bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Gossypol Acetate on Proliferation and Apoptosis in Lymphoblastoid Cell Line and Primary ALL and CLL Cells.
Authors: Zhao Yongqiang , Cheng Wei , Hua Baolai , Wang Shujie , Yang Dajun 
Published: October 2019

Abstract
Gossypol is a polyphenolic compound extracted from the cotton plant of Malvaceae family. It was originally demonstrated as a male antifertility agent. In the 1960’s, its anticancer efficacy was identified. In the last few years, gossypol has been approved to have antiproliferative and apoptosis-inducing effects on some kinds of cancer cell lines in vitro. Very recently, it has been showed that gossypol is a potent small-molecule inhibitor of Bcl-2 and Bcl-XL proteins. It can bind to Bcl-2/Bcl-XL and block the heterodimerization of Bcl-2/Bcl-XL with proapoptotic proteins, such as Bax, Bak and Bad. Such a blocking of antiapoptotic-proapoptotic protein heterodimerization in turn may inhibit the antiapoptotic function of Bcl-2/Bcl-XL, and induce apoptosis of cancer cells. Overexpression of Bcl-2 has been observed in B-cell lymphoma, B-ALL, B-CLL and correlates closely with the occurrence, progress, recurrence and multidrug-resistance of these malignancies. In this study, we found that gossypol acetate was able to inhibit the proliferation and survival of Raji cells in vitro at concentrations of more than 5 μmol/L. The effect was dose and time dependent. IC50 were 11.9±1.2 μmol/L at 24h and 6.8±0.4 μmol/L at 72h, which were 7.1 and 9.1 folds lower than that of normal MNCs, respectively. Gossypol acetate can remarkably increased pro-apoptosis activity of dexamethasone in Raji cells. Cell cycle analysis by flow cytometry (FCM) indicated that gossypol acetate could induce G0/G1 arrest. Apoptosis was demonstrated both morphologically by Wright-Giemsa staining, Hoechst 33342 staining and by DNA ladder formation in agarose-gel electrophoresis. By determination the percentage of subdiploid amount of DNA, apoptosis was quantified. The result showed that gossypol acetate could increase the percentage of cells with fragmented DNA in time and dose dependent manners at the concentrations of more than 1μmol/L. The fraction of annexin V+/PI− cells was also increased with dose. Colorimetric assay showed that an activation of caspase-3 was seen at 12h and peaked at 24h at the concentration of 25μmol/L. Protein expression of Bcl-2 decreased 13.6±3.8% at 12h and 69.5±6.2% at 24h. Gossypol acetate also had apoptosis-inducing effects in primary leukemia cells from patients with ALL (n=7) and CLL (n=3). The concentration inducing apoptosis in CLL cells was higher than that in ALL cells. It was concluded that gossypol acetate could inhibit the proliferation and induce the apoptosis of Raji cells and primary cultured ALL and CLL cells in a dose and time dependent manner. It seems that down-regulation of Bcl-2 protein expression may play a role in the apoptosis.
Inducing the Anti-Leukemia Effects of CD8+ T Cytotoxic Lymphocytes Derived from Umbilical Cord Cell.
Authors: Tan Huo , Liu Xin 
Published: October 2019

Abstract
Objective: The previous studies of our research group have proved that specific cytotoxic T lymphocytes(CTLs) can be generated from umbilical cord cells. The purpose of this study is to further demonstrate whether CD8+CTLs can be generated from cord blood (CB) and kill leukemia cells specifically ex vivo. And whether the leukemia cytotoxicity of CD8+CTLs and CD8−CTLs are different.
Method: First we induce adherent cells collecting from CB samples to generate dendritic cells (DCs) by culturing with GM-CSF, IL-4 and load lysate antigen of U937 cell to immature DCs. On day 6th, TNF-αand PGE2 was added to accelerate the maturation of DCs. After co-culturing DCs with the CTLs from the same CB, then we used MidiMACS to isolate CD8+ and CD8− T cell. The cytotoxicity of the CD8+ CTLs and CD8−−CTLs to U937 cell line was evaluated with Methyl thiazolyl tetrazolium(MTT) assay.
