bluuebunny/crossref_metadata_2025_split huggingface dataset:
X-Ray Cristallography Characterization of Human CD44 Epitopes Releasing Differentiation Blockage in Acute Myeloid Leukemia Blasts.
Authors: Lecomte Nicolas , Durand Laetitia , Roseto Alberto , Chomienne Christine , Kadouche Jean , Smadja-Joffe Florence 
Published: October 2019

Abstract
CD44 is a highly glycosylated type-1 transmembrane protein with multiple isoforms, including the standard one, CD44s (80kDa), which is the most abundant on hematopoietic cells, and alternatively spliced variant isoforms (CD44v). CD44 is strongly expressed on Acute Myeloid Leukemia (AML) blasts, which are immature myeloid cells which terminal differentiation is blocked at various stages, defining the distinct AML subtypes. In our laboratory, we have previously shown that several specific monoclonal antibodies (mAbs) directed to the CD44s isoform, can release the differentiation blockage of primary AML blasts (Charrad et al., Nature Medicine 1999), in all AML subtypes. These results indicated the possibility of using such mAbs to develop a CD44-targeted differentiation therapy that would be efficient in all AML subtypes. Our present aim is to characterize the molecular structure of the CD44 epitopes involved in the release of AML differentiation blockage, by using X-ray crystallography. In order to obtain high levels of soluble CD44 protein and to preserve its native conformational properties, we have constructed distinct fusion proteins comprising the glycosylated CD44s extracellular domain plus the human IgG1 Fc fragment (CD44-Fc). We have also added a polyhistidine tag, which allows to easily purify them by using Ni2+- and protein G affinity, and a TEV specific recognition sequence to cleave off the fusion partner. Eight distinct CD44-Fc constructions have been prepared, transiently expressed in COS-7 cells and the one displaying both the highest affinity for the anti-CD44 mAbs and the highest expression level, has been transfected into CHO-K1 cells. Using a stable cell clone, we have succeeded to produce large amounts of CD44-Fc (100 to 200 mg/L), in serum-free culture medium, using CELLine bioreactor flasks. This optimization of the CD44 production combined with an efficient three-step purification process (successive specific binding to G protein, to Ni2+, concluded by highly resolutive gel filtration), has allowed us to produce, for the first time, a sufficient quantity of highly pure and glycosylated CD44s extracellular domain for structural studies. SPR (Surface Plasmon Resonance) analysis have shown that the 2 differentiation-inducing anti-CD44 mAbs analyzed so far, differ in their dynamics of interaction with the CD44 protein. The molecular structure of the epitopes specifically mapped by differentiation-inducing anti-CD44 mAbs, as revealed by X-ray crystallography, will be presented and discussed, with the perspective of developing an efficient CD44-targeted differentiation therapy in AML.
Bcl-2 siRNA Induces Apoptosis and Enhances the Sensitivity to Arsenic Trioxide in NB4 Cells.
Authors: He Dongmei , Zhang Yuan , Liu Gexiu 
Published: October 2019

Abstract
Bcl-2 is the prominent member of a family of proteins responsible for dysregulation of apoptosis, prevention of cancer-cell death, and resistance to chemotherapy and radiotherapy. Overexpression of Bcl-2 protein was shown to result in resistance to a variety of apoptosis-inducing signals in acute leukemia. Arsenic trioxide (As203)induces clinical remission in acute promyelocytic leukemia with minimal toxicity and apoptosis in APL-derived NB4 cells at low (0.5 to 2 μmol/L) concentration. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. In this study, we studied the effect of transfection with siRNA targeting Bcl-2 in NB4 cells in terms of proliferation and induction of apoptosis. Further,we investigated whether the Bcl-2 siRNA may enhance the sensitivity of NB4 cells to arsenic trioxide. Synthetic siRNA was transferred against Bcl-2 into NB4 cells using Oligofectamine transfection protocol in the presence or absence of arsenic trioxide. RT-PCR and Western blotting analysis of Bcl-2 expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a modified MTT assay, counting cell number. Apoptosis was determined by morphological observation and flow cytometric analysis. When synthetic Bcl-2 siRNA was delivered into NB4 cells, no Bcl-2 transcripts were detected at 24h, and Bcl-2 protein levels were reduced by approximately 72% for 48h after siRNA transfer. Bcl-2 siRNA treatment for 72h led to 36% apoptosis of cells,as compared to control siRNA treatment. Control siRNA had no significant effect on growth and apoptosis of cells. Simultaneous administration of As203 and Bcl-2 siRNA was able to reduce the mRNA and protein expression of Bcl-2 significantly compared to Bcl-2 siRNA alone demonstrating a synergistic effect of combined As203 and siRNA treatment (P<0.05). Proliferation in NB4 cells was strongly inhibited (about 86%) following combination treatment for 72h as opposed to only 40% after administration of 0.5μmol/L As203 alone. When NB4 cells were incubated with 0.5μmol/L As203 for 72h after siRNA transfer, the percentage of apoptotic cells significantly increased (P<0.05), compared with combined As203 and control siRNA treatment or As203 treatment alone, respectively. No significant difference on apoptosis was seen in cells treated with As203 alone or treated with As203 and control siRNA. These results show that Bcl-2 siRNA could induce apoptosis and enhance the sensitivity to arsenic trioxide in NB4 cells. Our results suggest that the combination of arsenic trioxide and Bcl-2 siRNA might be an effective anti-leukemia protocol.
Comparative Effect of the Oral Iron Chelators ICL670A and CP20 on Cell Proliferation, Apoptosis and Polyamine Metabolism in Rat and Human Liver Cell Cultures.
Authors: Gaboriau François , Chantrel-Groussard Karine , Pasdeloup Nicole , Nick Hans Peter , Brissot Pierre , Lescoat Gérard 
Published: October 2019

