bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced Capacity of Monocytes from Patients with Essential Thrombocytemia to Generate Tissue Factor. Possible Mechanism of Thrombosis.
Authors: Kornberg Abraham , Koren-Michowitz Maya , Rahimi-Levene Naomi , Voltchek Yulia , Rapoport Micha 
Published: October 2019

Abstract
The main cause of thrombosis, a major complication of essential thrombocythemia (ET), is the high platelet count. In contrast, thrombosis is rare in reactive thormbocytosis (RT), indicating that thrombocytosis can not be the sole cause of thrombosis. Moreover, thrombosis occurs in ET even after reduction of the platelet count. Tissue factor (TF) is the most potent activator of coagulation. Monocytes are a main source of TF and monocyte TF is increased in various disorders including solid tumors and polycythemia vera that are associated with increased incidence of thrombosis. The decreased incidence of thrombosis that was reported recently in ET after treatment with hydroxyurea compared to anagralide was attributed to a decrease in leukocytes that occurs only after hydroxyurea. Based on this information, we studied the capacity of monocytes from patients with ET to generate TF. Peripheral blood mono-nuclear cells (PBMC) were isolated on Ficoll-hypague from 14 patients with ET and 10 with RT due to iron deficiency. Monocytes were counted by anti CD14 and were the same in both groups (18%). Monocytes were purified from lymphocytes by adherence to plastic surface. Cells were incubated for 16 hours with and without endotoxin.TF activity was measured in the cells by modified PT and TF antigen by ELISA. TF activity and antigen were 1.4–1.7x 10−5 U/monocyte and 21–31x10−5 pg/monocyte in unstimulated PBMC from normal controls (NC), RT and ET. Stimulated PBMC from NC and RT showed a similar increase of TF activity and antigen (2.5 and 3.8 fold).However, PBMC from ET generated 12.9 and 14.4 times more TF activity and antigen (p<.005). Similar increase was found in ET after reduction of platelet counts. Endotoxin-stimulated purified lymphocytes from four ET patients generated weak TF while purified monocytes generated TF levels similar to PBMC. Three ET patients compared to none of RT patients developed thrombosis. The increased capacity of monocytes to generate TF may be an additional mechanism of thrombus formation in ET and should be included in the list of hypercoagulability syndromes.
Down Regulation of Thrombin Procoagulant Activity by Prothrombin Activation Fragment 1.2.
Authors: Dasgupta Swapan K. , Thiagarajan Perumal 
Published: October 2019

Abstract
Prothrombin is the precursor of thrombin, the central enzyme in coagulation. Under physiological condition, prothrombin activation catalyzed by the fully assembled prothrombinase complex via an ordered, sequential reaction with primary cleavage at Arg320-lle321 followed by the second cleavage at Arg271-Thr272, forming fragment 1.2 and thrombin. The fragment 1.2 contains the amino-terminal gla domain and the two kringle domains. It has been shown that the kringle 2 domain in fragment 2 can bind to the exosite II of thrombin and inhibit allosterically its activity by inducing conformational changes at the active site. Nascent thrombin that is generated on platelet surface will remains non-covalenty bound to fragment 1.2 by kringle 2-exosite II interaction. To determine whether this interaction can modulate thrombin activity, we tested the effect of anionic phospholipid-bound fragment 1.2 (0–10 μM) on thrombin clotting activity. Phospholipid-bound fragment 1.2 inhibited fibrinogen clotting in a concentration-dependent manner but had no significant effect on esterase activity towards S2238 (D-Phe-Pip-Arg-pNA.2HCI) suggesting a competitive inhibition of fibrinogen binding. We also labeled thrombin at the active site with fluorescein-FPRCK (5-fluorescein-D-Phe-Pro-Arg chloromethyl-ketone) and monitored fluorescence changes following fragment 1.2 binding in a spectrofluorometer. Anionic phospholipid-bound fragment 1.2 induced changes in the active site similar to fragment 2 with half maximal effect at ~8 μM. We tested the effect of fragment 1.2 on fluorescein-FPR thrombin binding to platelets. Fragment 1.2 inhibited thrombin binding to platelet. Consistent with these findings fragment 1.2 inhibited thrombin-induced aggregation of gel filtered platelets in a concentration-dependant manner. These results suggest that membrane-bound prothrombin fragment 1.2 may play an important role in hemostasis by down regulating thrombin procoagulant activity because of its interaction with exosite 2.
