bluuebunny/crossref_metadata_2025_split huggingface dataset:
Secretable Human Megakaryocyte Factor V Endocytosed from Plasma in Complex with Multimerin: The Origin of Human Megakaryocyte Factor V.
Authors: Suehiro Youko , Veljkovic Dragoslava K. , Fuller Nola , Motomura Yasuaki , Masse Jean , Cramer Elizabeth , Hayward Catherine P.M. 
Published: October 2019

Abstract
Factor V (FV) is an essential coagulation cofactor that circulates in distinct plasma and platelet pools. Unlike plasma FV, platelet FV is stored complexed to the megakaryocyte (MK) synthesized α-granule protein multimerin, which is not found in plasma. The origin of platelet FV has been controversial and recent studies have suggested human platelet factor V originates from plasma. To determine if human MK could endocytose FV and generate the FV-multimerin complexes, we investigated FV endocytosis and storage by human MK derived from cord blood CD34+ cells. Cells were cultured with or without 10% plasma in 15% BIT/ IMDM containing 5ng/ml SCF and 50ng/ml TPO. Human plasma or purified plasma FV was added on day 7 of culture. Cells were harvested to analyze α-granule proteins on day 11. Enzyme-linked immunoassays were used to evaluate MK content of FV and multimerin, and their complexes. Control α-granule proteins analyzed included albumin, IgG and fibrinogen, as they are known to originate from plasma. Multimerin was detected in all MK whereas FV, albumin, IgG, fibrinogen and FV-multimerin complexes were only detected in MK cultured with plasma. The amounts of plasma-derived proteins in MK were orders of magnitude lower than in platelets. MK cultured with purified FV had dose-dependent FV uptake. With SFLLRN stimulation, MK cultured with plasma released multimerin, FV, albumin, IgG and fibrinogen, consistent with their storage in secretion granules. Like platelet FV, MK FV showed a proteolyzed pattern. Double immunostaining showed partial colocalization of intracellular multimerin and FV in MK cultured with FV. Immunoelectron microscopy confirmed that like bone marrow MK, MK cultured with FV contained FV within demarcation membranes, small vesicles and α-granules. Analyses of FV expression by cultured MK, using semi-quantitative RT-PCR, indicated there were no significant differences in FV expression by MK cultured with or without plasma, at any of the time points evaluated. However, there was a trend to increased FV expression with MK maturation, and for MK cultured in plasma, FV expression was significantly higher at 15 compared to 7 days, raising the possibility that some FV was synthesized at late stages of megakaryopoiesis. Our study illustrates that cultured human MK can proteolyze and target endocytosed plasma FV into α-granules and that they can generate complexes of FV with multimerin.
Formation of Tissue Factor-Factor VIIa-Factor Xa Complex Promotes Survival of Human Breast Cancer Cells by Inhibition of Apoptosis.
Authors: Jiang Xiaofeng , Guo Yan-Lin , Bromberg Michael E. 
Published: October 2019

Abstract
Tissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa (FVIIa). TF is constitutively expressed by a variety of tumor cells. Recently, TF has been shown to induce cellular signaling and promote tumor growth, angiogenesis, and metastasis. We showed previously that formation of TF-FVIIa-Factor Xa (FXa) complex induces cellular signaling in the Adr-MCF-7 cell line, a multidrug-resistant subline of the human breast cancer cell line, MCF-7 (Jiang et al. J. Thromb Haemost2: 2004; 93–101). This cell line has high endogenous expression of TF. Treatment of the Adr-MCF-7 cells with the combination of FVIIa (10 nM) and FX (150 nM) induces phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and protein kinase B (PKB), whereas no increase in the basal level of phosphorylation of these proteins occurs with treatment of the cells with FVIIa (10 nM) alone. In the present study, we investigated whether TF-FVIIa-induced signaling might alter apoptosis in human breast cancer cells. Apoptosis of the Adr-MCF-7 cells was induced by serum starvation for 5–7 days. Treatment of the cells with the combination of FVIIa (10 nM) and FX (150 nM), reduced apoptosis of the cells by nearly 50% compared with untreated, control cells using an ELISA that detects histone-DNA fragments. In contrast, FVIIa (10 nM) alone did not significantly prevent apoptosis. Pre-treatment of the Adr-MCF-7 cells with hirudin did not inhibit the anti-apoptotic effect of the combination of FVIIa and FX, whereas this effect could be completely blocked by either U0126 (10 μM), which inhibits p44/42 MAPK phosphorylation, or LY294002 (50 μM), which inhibits PKB phosphorylation. In addition, treatment of the Adr-MCF cells with the combination of FVIIa and FX led to a 50% increase in the level of the anti-apoptotic protein, survivin, compared with untreated cells by Western blot analysis. Results from this study indicate that formation of TF-FVIIa-FXa complex prevents apoptosis of breast cancer cells by a thrombin-independent pathway. Moreover, the anti-apoptotic effect of this signaling pathway involves both phosphorylation of p44/42 MAPK and PKB and might be mediated in part by an increase in cell survivin levels.
Long Term Survival Using Intensive Multiagent Chemotherapy for Relapses of Pediatric Acute Lymphoblastic Leukemia (ALL).
Authors: Rheingold Susan R. , Bunin Nancy J. , Aplenc Richard , Leahey Ann M. , Lange Beverly J. 
Published: October 2019