Abstract
Iron overload increases the mitotic index in rat hepatocyte cultures stimulated by EGF. Iron, implicated in tumoral cell proliferation, promotes the development of the hepatocellular carcinoma. Conversely, iron chelation decreases cell proliferation. Polyamines play also an important role in cell proliferation. The purpose of the present work was to compare the effect of the two oral iron chelators ICL670A and CP20 on cell proliferation, apoptosis and polyamine metabolism in rat and human liver cell cultures.
We used three experimental models: the rat hepatoma cell line FAO, the rat liver epithelial cell line RLEC and the human hepatoma cell line HUH7. Chelator cell uptake was analyzed by mass spectrometry. DNA replication and cell viability were measured by thymidine incorporation and mitochondrial succinate dehydrogenase activity. Cytotoxicity was evaluated by extracellular LDH activity. Cell cycle analysis was performed by flow cytometry. Apoptosis was quantified by DNA fragmentation and caspase 3 activity. Polyamine metabolism was analyzed by measuring their intracellular concentrations by mass spectrometry as well as the ornithine decarboxylase (ODC) and spermine-spermidine acetyl transferase (SSAT) mRNAs levels. ICL670A entered the cells four times better than CP20. We observed that ICL670A (20 μM) decreased DNA replication (−75%) and cell viability (−40%) while a comparable CP20 stoechiometric concentration (30 μM) had no significant effect. ICL670A exhibited also a higher cytotoxicity than CP20. The decrease in DNA replication induced by ICL670A was correlated to a cell cycle block in S phase; CP20 (30 μM) increased slightly the cell number in G2/M phase while a higher CP20 concentration (150 μM) blocked clearly cell cycle in G2/M phase. By measuring DNA fragmentation and the activity of the executioner caspase 3, we demonstrated a high apoptotic effect of ICL670A (20 μM) compared to CP20 (30 μM). In the HUH7 human cell line, we observed that ICL670A (20 μM) decreased the intracellular levels of polyamines which was correlated to a drop in ODC and SSAT mRNAs levels. Conversely, an increase in SSAT mRNA level was observed in the presence of CP20 (150 μM) leading to an enhancement of putrescine and spermidine levels.
In conclusion, ICL670A and CP20 inhibit cell proliferation by two distinct ways. ICL670A compared to CP20:
enters better the cells; exerts a higher antiproliferative and apoptotic effect; modulates polyamine metabolism by inhibiting ODC and SSAT enzyme activities.
Arsenic Trioxide Enhances STI571-Induced Apoptosis of K562 Cells through Downregulating Bcl-XL and Bcr-Abl.
Authors: Zhang Ri , Zhang Xuhui , Zhi YaJun , Zhu ZiLing , Wu De Pei 
Published: October 2019