Result: The purity of CD8+CTL was(95.73±1.50)%. Among the CD8−CTLs, the percentage of CD4+CTLs was (65.01±9.29)% and CD8+T lymphocytes was (4.9±4.46)%. When E:T ratios were 40:1, 20:1 and 10:1, the mean cytotoxic rate (MCR)of CD8+ CTLs to U937 cell were (66.36±12.43)%,(35.38±9.64)% and(19.04±6.15)% respectively; the MCR of CD8−CTLs to U937 cell were (34.47±8.19)%,(21.85±7.06)% and(12.26±4.87)% and the MCR of T cell from CB to U937 cell were(15.79±4.64)%,(9.6±3.71)% and (5.69±3.14)%. The MCR of CD8+ CTLs to U937 cell were significant higher than those of CD8−CTLs and T cell groups at the same E:T ratios(P<0.05). When E:T ratios was 40:1,the MCR of CD8+CTLs and CD8−CTLs to K562 cell line were (36.77±10.24)% and (26.95±3.06)% respectively, the cytotoxicity effect of CD8+ and CD8−CTLs to K562 cell are no difference (P>0.05).
Conclusions: The cytotoxicity of CD8+CTLs against U937 cell lines is more potent than that of CD8−CTLs, and this effect is specific.
Autophagic Cell Death in Leukemia Cells Regulated by Ceramide and Sphingosine-1-Phosphate through Mammalian Target of Rapamycin.
Authors: Harakawa Nari , Murata Ken , Yamaoka Shohei , Hori Toshiaki , Domae Naochika , Igarashi Yasuyuki , Umehara Hisanori , Okazaki Toshiro 
Published: October 2019

Abstract
Unlike conventional apoptosis, the mutually exclusive role of ceramide and sphingosine-1-phosphate (S1P) in autophagic cell death remains unclear in leukemia cells. Amino acids deprivation (AA(−))-induced autophagy and subsequent cell death of human leukemia HL-60 cells are preceded by the generation of intracellular ceramide with inhibition of mammalian target of rapamycin (mTOR) in a caspase-3-independent manner. S1P inhibits AA(−)- or N-acetylsphingosine (C2-ceramide)-induced autophagy through activation of an amino acids sensor mTOR, whereas C2-ceramide inhibits S1P-activated mTOR and overcomes inhibition of autophagy by S1P. Genetically activated mTOR inhibits AA(−)- or C2-ceramide-induced increases of autophagy and a mammalian homologue of yeast Apg8/Aut7, MAP-LC3. In contrast, overexpression of kinase-dead mTOR blocks S1P-induced inhibition of autophagy and LC3-II activation. We here suggest that in leukemia cells ceramide an S1P counteract as a novel mediator for autophagic cell death through regulation of mTOR-dependent MAP-LC3.
Effect of Arsenic Trioxide on E14 Murine Embryonic Stem Cells In Vitro.