Abstract
Relapses of childhood ALL that occur on therapy are associated with a dismal survival. To improve the prognosis for these patients, we developed an intensive multiagent chemotherapy protocol consisting of an induction with idarubicin, vincristine (VCR), dexamethasone (DEX), and peg-asparaginase (PEG). Consolidation included high-dose cytarabine (ARA-C), etoposide (VP-16), and PEG followed by VCR and methotrexate (MTX). After an interim maintenance (IM), induction and consolidation were repeated followed by maintenance therapy lasting two years. Maintenance and IM consisted of alternating two week cycles of VP-16, ARA-C, and PEG, with MTX ,VCR, DEX, and PEG. Between 1992 and 2002, 53 pts (32M, 21F) received treatment according to this protocol, 21 of whom were treated as part of the original study (Leahey AM et al., Med Ped Onco34(5):313–8, 2000). Median time to relapse was 37 months from diagnosis (range 12–86 mos). Twenty-one pts were on therapy at relapse and 25 pts were <36 months from diagnosis. Relapses included isolated bone marrow (BM) (32 pts), BM and central nervous system (CNS) (9 pts), BM and testicular (3 pts), and extramedullary (EM) (9 pts). By present day criteria 26 pts were standard risk (SR), 23 were high risk (HR), and 4 were infants. Two patients died in induction, and 2 never achieved a second remission. All others achieved remission by the end of induction (92%). Five-year event-free survival (EFS) and overall survival (OS) are both 56% +/−7% (CI 41%–68%), at a mean of 47 months from relapse (range 0–141 mos). Patients with a first complete remission (CR1) duration <36 months have an EFS of 40% +/−10% (CI 21%–58%); >36 months CR1 is associated with an EFS of 70% +/−9% (CI 50%–84%). Of the events in all pts who initiated therapy,13 were from refractory/recurrent ALL (25%), and 10 pts died of toxicity (19%). Four pts died from chemotherapy induced toxicity (8%), and 6 died from transplant (BMT) related toxicities (11%). Fourteen pts in second remission proceeded to BMT at a mean of 5 months from relapse (range 4.5–8 mos), and 7 of these pts remain in CR2. Of the 39 pts who continued on chemotherapy, 6 pts (35%) with CR1<36 months remain in CR2 and 16 patients (57%) with CR1>36 months remain in CR2 or 3. The 19 pts who were treated on modern standard risk (SR) protocols (CCG-1881 to present) were more salvagable than their 20 high risk (HR) counterparts (CCG-1882 to present). Five year EFS for SR and HR pts is 67% +/−8% (CI 48%–80%) and 38% +/−12% (CI 16%–59%) respectively. Intensive rotating therapy with reinduction and reconsolidation improves EFS. Children with early EM relapses of ALL, CR1 > 36 months, and SR patients all have very good long term survival. Novel therapies need to be integrated into intensive relapse protocols for children with early BM relapse and children treated upfront on HR protocols.
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Imatinib Mesylate Does Not Inhibit BCR-ABL Kinase Activity in CML Stem Cells In Vitro.
Authors: Elrick Lucy J. , Hamilton Ashley , Deininger Michael W. , Holyoake Tessa L. 
Published: October 2019

