bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Mechanism of Prothrombin G20210A Variant: Critical New Role of Exon Splicing Enhancer in Poly (A) Tailing.
Authors: Kuwahara Mitsuhiro , Kurachi Sumiko , Kurachi Kotoku 
Published: October 2019

Abstract
Although prothrombin G20210A variant was first found in 1996 as a genetic risk factor of thrombosis, the underlying molecular mechanism for elevation of circulatory prothrombin levels due to the variant has remained elusive. The position 20210 is at the poly (A) tailing site, and several research groups reported different, contradictive possibilities including enhancement in poly (A) tailing, change in RNA stability or no difference in prothrombin mRNA level between G (wild type) and A (variant) at position 20210. Such confusions appear to be due to no reliable assay model availability. To overcome these problems, we developed a minigene assay system composed of only autologous components of human prothrombin gene, including its promoter, coding regions, introns and 3′UTR with poly (A) signal.
A minigene containing the first intron and the last intron did not express at any appreciable levels, failing proper splicing of the last intron. Inclusion of upstream other introns was found essential for its appropriate splicing. Although it is generally accepted that the last intron enhances the efficiency of poly (A) tailing, we found that it is not the case for the prothrombin gene. Instead, an exon-splicing enhancer (ESE) of another upstream intron was found to be responsible for efficient poly (A) tailing. ESE, therefore, plays a critical role not only in alternative splicing but also in constitutive RNA processing, including poly (A) tailing. Etiology of prothrombin G20210A variant studied with culture cells and transgenic mice, supported an enhancement of poly (A) tailing, and not a change in mRNA stability.
Genetic Polymorphisms in the Promoter Region of the beta-2 Adrenergic Receptor Are Associated with the Early Response of Acute Lymphoblastic Leukemia to Chemotherapy.
Authors: Cheok Meyling H. , Ding Cong , Yang Wenjian , Das Somas , Campana Dario , Cheng Cheng , Cook Edwin H. , Pui Ching-Hon , Relling Mary V. , Evans William E. 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) in children is a paradigm of disseminated cancer that is curable with chemotherapy, yet current treatment fails to cure about 20% of patients, for reasons that remain unknown. In a genome-wide assessment of in vivo treatment-induced changes in gene expression in ALL cells using the Affymetrix U95A and U133A oligonucleotide microarray, we found that patients who eventually relapsed did not up-regulate expression of the pro-apoptotic beta-2 adrenergic receptor gene (ADRB2) in their ALL cells after initial treatment with methotrexate and mercaptopurine. After treatment we observed a 5-fold lower level of ADRB2 gene expression in leukemia cells of patients who ultimately relapsed. We found a common genetic polymorphism in the ADRB2 promoter that was significantly linked to high-dose methotrexate induced up-regulation in ADRB2 gene expression in ALL cells. Moreover, the ADRB2 promoter haplotype was significantly linked to poor early treatment response in ALL cells from 242 children (i.e., probability of event-free survival at two years, p=0.0275 stratified by risk groups). These findings have revealed a germline polymorphism that is linked to the early antileukemic effects of ALL chemotherapy and provide new insights into genetic determinants of ALL treatment efficacy.
Bone Mineral Density, Fracture Risk and Avascular Bone Necrosis in Childhood Acute Lymphoblastic Leukemia; an Up-Date of the Prospective DUTCH ALL9 Study.
Authors: Van den Heuvel-Eibrink Marry M. , Van der Sluis Inge M. , Leeuw Bert A. , Kardos Gaby , te Winkel Lizet M. , Revesz Tom , Korthof Elisabeth T. , Bokkerink Jos , Veerman Anjo J.P. 