Authors: Graham-Evans Barbara , Broxmeyer Hal 
Published: October 2019

Abstract
The ability of pluripotent murine embryonic stem (muES) cells to differentiate into all cell types makes them a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the growth and differentiation of the ES cells is a pre-requisite for selecting adequately the cell conditions which will be accessible for future use of ES cells in treating hematological malignancies. Pharmacological agents such as arsenic trioxide (As2O3) have shown to be an effective anticancer agent for acute promyelocytic leukemia but its effect on murine embryonic stem cells prior to differentiation and subsequent target organ toxicity is yet to be determined. This study was done to gain insight into the biological effects of As2O3 on muES cell line, E14, with respect to differentiation, proliferation, cytotoxicity and cell cycle status in vitro. MuEs cells were cultured in complete DMEM in the presence of LIF on gelatin coated plates for 24 hours prior to the addition of varying concentrations of As2O3 (0, 0.5,1, 5, and 10 μmol/L, respectively), and the cells were incubated for an additional 48 hours. The differentiation potential of As2O3 was determined by measuring Oct-4 levels by flow cytometry. Proliferation was measured by trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Cell cycle status was measured by Propidium Iodide using flow cytometry. Our results demonstrated that As2O3 at different concentrations did not induce differentiation. However, the proliferation of muES cells treated with As2O3 was inhibited in a concentration and time dependent manner. The cell-killing rate of As2O3 on muES cells was both dose and time dependent with an inhibitory concentration (IC50) of ~ 5±0.2 μM and ~3.75±0.7 μM at 24 and 48 hours respectively. Propidium iodide DNA staining revealed that after 48 hour incubation with arsenic, cells in S phase remained relatively constant (~62–67%) at low doses of As2O3 but this decreased by half (33%) at a high dose of 10uM As2O3. The percent of cells in the G2 phase of the cell cycle increased to 41±9% in 10μM treated cells compared to 6±3% in control. In conclusion, As2O3 does not induce the differentiation of muES cells into different lineages, but does inhibit growth and alter the cell cycle kinetics of these cells in a dose dependent manner.
Relationship between the As2O3 Induced Expression of NF-κB and Drug Resistance in K562/ADR Cells.
Authors: Zhang Xiao-hong , Su Li-da , Zhao Xiao-ying , Lv Qing-hua 
Published: October 2019

Abstract
Summery: Anti-apoptosis is one of drug resistance mechanisms in leukemic cells. It was found in our early study that As2O3 can induce apoptosis of K562 cells, and this effect involve the degradation of IκB-αand consequently the activation of NF-κB. The relationship between drug resistance of leukemic cells and the expression of both IκB-αand NF-κB associated with apoptosis induced by arsenic trioxide(As2O3) was studied in K562 and K562/ADR cells.
Methods: Apoptosis was induced in K562 and K562/ADR cells cultured with As2O3 in different concentrations. Western blot was used to analyze the expression of NF-κB in nuclear and IκB-α in cytoplasm of these cells. Apoptosis and degradation of IκB-αprotein were also observed by flow cytometry.
Results: The suppressive effect of As2O3 on proliferation of K562/ADR was lower than that in K562 cell, IC50 values were 19.07μmol/L and 5.26μmol/L, respectively. After exposure to As2O3, the ratio of apoptosis cells increased with the concentration of As2O3 in K562 cells, from(13.25±1.83)% to (50.56±8.62)% with variation of As2O3 from 1μmol/L to 4μmol/L(P<0.05). The ratio of apoptosis cells in K562/ADR cultured with 4μmol/L As2O3 was significantly lower than that in K562 cells, (8.00±1.47)% vs. (50.56±8.62)%, (P<0.05). The level of IκB-α in K562 cytoplasm was down-regulated from (88.07±0.99)% to (49.21±0.95)%, (P<0.01) after As2O3 stimulation, while NF-κB in nuclear was up-regulated, that was not found in K562/ADR cells.
Conclusion: As2O3 could induce apoptosis of K562 cells, associated with the degradation of IκB-αand the activation of NF-κB. There were resistance to As2O3 induced apoptosis and an abnormal regulation of NF-κB expression by As2O3 in K562/ADR cells.
Generation and Application of a CML-Specific Recombinant Adeno-Associated Virus (rAAV) Vector.