Abstract
We aimed to evaluate the effects of arsenic trioxide (ATO, Trisenox) on STI571 (Gleevec, imatinib mesylate)-induced apoptosis of a chronic myelogenous leukemia (CML) cell line, K562 cells. Cell prolifration and colony-forming assays were performed to determine the cytotoxicity of ATO alone and in combination with STI571. Apoptosis was analyzed by morphological changes, apoptosis rate and cell cycles. An Elisa assay was used to detect the levels of cytosolic cytochrome c (cyt c) and caspase-3 in K562 cells exposed to ATO and STI571 at graded concentrations. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assay the transcriptional levels of Bcl-XL and Bcr-Abl genes in K562 cells. Results showed that both the colony-forming assay and cell proliferation assay demonstrated additive to synergistic effects of ATO on STI571-induced apoptosis in K562 cells, a Bcr-Abl positive cell line. Caspase-3 was activated during apoptosis and there was an increase in cytosolic accumulation of cytochrome c. Treatment of K562 cells with STI571 alone led to down-regulation of transcriptional levels of Bcl-XL at 12 hours and Bcr-Abl at 96 hours after drug administration. Treatment with ATO alone only led to reduce the mRNA levels of Bcl-XL, but not Bcr-Abl. Combined treatment with ATO and STI571 down regulated the transcripts of Bcl-XL at 12 hours and Bcr-Abl 72 hours after drug administration. We conclude that ATO enhanced cytotoxic and proapoptotic actions of STI571 could be mediated by the down-regulation of Bcr-Abl and Bcl-XL genes in K562 cells. Therefore ATO in combination with STI571 could be a promising therapy for CML.
A Phase I Study of Temozolomide (Temodar®) in Pediatric Patients with Relapsed or Refractory Leukemia—A Children’s Oncology Group Study.
Authors: Horton Terzah M. , Dolan Eileen , Hegde Madhuri , Berg Stacey L. , Adamson Peter , Blaney Susan M. 
Published: October 2019

Abstract
A phase I study of oral temozolomide, administered daily for 5 days every 28 days, was performed in children with relapsed or primary refractory leukemias. The starting dose was 200 mg/m2/day with subsequent dose escalation to 260 mg/m2/day. Ten patients (5 male, 5 female), median age 9 years (range: 1–18 years), with leukemia (6 with acute lymphocytic leukemia [ALL], 4 with acute myelogenous leukemia [AML]) received a total of 14 cycles of temozolomide. Serum pharmacokinetic (PK) samples were obtained in 6 patients using a limited pharmacokinetic sampling strategy. Samples were also obtained from 8 patients for correlative biology studies, including analysis of methyl-guanine methyl transferase (MGMT) activity and microsatellite instability (MSI). MGMT activity was measured in 7 patients; all 3 patients with ALL tested for MGMT had elevated MGMT activity, with a median MGMT activity of 1240 fmol/mg protein (range: 356–1756). In contrast, 3 out of 4 patients with AML had MGMT activity below the limits of detection. Eight patients were tested for microsatellite instability by multiplex PCR using a panel of 13 MSI loci. Two patients (one with ALL and one with AML) demonstrated probable MSI instability. Temozolomide has been well tolerated, with no patients developing dose-limiting toxicity to date. One patient with AML, undetectable MGMT activity and no microsatellite instability had a partial response (PR) lasting 4 cycles (<10% myeloblasts in bone marrow). Further analysis is underway to determine the frequency of elevated MGMT activity in ALL and AML patients at diagnosis and following relapse.
HuMax CD20 Fully Human Monoclonal Antibody in Chronic Lymphocytic Leuchemia. Early Results from an Ongoing Phase I/II Clinical Trial.
Authors: Coiffier Bertrand , Tilly H. , Pedersen L. M. , Plesner T. , Frederiksen H. , van Oers M.H.J. , Wooldridge J. , Kloczko J. , Holowiecki J. , Hellmann A. , Walewski J. J. , Flensburg M.F. , Petersen J. , Robak T. 
Published: October 2019