Published: October 2019

Abstract
The high cumulative dose of dexamethasone, applied in the DCOG ALL9 protocol, prompted us to investigate the risk of osteoporosis, fractures and avascular necroses of bone (AVN) in children treated with acute lymphoblastic leukemia (ALL). Fracture risk and incidence of symptomatic AVN was assessed in 778 patients(482 boys, 297 girls), included in the ALL9 protocol since 1997. Total cumulative doses (TCD) of dexamethasone were 1370 mg/m2 and 1244 mg/m2 and of MTX 8.1g/m2 13.6g/m2 for NHR and HR patients respectively. No CNS-irradiation was applied. In children aged >3 years, lumbar spine bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DEXAscan), at diagnosis(T0), after 32 weeks(T1), at discontinuation of treatment at 109 weeks(T2), and one year after discontinuation of treatment(T3). Results were expressed as standard deviation scores (SDS). Symptomatic AVN was defined as on MRI confirmed AVN lesions in combination with non-vincristine related persistent pain in arms or legs. Fractures were reported in 82/778 (10.5%) patients. Most occurred after mild trauma. No difference was found in fracture incidence between boys and girls. BMD was measured in 387/427 (90.6%) eligible patients. Median BMD-SDS was significantly lower than zero at all times of evaluation, the lowest BMD values were found at T2 (−1.47 SDS). Fracture risk was 3.9 times higher as compared to healthy school children. Fracture incidence was correlated with BMD at T2 and T3(p=0.04 and p=0,04 respectively), but not at T0 and T1. A significant more rapid decline in BMD from T0 to T2 and to T3 was seen in patients with fractures as compared to patients without fractures. After discontinuation of therapy, BMD recovered faster in cases without fractures. Symptomatic AVN occurred in 33/778 (4.2%) of our patients (med age 14, range 6,5–18 years) showing irreversibility in 22 % of the cases. Differences found in the incidence between the centers may suggest underestimation of the risk of fractures and AVN in this prospective study. Children with ALL show a significantly increased fracture risk. Patients with a more severe reduction in BMD during treatment are more susceptible to fractures. The AVN incidence in this protocol did not exceed previous reports of prednisolone-based protocols.
The Heme Oxygenase-1-Targeting Compound PEG-ZnPP Inhibits Growth of Imatinib-Resistant BCR/ABL-Transformed Cells.
Authors: Mayerhofer Matthias , Aichberger Karl J. , Florian Stefan , Krauth Maria-Theresa , Derdak Sophia , Esterbauer Harald , Wagner Oswald , Pickl Winfried F. , Selzer Edgar , Deininger Michael W. , Druker Brian J. , Greish Khaled , Maeda Hiroshi , Sillaber Christian , Valent Peter 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the BCR/ABL oncogene and an increased survival of leukemic cells. The BCR-ABL tyrosine kinase inhibitor imatinib has successfully been introduced as a treatment of CML. However, resistance after an intitial response is common in patients with advanced disease, and it is not yet clear if responses in early disease phases will be durable. Therefore, current studies focus on novel potential drug-targets in CML cells. We have recently identified heme oxygenase-1 (HO-1) as a novel BCR/ABL-dependent survival-molecule in primary CML cells. In this study, we analyzed signal transduction pathways underlying BCR/ABL-induced expression of HO-1 and evaluated the role of HO-1 as a potential new target of drug therapy. We found that the PI3-kinase inhibitor LY294002 and MEK inhibitor PD98059 downregulate expression of HO-1 in CML cells. In addition, constitutively active Ras- and Akt -mutants were found to promote expression of HO-1 in Ba/F3 cells, further supporting the involvement of the PI3-kinase/Akt as well as the MAPK pathway in regulating HO-1 expression. To establish a role for HO-1 in survival of CML cells, expression of HO-1 was silenced by siRNAs which resulted in apoptosis of K562 cells. Next, HO-1 was targeted in CML cells by pegylated zinc protoporphyrin (PEG-ZnPP), a competitive inhibitor of HO-1. Exposure to PEG-ZnPP resulted in growth inhibition and induction of apoptosis in primary CML cells as well as in the CML-derived cell lines K562 and KU812 with IC50 values ranging between 1–10 μM. The growth-inhibitory effects of PEG-ZnPP were not only observed in CML cells responsive to imatinib, but also in imatinib-resistant K562 cells and Ba/F3 cells expressing various imatinib-resistant mutants of BCR/ABL (T315I, E255K, M351T, Y253F, Q252H, H396P). Moreover, imatinib and PEG-ZnPP were found to exert synergistic growth inhibitory effects on imatinib-resistant leukemic cells. Together, these data suggest that HO-1 represents a novel drug target in cells expressing BCR/ABL, including those with resistance to imatinib.
High Levels of CD34 Expression in Bone Marrow Trephines Predicts for Loss of Cytogenetic Response in Patients with in Chronic Phase Who Have Achieved Complete Cytogenetic Remission on Imatinib Therapy.