The Oncogenic Potential of the Homeobox Gene Cdx2 Is Depending on the MAPK Pathway and Can Be Antagonized by the MEK1/2 Inhibitor PD98059 in a Mouse Model of t(12;13)(p13;q12) Positive AML.
Authors: Rawat Vijay P.S. , Naidu Vegi M. , Schessl Christina , Cusan Monica , Humphries R.Keith , Lynch J. P. , Hiddemann W. , Feuring-Buske Michaela , Buske Christian 
Published: October 2019

Abstract
In AML the translocation t(12;13)(p13;q12) results in the ectopic expression of the homeobox gene Cdx2 and the expression of the ETV6-CDX2 fusion. We have recently shown that myeloid leukemogenesis is induced by the ectopic expression of the proto-oncogene Cdx2 and not by the ETV6-CDX2 fusion gene in a murine model of t(12;13) AML. To characterize the contribution of different Cdx2 motifs to the transforming capacity of the gene we generated different mutants, inactivating the DNA binding homeodomain (N51S-Cdx2), or the PBX1-interacting motif (W167A-Cdx2), or deleting the N-terminal portion of Cdx2 (N-Cdx2). Expression of Cdx2 and the different mutants were induced in primary murine bone marrow cells by retroviral gene transfer, using an MSCV based retroviral construct with an IRES-YFP cassette. Expression of Cdx2 and the W167A-Cdx2 mutant significantly increased primary colony formation (3-fold) (n=3;p<0.001) with a higher number of CFU-G/GM colonies (p<0.015). Furthermore, both constructs enhanced the replating capacity of clonogenic progenitors with an 80-100fold increase in secondary colonies (p<0.005). In addition, both constructs induced the outgrowth of blast colonies (2700fold; p<0.02). In contrast, cells transduced with N51S-Cdx2 and N-Cdx2 lost their clonogenic potential after replating. In vivo all mice transplanted with cells expressing Cdx2 or the W167A-Cdx2 mutant developed transplantable AML. However, in Cdx2 leukemic mice > 90% of the cells co-expressed Gr-1+ and Mac1+, whereas in W167A mice 40% of the leukemic population were Gr-1+ only. The N51S mutant induced a distinct leukemia phenotype with 90 % Gr-1+/c-Kit+. We extended structure-function analyses, inactivating the phosphorylation site (S60) in the Cdx2 transactivation domain, previously shown to be regulated by the MAPK family. We confirmed that oncogenic Cdx2 is phophorylated at the N-terminal in primary BM cells by Western blotting using a P-Cdx2-S60 specific antibody. S60 position mutation slightly reduced the hematopoietic activity of wild-type Cdx2. Incubation with the MEK1 inhibitor PD98059 inhibited phosphorylation, decreased the frequency of CFU-S 8fold (n=7; p<0.001) and blocked growth of leukemic Cdx2 transfected blasts in vitro. In contrast, the p38 inhibitor SB2059 did not prevent phosphorylation and was unable to antagonize Cdx2 induced transformation. These data demonstrate that the transforming activity of Cdx2 and the phenotype of Cdx2 induced leukemias is depending on the functional integrity of distinct Cdx2 domains. Furthermore, our data link the oncogenic potential of Cdx2 directly to the MAPK signaling, opening the possibility to counteract Cdx2 associated leukemogenesis by kinase inhibitors.
Epigenetic Suppression of the CTNNA1 Gene, Encoding the α-Catenin Protein, which Is Located in the 5q31 Critical Deleted Region in Malignant Myeloid Disorders with del(5q).