Abstract
The fully human monoclonal IgG1 antibody HuMax-CD20 targets a novel epitope of the CD20 molecule on B-cells. HuMax-CD20 stops growth of engrafted B-cell tumors in SCID mice more efficiently than Rituximab and i.v. infusion of HuMax-CD20 in cynomolgus monkeys leads to profound, long lasting, dose-dependent B-cell depletion. Data are presented from an open label, dose-escalation, multicenter phase I/II clinical trial. 3 cohorts of 3 (A), 3 (B) and an extended cohort of 27 (C) patients with relapsed or refractory chronic lymphocytic leukemia (B-CLL) received 4 weekly i.v. infusions of HuMax-CD20 and will be followed for 12 months. The first infusion administered was 100 mg, 300 mg and 500 mg in cohort A, B and C. The following 3 infusions were of 500, 1000 and 2000 mg, respectively. Patients were premedicated with oral acetaminophen and i.v antihistamine and received i.v. glucocorticoids before first and second infusions. The endpoints are B-cell depletion, adverse events, objective response according to the NCI working group guidelines for CLL, time to progression, duration of response, time to next anti-CLL treatment, and pharmacokinetics. Biopsies and CT images are evaluated centrally. Median age was 61 years; median time since diagnosis was 6.3 years. Maximum tolerated dose was not reached. Adverse events were predominantly observed on days of infusion and as expected most frequently symptoms of cytokine release e.g. rash, increased sweating, fatigue, rigors, pyrexia, and headache. 5 serious adverse events assessed as related to HuMax-CD20 treatment have been reported: hepatic cytolysis, herpes zoster, neutropenia (2 patients) and one death from pneumonia at week 4. In cohorts A and B, markedly reduced CD19+CD5+ cell counts were observed in 3 of 6 patients one week after final treatment and the depletion was sustained in one of these patients. In cohort C a pronounced CD19+CD5+ reduction was demonstrated by all patients; in fact the lymphocyte counts were ≤4.0 x 109/L in 21 patients. At week 11, ≥50% reduction in the product of diameters of lymph nodes was observed in 12 of 20 evaluable patients with enlarged lymph nodes. A response rate of 52% (11 of 21 evaluable patients in cohort C) was observed at week 11: 4 clinical CR (bone marrow and CT pending), 7 PR, 3 SD and 7 PD. In conclusion, this preliminary analysis of data from the first 33 CLL patients treated with HuMax-CD20 demonstrated significant depletion of CD19+CD5+ cells, a favorable safety profile and an indication of clinical efficacy. An updated report for all patients at week 19 will be presented.
Clinical and Interphase-FISH Studies of Deletion of Derivative Chromosome 9 in Patients with Chronic Myelogenous Leukemia.
Authors: Wu Wei , Xue Yong-quan , Wu Ya-fang , Pan Jin-lan , Shen Juan 
Published: October 2019

Abstract
Objective: To determine the frequency of the deletion of derivative 9 [der(9)] among chronic myeloid leukemia (CML) patients with classic Ph translocation and variant Ph translocation, and to assess the association between this deletion and clinical prognosis.
Methods: Cytogenetical analysis of bone marrow cells was performed by direct method and /or 24h culture method. RHG banding was used for karyotype analysis. Dual-color and dual-fusion DNA probe was used to perform interphase-FISH to investigate the deletion of der(9) in Ph+ CML patients and all patients were followed up.
Result: Cytogenetical studies showed typical Ph translocation in 76/105 and variant Ph translocation in 29/105. Interphase-FISH studies showed deletion of der(9) in 12 cases(15.8%) of 76 patients with classic Ph translocation and in 4 cases (13.7%) of 29 patients with variant translocation. The frequency of deletion was similar in classic and variant translocations (P>0.9). This result is contrary to previous reports which suggested that deletions are much more common in variant Ph translocation than in classic Ph translocation. When the deletion was seen in a patient, it was present in all the Ph+ metaphases and nuclei. Three patients with heterogeneous cell populations mixed with cells with single 5′-ABL or 3′-BCR deletion and with both 5′-ABL and 3′-BCR deletion. It may suggest clone evolution in the progression of deletion. Complete clinical information was available in 54 patients. There were no significant difference in peripheral leukocyte count, platelet count, hemoglobin and percentage of peripheral blood blast cells between patients with and without der(9)deletion. However, the results of following up showed that the median survival time of patients with der(9) deletion was significantly shorter than those without der(9) deletion (34 months vs 76 months; P<0.05, log-rank Test).
Conclusion: A deletion of der(9) is seen in about 1/6 Ph+ CML patients in china, with which Ph+ CML patients have shorter median survival time than those without it, indicating that it is a poor prognostic index. For evolution the prognosis of CML patients more precisely, it is best to perform cytogenetical analysis and FISH analysis for der(9) deletion simultaneously at diagnosis.
Evaluation of Molecular Response by Real-Time RT-PCR in Acute Promyelocytic Leukemia (APL): A Direct Comparison with Regular RT-PCR.
Authors: Hu Jiong , Liu Yuan-Fang , Zhu Yong-Mei , Zhang Shu-Jiang , Gu Bai-Wei , Shen Zhi-Xiang , Hu Jiong 
Published: October 2019