Abstract
Imatinib mesylate (IM, Gleevec, Novartis), a specific inhibitor of the tyrosine kinase BCR-ABL, promotes an impressive complete cytogenetic remission rate in patients with chronic phase Chronic Myeloid Leukaemia (CML). In most cases however, molecular disease persists and may lead to eventual relapse. IM selectively interacts with BCR-ABL thereby inhibiting activation of downstream targets, in particular phosphorylation of CrkL. We have previously demonstrated the sensitivity of rare Ph+ CD34+ CML stem cells (<5% of total CD34+ cells) to the anti-proliferative but, not the pro-apoptotic effects of IM, hence their persistence in vitro. We hypothesised that these rare cells may also behave differently from total CD34+ cells in terms of their kinase response to IM. The effect of IM on the phosphorylation status of CrkL was therefore investigated, in total CD34+ and in primitive CD34+/38- cells from 6 untreated chronic phase CML patients. Ph+ CD34+ and CD34+/38- cells were cultured in serum free medium for up to 72h, in the presence and absence of IM (5mM). After 16 and 72h the phosphorylation status of CrkL was measured with an antibody specifically designed to recognise only the phosphorylated form of the protein. After 16h total CD34+ cells showed a significant dephosphorylation of CrkL in response to IM. However, after 72h the remaining viable BCR-ABL+ cells failed to show any de-phosphorylation of CrkL, consistent with an IM selected, resistant population of stem cells. Primitive CD34+/38- cells from the same patients exhibited no de-phosphorylation of CrkL after either 16 or 72h, confirming their innate/inherent resistance to the drug. These data show, for the first time, that very primitive CML cells exhibit BCR-ABL dependent resistance (i.e. BCR-ABL kinase remains active during drug exposure) and suggest that the drug is not reaching or binding its target at inhibitory concentrations. Novel approaches to eliminate these cells will be critical to the development of curative strategies for CML.
The SRC/ABL Inhibitor BMS-354825 Overcomes Resistance to Imatinib Mesylate in Chronic Myelogenous Leukemia Cells through Multiple Mechanisms.
Authors: Donato Nicholas J. , Wu Ji , Kong Ling Y. , Lee Francis , Talpaz Moshe 
Published: October 2019

Abstract
BCR-ABL is an oncogenic tyrosine kinase expressed in chronic myelogenous leukemia (CML) cells and is the main target of the tyrosine kinase inhibitor imatinib mesylate. Imatinib-based CML therapy induces hematological and cytogenetic remission in early phase CML patients whereas more advanced patients frequently develop resistance to imatinib by multiple mechanisms, including mutations in the BCR-ABL kinase domain and over-expression of tyrosine kinases that are not inhibited by imatinib. These observations suggest that dual inhibition of src and abl kinases may circumvent imatinib resistance and provide more effective therapy for CML. BMS-354825 is a novel tyrosine kinase inhibitor that inhibits both abl and src kinases at low nM concentrations and is currently being clinically evaluated in imatinib resistant or intolerant CML patients. Our earlier studies demonstrated that increased expression of the src-related kinase Lyn in BCR-ABL expressing K562 cells was associated with imatinib resistance in this cell model and some CML patients. To determine whether inhibition of SRC/ABL kinases differentially affects imatinib sensitive K562 (BCR-ABL +, Lyn −) and resistant K562R (BCR-ABL +, Lyn +) cells were treated with imatinib or BMS-354825 before analysis of cell growth, survival and signaling. BMS-354825 induced apoptosis in both K562 and K562R cells which correlated with inhibition of both Lyn activation and BCR-ABL signaling (CrkL). BMS-354825 effectively reduced both K562 and K562R tumor growth in nude mice whereas imatinib had minimal effects on K562R tumors. Clinical specimens from imatinib resistant CML patients (with and without BCR-ABL kinase mutations) were treated with imatinib or BMS-354825 and analyzed for changes in Lyn and Hck activation. While imatinib had minimal inhibitory effects on Lyn/Hck activation, BMS-354825 completely suppressed Lyn/Hck phosphorylation which correlated with its greater anti-tumor activity in CML samples. BCR-ABL tyrosine phosphorylation was not inhibited by imatinib in Cos cells co-expressing BCR-ABL and Lyn kinase and loss of imatinib sensitivity was totally dependent on Lyn kinase activity. BMS-354825 reduced both Lyn and BCR-ABL activation in co-expressing cells, suggesting that Lyn-mediated phosphorylation plays a direct role in imatinib resistance. We conclude that dual inhibition of SRC/ABL kinases in CML cells by BMS-354825 overcomes resistance to imatinib in vitro and in vivo and induces anti-tumor effects in CML patient specimens resistant to imatinib through expression of imatinib-inactivating BCR-ABL kinase mutations as well as other resistance mechanisms.
Co-Administration of SAHA and 17-AAG Synergistically Induces Apoptosis in Bcr-Abl+ Cells Sensitive and Resistant to STI-571 in Association with Down-Regulation of Bcr-Abl, Abrogation of STAT5 Activity, and Bax Conformational Change.
Authors: Rahmani Mohamed , Reese Erin , Dai Yun , Bauer Cheryl , Kramer Lora , Huang Mei , Jove Richard , Dent Paul , Grant Steven 
Published: October 2019