Authors: Stiefelhagen Marius , Veldwijk Marlon R. , Jauch Anna , Eckstein Volker , Laufs Stephanie , Kleinschmidt Juergen A. , Wenz Frederik , Zeller Jens W. , Ho Anthony D. , Fruehauf Stefan 
Published: October 2019

Abstract
Introduction: Chronic myelogenous leukemia can be controlled but in most patients not be cured by tyrosine kinase inhibition. Direct targeting using gene therapy vectors combined with vaccination strategies may allow to eradicate residual leukemic progenitors. Adeno-associated virus (AAV) vectors are stable DNA vectors which were proven to be effective in the clinical gene therapy for e.g. coagulation disorders. The various AAV serotypes lack specificity for BCR-ABL+ leukemia cells. Recently developed AAV-library techniques allow a retargeting of vectors. We generated a set of rAAV vectors specific for BCR-ABL+ cells.
Material and Methods: After four selection rounds on BCR-ABL+ cells, the peptide sequences of the persisting clones were cloned into an AAV helper plasmid. rAAV-GFP stocks (2 K562-specific, 1 random) of each of the mutants were produced. Titers were determined using real time PCR-based titration assay. Both, a panel of leukemic cell lines and CML primary material were transduced with these vectors and gene transfer was determined by FACS analysis. Specificity was tested in a competitive transduction assay using BCR-ABL+ and BCR-ABL− leukemic cell lines. Transduction of primary CML cells was confirmed using FACS-sorted GFP+ cells and subsequent BCR-ABL-FISH.
Results: Using the CML-specific rAAV clone on a panel of BCR-ABL+ cell lines, gene transfer rates of >60% could be obtained (random clone: <1%; rAAV-2: <5%), whereas the BCR-ABL− cell lines were not susceptible to these vectors (gene transfer < 1 %). Admixing BCR-ABL− to BCR-ABL+ cells did not result in a significant drop of the gene transfer rates in the BCR-ABL− cell lines, suggesting that vector particles were not blocked by unspecific binding. Using primary material, significant gene transfer was observed (>5%; 6x more efficient than rAAV-2). In those cells, the CML-genotype was confirmed by BCR-ABL-FISH.
Conclusion: In this study, we were able to generate and apply a CML-specific rAAV vector on CML cell lines and primary material. Efficient and selective gene transfer in these cells could be obtained compared to standard rAAV-2 vectors and randomly generated clones. These data hold promise for future developments.
Use of Peripheral Blood Grafts Is Associated with Increased Acute and Chronic Graft-Versus-Host Disease without Improved Survival after Unrelated Donor Transplantation.
Authors: Eapen Mary , Haagenson Michael , Logan Brent , Confer Dennis , Horowitz Mary , Wagner John , Weisdorf Daniel , Anasetti Claudio 
Published: October 2019

Abstract
Data from the CIBMTR indicate that approximately 70% of unrelated donor hematopoietic stem cell transplants (HCT) in the U.S. utilize peripheral blood (PB) rather than bone marrow (BM) as a graft source. Comparative studies verifying its benefit, however, are lacking. We, therefore, performed a retrospective analysis comparing the results of 275 unrelated PB and 620 unrelated BM transplants in adults 18–60 years of age with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) or chronic myeloid leukemia (CML), transplanted in 2000–2002. 