Authors: Marin David , Horncastle D. , Howard Jill , Andreasson Catherina , Elliot V. , Goldman Jonh M. , Apperley Jane F. , Naresh Kikkeri , Lampert Irvin 
Published: October 2019

Abstract
We studied the expression of CD34 in bone marrow trephines in 71 patients with CML in CP who had achieved complete cytogenetic remission (CCyR) on imatinib in order to predict the durability of the cytogenetic response. 26 patients received the imatinib shortly after diagnosis while 45 were treated after interferon failure. Imatinib was given at a dose of 400 mg daily. Imatinib dosage was adjusted depending on tolerance and response. Bone marrow examinations were performed every 3-6 months. The CD34 stain was performed in paraffin sections with the monoclonal antibody QBEND10. A minimum of 500 nucleated cells were counted per trephine section. Results were expressed as percentage of CD34+ vs all nucleated marrow cells. The expression of CD34 by the endothelial cells was used as an internal positive control. The median follow up was 1120 days (percentile 25 and 75: 960 and 1254 days). The median time to the achievement of CCyR was 254 days (percentile 25 and 75: 167 and 380 days). Eight patients lost CCyR at a median time of 981 days (range 546 to 1428 days). For the study we used the first evaluable bone marrow taken after the start of the imatinib therapy (median time 242 days; percentile 25 and 75: 110 and 342 days. The median value of CD34 was 0.9% (percentile 25 and 75 percentiles: 0.8 and 1%). Univariate and multivariate analysis for loss of CCyR was performed. The variables included in the analysis were: age, sex, Sokal and Euro risk groups, percentage of blasts in the bone marrow aspirate, new patient vs IFN intolerant, time from diagnosis to the start of the imatinib therapy and the presence of additional cytogenetic abnormalities. The only variable found to be significant in the univariate and in the multivariate analysis was the level of CD34, RR=2.45 (95CI 1.29–4.64, p=0.006). When the variable was categorised as CD34 level <=2% and CD34 level >2% the cumulative incidence of loss of CCyR at 4 years was 10.58% (95CI 3.8–10.6%) in the patients with a CD34 <=2% (n=64) vs 75% (95CI 31.9–95.1%) in the patients with a CD34 >2% (n=7), p<0.0001. BCR-ABL positive hematopoietic progenitors resistant to imatinib are detectable in all the patients who have achieved cytogenetic remission (Bhatia, 2003. Graham 2002) and may be important in disease relapse. We have shown that patients in CCyR whose percentage of CD34 positivity in trephine biopsies. taken shortly after commencing imatinib, is in excess of 2% are much more likely to lose cytogenetic remission than those with lower levels. We speculate that an increase in CD34 expression could reflect an increase in leukemic precursors in some patients.
Clinical Useful Prognostic Index for Adult Patients with Acute Myeloid Leukemia in First Relapse.
Authors: Breems Dimitri A. , Van Putten Wim L.J. , Lowenberg Bob 
Published: October 2019

Abstract
The treatment of acute myeloid leukemia (AML) in first relapse is associated with unsatisfactory rates of complete responses that are usually short lived. Previously proposed prognostic classification methods serving therapeutic decisions and evaluation of investigational treatment strategies at relapse of AML have been based on the duration of the relapse free interval and have largely neglected the influence of other known prognostic factors. Here we present an improved clinically useful prognostic index. This index has been developed from a multivariate analysis of 667 AML patients in first relapse among 1540 newly diagnosed non-M3 AML patients of age 15 to 60 years entered into three successive HOVON/SAKK Collaborative Group trials. The score, which has a range of 0 to 14 points, uses four relevant parameters. The parameters are: length of relapse free interval after first complete remission (more than 18 months: 0 points; 7 to 18 months: 3 points; 6 months or less: 5 points), cytogenetics at diagnosis (t(16;16) or inv(16): 0 points; t(8;21): 3 points; other cytogenetics: 5 points), age at relapse (35 years or younger: 0 points; 36 to 45 years: 1 point; older than 45 years: 2 points) and whether or not a previous stem cell transplantation (SCT) has been undertaken in first complete remission (no SCT: 0 points; previous SCT: 2 points). These points were assigned following estimations of the relative values of each of these factors contributing to outcome. Ultimately, three risk groups were defined: a favorable prognostic group A (0 to 6 points; overall survival of 70% at one year and 46% at five years), an intermediate risk group B (7 to 9 points; overall survival of 49% at one year and 18% at five years), and an unfavorable risk group C (10 to 14 points; overall survival of 16% at one year and 4% at five years). Thus, four commonly applied clinical parameters may identify among patients with AML in first relapse those for salvage or investigational therapy.
Distinctive Efficacy of IVIG in Ameliorating Thrombocytopenia Induced by Anti-Platelet GPIIbIIIa and GPIbα Antibodies.