Authors: Liu Ting Xi , Becker Michael , Hsu Karl , Jelinek Jaroslav , Deng Min , Bloomfield Clara , Issa Jean-Pierre , Clarke Michael F. , Look A. Thomas 
Published: October 2019

Abstract
Recurring interstitial loss of all or part of the long arm of chromosome 5, del(5q), is associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Although the affected genes have not been identified, two critical deleted regions (CDR) on 5q have been established, a proximal CDR located in 5q31, and another distal CDR in 5q32–33. We investigated the expression of 28 genes located in the 5q31 CDR, in enriched populations of normal hematopoietic stem cells (HSCs) and leukemic initiating cells (L-ICs) by the reverse transcription polymerase chain reaction (RT-PCR). Of the 28 5q CDR genes investigated, 12 (42%) were expressed in HSCs (CD34+Thy-1+CD38-Lin-), including HNRPA0 (Hs.77492), LOC51306 (Hs.82035), SMAP (Hs.5464), CDC23 (Hs.153546), LOC51307 (Hs.102469), LOC51780 (Hs.24125), EGRI (Hs.326035), ETF1 (Hs.77324), HSBPA9B (Hs.3069), CTNNA1 (Hs.178452), MATR3 (Hs.223745) and UBE2D2 (Hs.108332). We also isolated L-ICs (CD34+CD123+CD38-Lin-) from 11 cases of MDS/AML with 5q-, and investigated their expression levels of these 12 HSC genes. Expression of CTNNA1 was not detected in the L-ICs of 8 of the 11 cases of del(5q) MDS/AML using standard RT-PCR, while the other 11 genes were expressed in each patient at levels comparable to those of HSCs. Quantitative analysis of CTNNA1 expression in L-ICs by real-time RT-PCR confirmed expression levels <30% of HSC levels in 4 of the 6 cases tested with del(5q) MDS/AML. Analysis of protein expression by immunofluorescence also verified a lack of detectable CTNNA1 in L-ICs for the two del(5q) cases tested. CTNNA1 expression levels were normal in each of the 11 non-del(5q) MDS/AML cases tested. Methylation of the CTNNA1 promoter region correlated with the downregulation of CTNNA1 transcript in the del(5q) HL-60 cell line, in which CTNNA1 expression was partially restored following treatment with 5-aza-2-deoxycytidine. Our results suggest that epigenetic suppression of the expression of the intact CTNNA1 allele may contribute to the pathogenesis of MDS/AML with del(5q). We are currently using mouse and zebrafish models to study the role of CTNNA1 loss-of-function in del(5q) MDS/AML leukemogenesis.
cDNA Microarray Analysis of Genes Up-Regulated by Treatment with 5-AZA-2′-Deoxycytidine in Combination with Trichostatin A Identifies Aberrant Metallothionein 1H Promoter Methylation at a High Frequency Iin Human AML.
Authors: Scott Stuart A. , Pearson Derek S. , Bainbridge Matthew N. , Dong Wei-Feng , Takahashi Naoto , Sheridan David P. , Ichinohasama Ryo , Geyer C. R. , DeCoteau John F. 
Published: October 2019

Abstract
Gene promoter methylation is a major mechanism of transcriptional silencing in cancer cells that is mediated by CpG methylation and histone deacetylase activity, with methylation being dominant. Importantly, pharmacological treatment of cancer cells with inhibitors of DNA methylation and histone deacetylation have been shown to synergistically reactivate transcription of previously silenced genes. In an effort to identify novel genes silenced by DNA methylation in human acute myeloid leukemia (AML), we used cDNA microarray technology to screen for genes upregulated in the human cell line AML193 following treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, Trichostatin A (TSA). Analysis of 1,700 gene microarrays in triplicate revealed seven genes consistently upregulated by combined 5-Aza-dC and TSA treatment, all of which were confirmed by semi-quantitative RT-PCR. Interestingly, four of these genes (MT1H, MT1E, MT2A, MT1G) are members of the metallothionein family of cysteine-rich small molecular weight proteins. These proteins are considered to be important mediators of cellular detoxification and are induced by toxic heavy metals, UV irradiation and reactive oxygen species. As well, MT1G promoter methylation has been implicated in the pathogenesis of sporadic papillary thyroid carcinoma. The methylation status of candidate metallothionein genes reactivated by 5-Aza-dC and TSA treatment in AML193 cells was assessed by methylation specific PCR (MSP) in seven human AML cell lines. Methylated promoter alleles were detected in MT1H, MT1E and MT1G in 71%, 29% and 29% of cell lines, respectively, and there was no methylation of MT2A in any of the cell lines tested. To investigate for the occurrence of MT1H, MT1E and MT1G methylation in human AML in vivo, we analyzed AML patient blasts (n=39) and non-leukemic controls (n=13) by MSP. MT1H, MT1E and MT1G methylation was detected in 51%, 0% and 3% of AML patient samples, respectively, and there was no methylation of any of these genes in control samples. Our findings implicate MT1H promoter methylation in the pathogenesis of human AML and suggest that the use of cDNA microarray technology following pharmacological manipulation is a useful approach for identifying novel epigenetically silenced genes in this disease.