73% of PB grafts were matched at HLA A, B, C (low resolution) and DRB1, 21% were mismatched at a single locus and 6% were mismatched at ≥ 2 loci. 69% of BM grafts were matched, 26% were mismatched at a single locus and 5% were mismatched at ≥ 2 loci. Median follow-up was 24 (range, 6–48) and 34 (range, 6–54) months for PB and BM recipients, respectively. Groups were similar except PB recipients were less likely to have CML, were more likely to have MDS and were transplanted more recently. Incidences of neutrophil recovery (95% vs. 90% at day 100, p=0.01) and platelets ≥20,000/ul (81% vs. 66%, at 1-year, p <0.0001) were significantly higher after PB than BM transplants. Incidences of grades 2–4 but not grades 3–4 acute graft-versus-host disease (GVHD) were significantly higher after PB than BM transplants (56% vs.45%, at day 100, p=0.003). Chronic GVHD was also significantly higher after PB transplants (54% vs. 39%, at 3 years, p<0.0001). Despite higher rates of grade 2–4 acute and chronic GVHD after PB transplantation, incidence of relapse was similar in the two groups for both early and advanced leukemia. In multivariate analysis, risks of treatment-related mortality (TRM), treatment failure (relapse or death) and overall mortality during the first 9 months after transplantation were similar. However, among patients surviving the first 9 months, subsequent risks of TRM (relative risk [RR] 1.90, 95% confidence interval [CI], 1.14–3.17, p=0.01) and treatment failure (RR 1.60, 95% CI 1.06–2.44, p=0.03) were significantly higher in the PB cohort. Three-year adjusted (from multivariate models) probabilities of leukemia-free survival were 29% and 31%, p=0.5, after PB and BM transplantation, respectively; corresponding probabilities of overall survival were 31% and 32%, p=0.8. While these data do not indicate a survival advantage with either stem cell source by disease or risk group, PB is associated with earlier engraftment. This advantage is offset by higher rates of grades 2–4 acute and chronic GVHD, leading to a higher risk of late adverse events. Randomized clinical trials are necessary to better define the relative risks and benefits of PB in the setting of unrelated donor HCT.
CD44 Ligation Induces Apoptosis Via Caspase- and Serine Protease-Dependent Pathways in Acute Promyelocytic Leukemia Cells.
Authors: Maquarre Eliane , Artus Cedric , Gadhoum Zeineb , Jasmin Claude , Smadja-Joffe Florence , Robert-Lézénès Jacqueline 
Published: October 2019

Abstract
We have recently reported that ligation of the CD44 cell surface antigen with A3D8 monoclonal antibody (mAb) triggers incomplete differentiation and apoptosis of the acute promyelocytic leukemia (APL)-derived NB4 cells. The present study characterizes the mechanisms underlying the apoptotic effect of A3D8 in NB4 cells. We show that A3D8 induces activation of both initiator caspase -8 and -9, and effector caspase-3 and -7 but only inhibition of caspase-3/7 and caspase-8 reduces A3D8-induced apoptosis. Moreover, A3D8 induces mitochondrial alterations (decrease in mitochondrial membrane potential ΔΨm and cytochrome c release) which are reduced by caspase-8 inhibitor suggesting that caspase-8 is primarily involved in A3D8-induced apoptosis of NB4 cells. However, the apoptotic process is independent of TNF-family death receptor signalling. Interestingly, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) decreases A3D8-induced apoptosis and when combined with general caspase inhibitor displays an additive effect resulting in complete prevention of apoptosis. These results suggest that both caspase-dependent and serine protease-dependent pathways contribute to A3D8-induced apoptosis. Finally, A3D8 induces apoptosis in ATRA-resistant NB4-derived cells and in APL primary blasts, characterizing the A3D8 anti-CD44 mAb as a novel class of apoptosis-inducing agent in APL.
2-Methoxyestradiol Is Exclusively Effective on Both Retinoic Acid-Sensitive and -Resistant Acute Promyelocytic Leukemia through the Inhibition of NF-κB.
Authors: Huang Ming-jer , Cheng Yunchi , Lin Shufan , Chen Zhilone , Liu Hsingjin E. 