Abstract
Interactions between the histone deacetylase inhibitors (HDACIs) suberanoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) and the heat shock protein 90 (Hsp90) antagonist 17-AAG have been examined in Bcr-Abl+ human leukemia cells (K562 and LAMA84), including those sensitive and resistant to STI571. Co-administration of 17-AAG with SAHA or SB for 24 h synergistically induced mitochondrial dysfunction (cytochrome c and AIF release), caspase-3 and -8 activation, apoptosis, and growth inhibition. Similar effects were observed in LAMA84 cells and K562 cells resistant to STI-571 as well as in CD34+ leukemic cells obtained from patients with CML including those who had developed a resistance against STI-571. These events were associated with increased association of Bcr/abl, Raf-1, and Akt with Hsp70, whereas the total level of these proteins markedly decreased. A pronounced inactivation of ERK1/2, and activation of JNK were also observed. In addition, 17-AAG/SAHA abrogated DNA binding and transcriptional activities of STAT5 in K562 cells, including those ectopically expressing a constitutively active STAT5A construct. Co-administration of 17-AAG and SAHA also induced downregulation of Mcl-1, Bcl-xL, B-Raf, and p21CIP1, upregulation of Bak, cleavage of 14-3-3 proteins, and a profound conformational change in Bax accompanied by translocation to the membrane fraction. Together, these findings raise the possibility that combining HDACIs with the Hsp90 antagonist 17-AAG may represent a novel strategy against Bcr-Abl+ leukemias, including those resistant to STI571.
Newly Discovered Polymorphism in the CD34+ Stem Cell Specific AC133-P1 Promoter Linked to Leukemias.
Authors: Shmelkov Sergey V. , St.Clair Ryan , Jun Lin , Calderone Agata , Karajannis Matthias A. , Jensen Kristian K. , Young Lauren M. , Walsh Kathryn , Shido Koji , Jin David , Zhang Fan , Cheng Joseph , Lam George , Tejada Rafael , Avecilla Scott T. , Vincent Loic , Kopp Hans-Georg , Akiri Gal , Lyden David , Rafii Shahin 
Published: October 2019

Abstract
AC133 (CD133, human Prominin-1) is a cell-surface glycoprotein which is expressed on several types of stem cells, including hematopoietic stem cells and endothelial progenitor cells. AC133 is also expressed on acute and chronic myeloid leukemias, as well as lymphoblastic leukemias. AC133 expression is correlated with a poor prognosis in cases of leukemia; in addition, preliminary observations suggest that AC133 may define a population of leukemic stem cells. We previously identified and characterized the AC133 promoter region, and demonstrated that transcription of AC133 is driven by five alternative promoters in a tissue-dependent manner, resulting in the formation of multiple alternatively spliced isoforms. CD34+ hematopoietic stem cells were found to utilize only one of the five promoters, AC133-P1. Furthermore, preliminary data suggest that hematopoietic stem cells express only one AC133 isoform. We identified four allelic variants in the AC133-P1 promoter, 250 bp upstream of the transcription initiation point, which differ by the number of short tandem repeats as well as by a single nucleotide polymorphism (SNP). One allele, (CAA)3CAG, is found in patients with AML, chloroma, and myeloma. Two different alleles, (CAA)4 and (CAAAA)5, are found in cancer cell lines derived from AML, retinoblastoma, colon cancer, and sarcoma. One allele, (CAA)5, is found only in healthy human subjects. Our preliminary results show that these different alleles may affect the activity of the AC133-P1 promoter. These data might lay a foundation for testing a risk of susceptibility to AC133+ leukemias. Additional investigations are required to establish the role of AC133 in emergence, development and survival of leukemias, as well as to explore the viability of AC133 as a primary therapeutic target in patients with aggressive AC133+ leukemia.
Vascular Endothelial Growth Factor 121 (VEGF121) Isoform mRNA Is Predictive of Poor Prognosis in Acute Myeloid Leukemia (AML) .
Authors: Calvo Fabien , Rousselot Philippe , Dombret Herve , Medioni Jacques , Mourah Samia 
Published: October 2019