Differential Effects on Cell Cycle Regulators by AML1-ETO and a C-Terminal Truncated AML1-ETO (From Tumor Suppressor to Oncogene).
Authors: Peterson Luke F. , Yan Ming , Boyapati Anita , Zhang Dong-Er 
Published: October 2019

Abstract
The fusion protein AML1-ETO created by the 8;21 translocation involved in de novo acute myeloid leukemia requires secondary mutational events to promote leukemia. Here, we report that the loss of the molecular events associated with AML1-ETO C-terminus including an NCoR/SMRT interacting domain transforms AML1-ETO into a potent leukemogenic protein in mice. Furthermore, we present evidence of aberrant protein expression of cell cycle regulators in a hematopoietic cell line expressing AML1-ETO compared to the leukemogenic truncated form of AML1-ETO (AML1-ETOtr). Our studies show that AML1-ETO and AML1-ETOtr are biochemically isolated from the same cellular compartments, that they can oligomerize, and that they can both efficiently immunoprecipitate the transcription factor Gfi-1. However, contrary to AML1-ETO, AML1-ETOtr does not promote growth arrest. Western analyses show that cell cycle promoting factors cyclin D3 and cyclin A are decreased by AML1-ETO, while their RNA levels remain the same. In addition, an increase in the cdk-inhibitor p21WAF1 is observed in the presence of both proteins, while only AML1-ETO induces high expression of the cdk-inhibitor p27KIP1. These changes are associated with a deregulation of the SCF ubiquitin E3 ligase component Skp2 that is involved in controlling the levels of both cyclins and cdk-inhibitors. These observations suggest that AML1-ETO has tumor suppressor activity. Additional mutations to bypass this effect can change it into an oncogenic protein. Therefore, our results lead to a new model of AML1-ETO in leukemogenesis, i.e., the gain/loss of function of cell cycle regulators to promote cell cycle progression or disruption of molecular events associated with AML1-ETO C-terminal NCoR/SMRT interacting domain are required for AML1-ETO involved leukemogenesis. The disrupted molecular events may include 1) the loss of factor(s) physically interacting with the C-terminal domain of AML1-ETO, 2) the alteration by mutagenesis of signaling pathways downstream of AML1-ETO C-terminal domain, and 3) the dysfunction within AML1-ETO C-terminal domain by truncations or mutations.
A Prospective Randomized Trial of Oral Melphalan, Prednisone, Thalidomide (MPT) vs Oral Melphalan, Prednisone (MP): An Interim Analysis.
Authors: Palumbo Antonio , Bertola Alessandra , Musto Pellegrino , Caravita Tommaso , Callea Vincenzo , Cangialosi Clotilde , Liberati Anna Marina , Niscola Pasquale , Catalano Lucio , Grasso Mariella , Lauta Vito Michele , Petti Maria Concetta , Morandi Sergio , Galli Monica , Bringhen Sara , Cavallo Federica , Falco Patrizia , Boccadoro Mario 
Published: October 2019

Abstract
In newly diagnosed multiple myeloma (MM) patients, the combination melphalan, prednisone and thalidomide induces a fast tumor response with a high complete remission rate. In a prospective randomized trial, we compare the efficacy and toxicity of oral MPT and MP.
