation antigen was inhibited in these cells. Moreover, the endocytic activity of DC generated in the presence of LPS was dramatically reduced. DC desensitized with LPS were potent stimulators of T-cell proliferation but poor inducers of interferon-γ (IFN-γ) production in the allogeneic mixed leukocyte reaction. TNF-α and prostaglandin E2, two major products of LPS stimulation, could replace LPS for the induction of tolerance to LPS. Moreover, treatment of desensitized DC with TNF-α plus prostaglandin E2 fully restored CD83 expression and partially restored IL-12 production as well as the IFN-γ–inducing activity of DC in the mixed leukocyte reaction. Our data show that human DC are highly susceptible to the induction of LPS tolerance, which seems to be a state of differential deactivation in which some functions are impaired whereas others are retained. Tolerization at the level of the professional antigen-presenting cell by inflammatory mediators may play an important role in septic disease and in the origin of cancers associated with chronic inflammation.
Expression of a Knocked-In AML1-ETO Leukemia Gene Inhibits the Establishment of Normal Definitive Hematopoiesis and Directly Generates Dysplastic Hematopoietic Progenitors
Authors: Okuda Tsukasa , Cai Zhongling , Yang Shouli , Lenny Noel , Lyu Chuhl-joo , van Deursen Jan M.A. , Harada Hironori , Downing James R.
Published: October 2019

The t(8;21)-encoded AML1-ETO chimeric product is believed to be causally involved in up to 15% of acute myelogenous leukemias through an as yet unknown mechanism. To directly investigate the role of AML1-ETO in leukemogenesis, we used gene targeting to create anAML1-ETO “knock-in” allele that mimics the t(8;21). Unexpectedly, embryos heterozygous for AML1-ETO(AML1-ETO/+) died around E13.5 from a complete absence of normal fetal liver–derived definitive hematopoiesis and lethal hemorrhages. This phenotype was similar to that seen following homozygous disruption of either AML1 orCBFβ. However, in contrast to AML1- or CBFβ-deficient embryos, fetal livers from AML1-ETO/+ embryos contained dysplastic multilineage hematopoietic progenitors that had an abnormally high self-renewal capacity in vitro. To further document the role of AML1-ETO in these growth abnormalities, we used retroviral transduction to express AML1-ETO in murine adult bone marrow–derived hematopoietic progenitors. AML1-ETO–expressing cells were again found to have an increased self-renewal capacity and could be readily established into immortalized cell lines in vitro. Taken together, these studies suggest that AML1-ETO not only neutralizes the normal biologic activity of AML1 but also directly induces aberrant hematopoietic cell proliferation.
The P2X1 Receptor, an Adenosine Triphosphate–Gated Cation Channel, Is Expressed in Human Platelets but not in Human Blood Leukocytes
Authors: Clifford Erin E. , Parker Karen , Humphreys Benjamin D. , Kertesy Sylvia B. , Dubyak George R.
Published: October 2019

Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+influx/mobilization demonstrated that the platelet P2X1receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.
Interleukin-3–Induced Activation of the JAK/STAT Pathway Is Prolonged by Proteasome Inhibitors
Authors: Callus Bernard A. , Mathey-Prevot Bernard
Published: October 2019

One facet of cytokine receptor signaling involves the activation of signal transducers and activators of transcription (STATs). STATs are rapidly activated via tyrosine phosphorylation by Janus kinase (JAK) family members and subsequently inactivated within a short period. We investigated the effect of proteasome inhibition on interleukin-3 (IL-3) activation of the JAK/STAT pathway following stimulation of Ba/F3 cells. Treatment of Ba/F3 cells with the proteasome inhibitor,N-acetyl-l-leucinyl-l-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-3 receptor, beta common (βc), and STAT5 following stimulation. The effects of LLnL were not restricted to the JAK/STAT pathway, as Shc and mitogen-activated protein kinase (MAPK) phosphorylation were also prolonged in LLnL-treated cells. Further investigation showed these stable phosphorylation events were the result of prolonged activation of JAK2 and JAK1. These observations were confirmed using pharmacologic inhibitors. In the presence of LLnL, stable phosphorylation of STAT5 and βc was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on STAT5 phosphorylation could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting phosphorylated STAT5 could be stabilized by phosphatase, but not by proteasome inhibition per se. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the JAK/STAT pathway by regulating the deactivation of JAK.
Interactions of Erythropoietin, Granulocyte Colony-Stimulating Factor, Stem Cell Factor, and Interleukin-11 on Murine Hematopoiesis During Simultaneous Administration
Authors: Roeder I. , de Haan G. , Engel C. , Nijhof W. , Dontje B. , Loeffler M.
Published: October 2019

We investigated how in vivo effects of single hematopoietic cytokines change if given in combination for a prolonged time. Mice were treated with every combination of recombinant human (rh) erythropoietin (EPO), rh granulocyte colony-stimulating factor (G-CSF), recombinant rat (rr) stem cell factor (SCF), and rh interleukin (IL)-11 by continuous infusion over 7 days (full factorial design with three dose levels for each cytokine). Burst-forming unit–erythroid (BFU-E), colony-forming unit–erythroid (CFU-E), and colony-forming unit–granulocyte-macrophage (CFU-GM) were determined in bone marrow and spleen, reticulocytes, hematocrit, granulocytes, and thrombocytes in the peripheral blood. An analysis of variance (ANOVA) and multiple comparison of means was used to evaluate the data. For several cell types, cytokine effects superimposed in an additive way if combined. However, in a large number of circumstances, nonadditive pairwise interactions were found. They differed in type and magnitude involving high-dose saturation, high-dose antagonistic effects, and even effect reversals (qualitative interactions). Hence, in general, it was not possible to foresee the combination effects on the basis of existing knowledge of single effects. On the other hand, the cytokine network was robust and no system hazards were observed under multiple cytokine combinations. The results illustrate that the cytokine network has nonlinear dynamic properties in vivo with dose-response characteristics of one cytokine being continuously modified by other cytokines.