Published: October 2019

Abstract
The superoxide dismutase inhibitor, 2-methoxyestradiol (2ME), has recently been found to have anti-leukemic activity. As both 2-ME and ATO induce apoptosis of leukemic cells through similar mechanisms- mitochondrial damage through increased intracellular ROS, we hypothesized that 2-ME, like ATO, might be particularly effective on acute promyelocytic leukemia (APL). Using MTT assays, we found that the dose of 2-ME required to inhibit 50% of cell growth (IC50=0.25 μM) was 5 times less for NB4 cells than for other types of leukemic cells such as HL-60, U937, K562, THP-1 and KG1a (Fig 1). The exclusive sensitivity correlated with the increase of cellular superoxide and the decrease of cellular glutathione upon treatment but not with the endogenous levels of superoxide or glutathione. 2ME induced apoptosis of NB4 cells through upregulation of pro-apoptotic Bax, and Bak proteins, and induced apoptosis through mitochondrial pathways, shown by the release of cytoplasmic cytochrome C, and cleavage of caspase 9 and 3, and 6. Of note, 2-ME also induced neutrophilic differentiation, confirmed by NBT assays, and the expression of CD11b. We showed that, although 2-ME did not affect the PML/RARα transcription, it decreased the levels of PML/RARα in the cells. Like ATO, 2-ME specifically inhibited the activation of NF-κB in NB4 cells but not in HL-60 cells, shown by the EMSA assay (Fig 2). The effectiveness of 2-ME on NB-4 cells could also be observed in both all-trans retinoic acid (ATRA)- resistant and ATO-resistant cells. Furthermore, a combination of 2-ME with ATO was more effective on NB4 cells than either agent alone. Therefore, 2-ME could be potentially useful for eradication of APL or as a salvage agent for ATRA-and ATO-resistant APL.
Fig 1, Fig 2 Fig 1, Fig 2.
Single Stranded Polydeoxyribonucleotides Compete with the Incorporation of [3H] Thymidine or [3H]dAMP into DNA.
Authors: Yang Ting , Buhmann Raymund , Svihla Michaela , Kolb Hans-Jochem 
Published: October 2019

Abstract
Defibrotide, the sodium salt of a single-stranded polydeoxyribonucleotide is prepared by a controlled depolymerisation of deoxyribonucleic acid (DNA) which is obtained from mammalian organs. In previous studies it was shown to mediate immunosuppressive effects (Ferraresso et al. 1993)1.
In the current survey we investigated whether randomly chemically synthesized single-stranded polydeoxyribonucleotides of different length and composition could provide similar effects. For this purpose, purified T-cells were stimulated in the presence of dNTP’s or single-stranded polydeoxyribonucleotides with irradiated, allogeneic PBMC’s, PHA or anti-CD3/CD28 Dynabeads. Cellular proliferation was assessed by incorporation of tritium-labelled thymidine ([3H]thymidine), respectively [3H]dAMP or by staining with CFSE (carboxyfluorescein succinimidyl ester). After 72h or 120h of incubation, the incorporation of [3H]thymidine, or [3H]dAMP as well as the CFSE distribution was assessed. Cell viability was measured by trypan blue exclusion. T-cell activation was measured after 72h by quantifying the number of CD3+ T-cells expressing the activation markers CD25 and CD69. Cellular uptake of fluorochrome-labelled oligos was detected by fluorescence microscopy using an AxioCam HR and visualized by AxioVison. Each experiment was performed at least three times. Polydeoxyribonucleotides of different length, composition or concentrations (up to 5mM) did not cause cytotoxic effects to lymphocytes. But the incorporation of [3H]thymidine or [3H]dAMP was competed by polydeoxyribonucleotides. These effects were found to be dependent on length, concentration and base-composition of the nucleic acids. The proliferative capacity of the T-cells, as assessed by CFSE-staining, seemed to be unaffected. In context with data obtained by fluorescence microscopy, we hypothesise that there might be a cellular uptake of the polydeoxyribonucleotides followed by a subsequent cytoplasmatic degradation and a intracellular reutilisation via the salvage pathway for synthesis of nucleotides. The standard approach to detect cellular proliferation by incorporation of tritium-labelled nucleotides or derivatives is not useful to assess changes in cellular metabolism or proliferation in context with polydeoxyribonucleotides. Moreover, and that might be even more important, treatment approaches using nucleoside analogues like fludarabine, cytarabine e.g. in context with polydeoxyribonucleotides might be critical and diminish the efficacy of these drugs. Further experiments are necessary to elucidate the precise mechanism of these effects.