Authors: Sayeh Ebrahim , Crow Min , Nieswandt Bernhard , Freedman John , Ni Heyu 
Published: October 2019

Abstract
Idiopathic autoimmune thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibody-mediated platelet destruction. The major platelet antigens that autoantibodies are directed against in ITP are GPIIbIIIa and GPIbα. Intravenous immunoglobulin G (IVIG) is a treatment used in ITP; however, some patients are refractory to this treatment and the mechanisms of action of IVIG in this disease are not fully understood. We tested the hypothesis that IVIG may not be equally effective in preventing ITP caused by GPIIbIIIa versus GPIbα anti-platelet antibodies. Thrombocytopenia was induced in Balb/c mice using three monoclonal antibodies against either mouse platelet GPIIbIIIa (JON1, 2, −3) or against GPIbα (p0p 3, 4, −5). Mouse platelet counts were performed daily from day one (pre-treatment) until day five using a flow cytometric assay. The efficacy of IVIG in inhibiting monoclonal antibody-induced platelet destruction in mice was assessed by intraperitoneal injection of IVIG (2 g/kg) 24-hours before anti-platelet antibody injections (7 mg/mouse, i.v.) on day two. We found that pre-treatment with IVIG resulted in a significantly higher platelet count on day three in all groups of anti-GPIIbIIIa-injected animals (n=3/antibody; for JON1, P=0.002; JON2, P=0.015; JON3, P=0.002) as compared to albumin-treated controls. In contrast, IVIG failed to prevent platelet clearance and thrombocytopenia on day three in two anti-GPIbα antibody-treated groups (p0p3 and p0p5, n=3/antibody), but did inhibit platelet destruction in mice that were injected with p0p4 antibody (P=0.0003). Our results indicate that the efficacy of IVIG treatment in preventing ITP induced by these anti-platelet antibodies is affected by their antigen specificities. These data also suggest that patients with ITP mediated by GPIbα may be less responsive to IVIG treatment than those with ITP mediated by GPIIbIIIa antibodies.
A 3′ UTR Single Nucleotide Polymorphism 829C→T in Dihydrofolate Reductase Gene Results in an Increase in DHFR Protein Level and MTX Resistance.
Authors: Mishra Prasunkumar J. , Longo Giuseppe S.A. , Menon Lata G. , Abali Emine , Banerjee Debabrata , Bertino Joseph 
Published: October 2019

Abstract
Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate (THF) required for the synthesis of thymidylate and purines. Methotrexate (MTX) acts as a tight-binding inhibitor of DHFR and remains an important chemotherapeutic agent for treatment of leukemias and lymphomas. Increased DHFR confers resistance to antifolates in target cells. A previously reported single nucleotide polymorphism (SNP) 829C/C→829T/T (829C→T) found in the 3′- untranslated region of DHFR gene transcript (between the first and second polyadenylation site) was associated with higher expression of the DHFR transcript. The SNP was identified in 5.4% of the cases and 6.0% in the controls of Japanese patients with childhood leukemia/lymphomas (Goto et al. 2001, Clinical Cancer Research, Vol. 7, 1952-1956). The objective of the present study was to determine the role of the 3′ UTR SNP 829C→T in DHFR gene expression, DHFR protein level and resistance to MTX. The mutation 829C→T in the 3′ UTR of wild type DHFR was introduced by site directed mutagenesis and the mutant cDNA expressed in DHFR deficient CHO cells (DG-44), wild type DHFR and vector alone constructs were also transfected into DG44 as controls. After two weeks of selection in G418 containing media, several well-isolated surviving colonies were picked and expanded as cell lines in media containing G418. Real-time quantitative PCR was used to compare mRNA and genomic DNA level of the clones while Western blotting was used to compare the protein levels. MTX cytotoxicity assay was carried out in media lacking thymidine. Clones expressing the mutant 829C→T showed greater than two fold enhanced expression of DHFR transcripts as compared to wild type clones. Corresponding to the high mRNA levels, an increase in DHFR protein level was observed in the mutant clones without an increase in DHFR gene copy number. Cytotoxicity studies showed that cell lines with increased levels of DHFR were significantly more resistant to MTX than cells with wild type 3′ UTR. Of interest clonogenic efficiency of the mutants in medium lacking thymidine was greater than wild type and was directly proportional to the level of DHFR expressed in the clones. This study demonstrates that when SNP 829C→T is introduced in the 3′ UTR of wild type DHFR, the expression of the DHFR mRNA is enhanced with a corresponding increase in the protein level. The presence of a SNP 829C→T in patients with ALL may contribute to treatment failure, as MTX is a key drug in curative regimen for this disease. Future studies are directed toward determining the abundance of this SNP in other populations, and the correlation between this SNP and clinical methotrexate resistance and or decreased MTX toxicity.
Heme Affects Sensitivity to Imatinib through Regulation of Nrf2 Activity in BCR/ABL-Positive Cell Lines.