An interim analysis was conducted after the first 200 newly diagnosed myeloma patients, median age 72, range 56–85, entered the study, between January 2002 and June 2004. At present, 116 patients were evaluated for toxicity and 102 patients for response on an intent to treat basis. The MPT regimen included 6 monthly courses of oral melphalan 4 mg/sqm and prednisone 40 mg/sqm for 7 days every month plus thalidomide 100 mg/day continuously until any sign of progressive disease or relapse. The dose of thalidomide was reduced to 50% when grade II toxicity occurred, and suspended for any grade III. On December 2003, the protocol was amended and enoxaparin prophilaxys was added. The MP regimen was as MPT without thalidomide. The end points of the study were: response, EFS, OS and toxicity.
The response rate among patients who received MPT was: 25.9% immunofixation negative CR (CR), 5.5% immunofixation positive near CR (nCR) 48.2% partial remission (PR) (M-protein reduction 50–99%), 9.3% stable disease (SD) (M-protein reduction 0–49%) and 11.1% progressive disease (PD). The response rate after MP was 4.2% CR, 0% nCR, 43.6% PR, 23% SD and 29.2% PD. Response was followed by significant improvement of performance status, skeletal pain, anemia and transfusion requirement.
After a median follow up of 15 months, 38 patients relapsed: 11 (29%) after MPT and 27 (71%) after MP. The EFS @ 26 months was 67.8% for MPT and 32.4% for MP (P <0.001). The median OS has not been reached.
Treatment-related mortality was 5% after MPT (1 septicemia, 1 pulmonary thrombo-embolism and 1 heart failure), and 1.9% after MP (1 myocardial infarction). The major adverse events of MPT vs MP were: deep-vein thrombosis (19.3% vs 1.9%), grade III-IV infections (12.9% vs 1.9%), grade I-II neurotoxicity (35.5% vs 5.5%), grade III-IV hematologic toxicity (22.6% vs 27.6%). Thalidomide discontinuation was recorded in 33.8% of patients (8 thrombo-embolic events, 4 neurotoxicities, 4 constipations, 2 infections, 3 miscellaneous); dose-reduction in 24.2% of patients (8 neurotoxicities, 3 constipations, 4 miscellaneous).
MPT significantly improves response rate and EFS in elderly myeloma patients with a median age of 72 years. These results are similar to those observed after transplant.
An update of these data will be presented.
Mutation of Cellular Retinoic Acid-Binding Protein-II (Crabp-II) Does Not Account for Resistance to All-Trans Retinoic Acid (ATRA) in Relapse Acute Promyelocytic Leukemia (APL).
Authors: Sankoorikal Binu-John , Zhou Da-Cheng , Wiernik Peter H. , Gallagher Robert E. 
Published: October 2019

Abstract
An increase in CRABP-II has frequently been invoked as a cause of resistance to ATRA in APL due to cytoplasmic sequestration and catabolism of ATRA. However, recent evidence indicates that CRABP-II has a positive rather than a negative role in ATRA activity by facilitating delivery of ATRA to retinoic acid receptor-alpha (RARα) associated with ATRA target genes in the cell nucleus or/and by serving as a co-activator of RARα-regulated transcription. This implies that if CRABP-II has a role in the development of ATRA resistance in APL, this would more likely occur through a deficiency rather than from an increase in CRABP-II. We previously reported that CRABP-II is constitutively expressed at similar levels in pretreatment and relapse APL cells (Zhou, et al, Cancer Res58, 5770, 1998), suggesting that putative CRABP-II deficiency is not due to the loss of CRABP-II expression. To investigate the alternative possibility that CRABP-II deficiency might arise through inactivating mutations, we sequenced the entire coding region of CRABP-II from 18 cases of APL who had relapsed from prior ATRA-containing treatment regimens. In 8 cases RNA was transcribed by reverse transcriptase to cDNA and amplified by polymerase chain reaction (PCR), using primers anchored in the 5′ and 3′ untranslated region of mRNA; in 10 cases, genomic DNA was PCR amplified for sequence analysis, using primers anchored in the introns between the 4 exons of the gene. No CRABP-II mutations were identified. The samples tested included 11 first-relapse cases, 2 of whom were refractory to reinduction therapy with intravenous liposomal-ATRA, and 7 multiple relapse cases, all of whom were clinically refractory to ATRA and had, additionally, relapsed from arsenic trioxide therapy. Also, no mutations were found in 3 APL patients who had relapsed from chemotherapy-induced remissions or in 3 APL cell lines (NB4, UF-1 and AP-1060). Two heterozygous base substitutions were incidentally identified in CRABP-II intron 2 in a chemotherapy-only treated patient. These results indicate that CRABP-II mutations rarely, if ever, contribute to ATRA-resistance or disease relapse in APL.