Abstract
Bone marrow microvascular organisation has been shown to play an important role in Acute Myeloid Leukemia (AML). VEGF has four main isoforms produced by alternative splicing: VEGF121, VEGF165, VEGF189 and VEGF206. The VEGF121 protein which lacks the basic amino acids responsible for heparin binding, is the more soluble and potent isoform. The present work aimed at quantitatively measure the shortest transcript for soluble VEGF121 isoform by quantitative RT-PCR in peripheral blood mononuclear cells (PBMC) from AML patients and to evaluate its value as a prognostic marker for response to therapy and survival. We conducted a single institution prospective study in 67 consecutive AML patients at diagnosis. VEGF121 transcript levels in AML PBMC were significantly higher than in normal controls (25.9 copies/1000 copies of β2 microglobulin (β2m) compared to 1.9 copies in normal participants, p<0.001). No relation was found between VEGF121 level and sex, age, white blood cells counts or the dose intensity of aracytine during induction treatment. Both univariate and multivariate analysis of overall survival showed that high levels of VEGF121 transcripts (VEGF121 in AML patients > 5 copies of VEGF121/1000 copies of β2m; 25th centile, this cut-point were designed after systematic searches) were significantly related with a worse prognosis (p<0.0001 for univariate analysis and OR=11.6 [2.76–48.6, p=0.008 for multivariate analysis). Neither sex (p=0.08) nor age (p=0.90) nor WBC (p=0.96) nor caryotype (p=0.29), nor AraC dose intensity (p=0.29) were related with a bad prognosis in this group of patients. Analysing disease free survival, only high levels of VEGF121 transcripts were significantly related to a worse prognosis (p<0.0001, using univariate analysis). Of note, 94% of the patients who relapsed had an initial high level of VEGF121 transcripts. Our findings support the use of this test as a predictive and prognostic tool, helping the clinician to identify patients who should benefit for alternative therapeutic strategies. This test has advantages over ELISA or RIA monitoring cellular or plasma VEGF protein, since it is more specific of this isoform, independant of the number of circulating cells, of binding to α2 macroglobulin and of contaminating sources of VEGF such as platelets which sequester this cytokine. Monitoring of antiangiogenic treatment through QRT-PCR of VEGF121 could therefore be useful in these patients.
Proteomic Analysis of Acute Promyelocytic Leukemia Reveals That PML-RARalpha Induces Cell Cycle Progression and Mitotic Exit by Increased Expression and Decreased Ser63 Phosphorylation of OP18.
Authors: PeerZada A. , Geletu M. , Pullikan J. , Reddy V. , Hiddemann W. , Behre G. 
Published: October 2019

Abstract
We applied a mass spectrometry based approach to explore the proteins differentially regulated by PML-RARalpha, a translocation characteristic of acute promyelocytic leukemia (APL). Bioinformatic pathway analysis placed the 46 identified PML-RARalpha regulated proteins into three major networks, OP18-MAPK1, HSP-STAT3 and CCT-MYC. Using this approach, we were able to generate a common cell cycle network of the proteins in these pathways. Further analysis indicated that mRNA expression of OP18, which belonged to this network, was elevated in APL patients and the increased OP18 protein expression upon PML-RARalpha induction was overcome by retinoic acid treatment. Here we also report, for the first time a novel role of PML-RARalpha in cell cycle progression and mitotic exit. RNA interference experiments revealed that siRNA against OP18 overcomes PML-RARalpha effects on cell cycle progression. In addition to increased OP18 expression by PML-RARalpha, 2D gel electrophoresis revealed an isomer of OP18, subsequently confirmed by 2D-western as ser63 phosphomer to be downregulated by PML-RARalpha. Based on these findings, point mutation experiments indicated that decreased phosphorylation of ser63 in OP18 is important for PML-RARalpha mediated cell cycle and mitotic index effects since a constitutive phosphorylated mutant (ser63/asp) of OP18 overcame the PML-RARalpha effects in U9/PR cells, NB4 and APL patients. In summary, our results demonstrate that the effect of PML-RARalpha on cell cycle progression and mitotic exit is via two mechanisms: increasing the expression of OP18 and decreasing the phosphorylation of OP18 at ser63.