Authors: Miyoshi Takuji , Nagai Tadashi , Nakamura Makiko , Ohmine Ken , Fujita Hiroyoshi , Taketani Shigeru , Komatsu Norio , Ozawa Keiya 
Published: October 2019

Abstract
Heme has been recognized as an important modulator of the functions of various cellular molecules. Recent work showed that heme binds to Bach1 transcription factor, resulting in the loss of the repressor activity of Bach1. Because Nrf2 transcription factor and Bach1 may competitively recognize the common gene regulatory elements, heme may have an effect on the Nrf2-madiated gene expressions. We recently reported that Nrf2 is involved in the resistance to imatinib in BCR/ABL-positive cells. Here we investigated the role of heme in the sensitivity to imatinib in human BCR/ABL-positive cell lines. Addition of hemin to KCL22 and KU812 cells resulted in significant increases in the IC50 values of imatinib in accordance with the abrogation of imatinib-mediated induction of apoptosis-related molecules, cleaved caspase 3 and cleaved PARP. The levels of phosphorylated BCR/ABL and phosphorylated ERK1/2 MAP kinase (active form) were both decreased by imatinib even when hemin was added, indicating that hemin does not interfere with imatinib-mediated inhibition of BCR/ABL function and that mechanisms of hemin-induced acquisition of imatinib-resistance is not related to BCR/ABL kinase activity. Hemin increased Nrf2/DNA complex formation at the antioxidant response element of the human γ-glutamylcystein synthetase (γ-GCS) light subunit gene promoter. Because γ-GCS is a rate-limiting enzyme of the synthesis of glutathione (GSH), which has been shown to be involved in resistance to imatinib, we next examined the level of GSH and found that the intracellular amount of GSH was increased by hemin treatment of these cells. In addition, the protein level of heme oxygenase-1, which is important for survival of chronic myeloid leukemia cells and the gene expression of which can be also regulated by Nrf2, was increased after hemin treatment. Addition of ascorbic acid, which suppresses Nrf2-dependent gene expression, partially restored the sensitivity to imatinib in hemin-treated cells. We also showed that inhibition of intracellular heme synthesis by succinyl acetone increased the sensitivity to imatinib in imatinib-resistant cell lines, KCL22/SR and KU812/SR. The results suggest that heme is critically involved in the sensitivity to imatinib, at least in part, through regulation of Nrf2 function.
Persistence of eGFP Marked Bone Marrow Cells in Long-Term Hematopoiesis.
Authors: Pilz Ingo H. , Schmidt Manfred , Ball Claudia , Glimm Hanno , von Weizsäcker Fritz , von Kalle Christof 
Published: October 2019

Abstract
To study transplanted unperturbed and mobilized long-term hematopoiesis after selection with an alkylating agent, bone marrow (BM) from 5 C57BL/6J mice was pooled, repeatedly transduced with retroviruses encoding the alkylating agent resistance protein O6-Methylguanine-DNA and enhanced green fluorescent protein (eGFP) as an easily traceable marker. Between 1 to 9x105 transfected BM cells were transplanted into 15 myeloablatively irradiated sex-mismatched C57BL/6J mice. Subsequently, 3 to 4 selection rounds with BCNU/O6-BG were carried out, enriching eGFP marked hematopoiesis in these mice up to 70–90%. Between 1 and 7x107BM cells of different mice were transplanted according to marrow location into groups of 5 sex-matched Bri44[1] mice. Two mice each received BM from the hind limbs, two from the pelvis and one received cells from the spleen, only, respectively. Altogether the study comprised 15 groups divided into 6 female and 9 male groups. Of these, 4 male and 3 female groups received 3 HSC-mobilization courses with G-CSF at intervals of 2 months starting 3 month after transplantation. Hematopoiesis in the other fraction remained unperturbed. During the observation period of 11–14 months in these tertiary recipients, repeated FACS analyses as well as linear amplification mediated (LAM) PCRs were carried out to track the clonal contributions. A decrease in the percentage of eGFP expressing marked hematopoiesis was observed in most cases. However, eGFP expression never disappeared altogether and could still be detected in the different hematopoietic lineages and successfully sorted for further analyses by MoFlo (Dako-Cytomation). Assessment of the clonal status of the Bri44 by LAM-PCR displayed interesting results. In some mice a decline in clone numbers was observed, whereas clone numbers remained stable in others. Tertiary transplantation with long-term follow-up indicates that this observation may be related to the transplantation of limited long-term repopulating clone numbers and progenitor cell exhaustion over time.