Abstract
PURPOSE: In this study, we systemically compare the molecular response by regular reverse transcript polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR in newly diagnosed patients with acute promyelocyte leukemia (APL).
PATIENTS AND METHODS: A total of 31 newly diagnosed patients with APL who entered remission by induction therapy with ATRA plus As2O3 were included in this study with median follow-up of 38 months (27–46 months). Serial bone marrow samples were obtained before and after the induction, consolidation chemotherapy and every 3 to 6 months during maintenance therapy. Regular RT-PCR and real-time RT-PCR were carried out to evaluate the molecular response. The PML-RARa fusion transcript level was calculated in two ways: normalized PML-RARa level as DoseN and log-reduction compared to the pre-treatment level.
RESULTS: A total of 29 patients remained disease free for a median of 27 to 46 months. Comparing two PCR assays, the sensitivity limit for real-time RT-PCR was 5.7 log DoseN reduction while the limit for regular RT-PCR was about 3 log. Furthermore, we established criteria for evaluation of molecular response by real-time RT-PCR: ≥3.0 log reduction of PML-RARa transcript level as minor molecular response; ≥5.0 log reduction as major molecular response and undetectable level of PML-RARa transcript as complete molecular responses. Minor molecular response by real-time RT-PCR was achieved in 22.6% and 96.8% of patients after induction and consolidation chemotherapy, which was equivalent to molecular response by regular RT-PCR (35.5% and 96.8% respectively). The major molecular response rate was 12.9% and 90.3% after induction and consolidation whereas the complete response was at 3.23% and 77.4% respectively. All patients achieved major and complete molecular remission during and after maintenance therapy. Loss of either major or complete molecular response occurred in two patients while both patients experienced subsequent loss of minor molecular response, which coincided with CNS leukemia and bone marrow relapse.
CONCLUSION: Real-time RT-PCR was more sensitive than regular RT-PCR and the evaluation criteria for real-time RT-PCR was established and proved to be clinically relevant.
Serum Tryptase Levels in Acute Leukemia at Diagnosis: Correlation with CBF AML.
Authors: Cairoli Roberto , Ripamonti Carla B. , Granata Simonetta , Folli Daniele , Brasca Paola , Grillo Giovanni , Bertani Gianbattista , Intropido Liliana , Marenco Paola , Nosari Annamaria , Pezzetti Laura , Morra Enrica 
Published: October 2019

Abstract
Α and β tryptase genes cluster on the short arm of human chromosome 16 and encode lineage-associated serine proteases that are abundantly expressed in mast cells and, in trace amounts, in basophils. Under physiologic conditions no other myeloid cells express tryptases. However, in several myeloid leukemia cell lines and in AML blasts, the level of tryptase is elevated. In vitro, AML blasts are capable to produce and release the α-protryptase constitutively. Recently, elevated levels of serum tryptase have been detected in certain FAB subtypes of AML, particularly in AML-M4eo. In an attempt at correlating the levels of tryptase with FAB classification and cytogenetics, we have analyzed serum samples collected at diagnosis, from 103 AML and 57 ALL patients referred to our Institution. The total serum concentration was determined by UniCAP 100 and UniCAP Tryptase Fluorenzyme Immunoassay Kit (Pharmacia-Upjohn, Uppsala, Sweden). The median value of tryptase level in the control group (50 healthy people; mean age 35 y, range 20–50; M/F= 26/24) amounted to less than 5 ng/ml. We found elevated tryptase levels (more than 15 ng/ml) in 46 out of 103 AML-patients (44.6%) and in 1 out of 57 ALL-patients (1.75%) (p = <0.0001). In AML, the patients showing elevated tryptase levels resulted equally distributed among the FAB subtypes (M0, M1, M2, M3, M4, M4Eo, M5) (p = 0.269). In the group of 18 AML with t(8;21) or inv(16), we recorded the highest frequency (89%) of cases with tryptase more than 15 ng/ml (p =< 0.001) and the highest median serum tryptase levels (>89.3 ng/ml) (p = 0.0001), in comparison with other cytogenetic groups (diploid, n= 43; t(15;17), n=10; other, n=29). Our data suggest that elevated serum tryptase levels at diagnosis may play a role as a marker for CBF AML subtypes.