Identification and Effects of Novel Promoter Region Haplotypes in the Human Equilibrative Nucleoside Transporter, hENT1.
Authors: Myers Scott N. , Goyal Rakesh K. , Roy Jennifer D. , Ferrell Robert E. 
Published: October 2019

Abstract
Front-line induction chemotherapy regimens containing cytosine arabinoside (Ara-C) and anthracyclines result in 80% complete remission rate in childhood acute myeloid leukemia (AML) but their cure rate is about 35 – 50%, one of the lowest of all childhood cancers. Understanding the factors that contribute to emergence of chemoresistant leukemic cells is crucial to improving treatment outcome in children with AML. We are interested in studying the role of variation in Ara-C transport and biotransformation pathway genes in the efficacy and toxicity of treatment of childhood AML. To permeate the cell membrane, Ara-C is mainly dependent on human equilibrative nucleoside transporter 1 (hENT1; SLC29A1; gene localized to 6p21.1). Several studies have suggested an important role for altered levels of hENT1 in the chemosensitivity of AML blasts to Ara-C (Galmarini et al. Leukemia2001; 15(6):87; Gati et al. Leuk Lymphoma1998; 32(1–2):45). Osato and colleagues identified two single nucleotide polymorphisms (SNPs) in the hENT1 coding sequence that led to missense changes, but their in vitro analysis did not detect differences in the activity of variant alleles in a yeast transfection system (Osato et al. Pharmacogenetics2003;13(5):297). To identify variation in hENT1 that might influence its expression, we sequenced 1.6Kb of the proximal 5′-flanking sequence of the gene in 42 unrelated individuals and identified three SNPs at positions C-1345G, G-1050A, and G-706C. TRANSFAC analysis (www.genomatix.de) predicted that two of these (C-1345G & G-706C) would alter consensus transcription factor binding site sequences. We cloned four naturally occurring haplotypes (CGG, CAG, CGC, and GAG) using the TOPO-TA cloning kit, then transfected Cos-1 cells using the Lipofectamine 2000 protocol. Gene expression was assayed using the Promega Dual-Luciferase Reporter Assay System and read on a Molecular Devices HT Analyzer. Luciferase activity was measured at 24 and 48 hours after transfection for six replicates of every condition during three separate transfections. To correct for differences in transfection efficiencies, experimental (Photinus pyralis) luciferase activities were normalized by co-transfection with control (Renilla reniformis) luciferase plasmid. Compared to the wild type CGG haplotype, variant haplotypes CAG, CGC, and GAG drive luciferase expression at approximately 2x (p <0.0001), 1.4x (p <0.001) and 1.2x (p =0.08), respectively. This leads to the hypothesis that individuals carrying CAG or CGC haplotypes (17% of the population) exhibit higher levels of hENT1 expression and are more sensitive to Ara-C exposure. Experiments are underway to quantify gene transcripts in people of known hENT1 haplotypes. We also plan to genotype a large cohort of children with de novo AML for these three SNPs in hENT1 and correlate clinical outcomes in individuals carrying the low- versus the high-expressing haplotypes.