bluuebunny/crossref_metadata_2025 huggingface dataset:
Inducing the Anti-Leukemia Effects of CD8+ T Cytotoxic Lymphocytes Derived from Umbilical Cord Cell.
Authors: Tan Huo , Liu Xin
Published: October 2019

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

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

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

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

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

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

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

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

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.
MDM2 Inhibitor Nutlin-3a Induces p53-Dependent Apoptosis Via Transcription-Dependent and -Independent Pathways and Overcomes Fludarabine-Resistance in CLL.
Authors: Kojima Kensuke , Konopleva Marina , Samudio Ismael J. , McQueen Teresa , Tsao Twee , Zeng Zhihong , Plunkett William K. , Andreeff Michael
Published: October 2019

Fludarabine containing combination therapies are the current standard for CLL therapy, in which p53-mediated induction of apoptosis contributes to leukemia cell killing. Although TP53 mutations occur in only 5–10% of patients with CLL, inactivation of the p53 pathway also occurs through Mdm2 overexpression or Atm deficiency. We investigate if recently developed potent and selective small-molecule antagonists of Mdm2, Nutlins, can overcome functional p53 inactivation associated with Mdm2 overexpression or Atm deficiency in CLL. Nutlin-3a caused a dose- and time-dependent increase in the percentage of Annexin V-positive cells in 20 primary CLL samples, irrespective of Mdm2 or Atm status. Samples with low Atm levels (n=3) were resistant to fludarabine. In addition to the transcriptional activation of target genes in the nucleus, cytoplasmic p53 can trigger transcription-independent apoptosis at the mitochondria. Samples with a cytoplasmic p53 localization pattern (n=7) showed a higher percentage of Nutlin-induced apoptosis than those with a nuclear pattern (n=8; P < .05). Furthermore, the inhibitory effect of cycloheximide pretreatment showed a negative correlation with the degree of Nutlin-induced apoptosis (r = - 0.617, P < .05). These findings suggest that the transcription-independent pathway may have stronger apoptogenic activity than the transcription-dependent pathway. The degree of Nutlin-3a-induced apoptosis directly correlated with apoptosis induced by the same concentrations (1–10 μM) of fludarabine in the early (24 hr) time period, and the Nutlin-3a/fludarabine combination induced synergistic apoptosis [averaged combination index (CI): 0.68]. Interestingly, this synergism was not affected by Mdm2 overexpression (CI: 0.59, n=12) or Atm deficiency (CI: 0.79, n=3). We conclude that (1) Mdm2 inhibitor Nutlin-3a efficiently induces p53-dependent apoptosis in wild-type p53 CLL cells, independent of Mdm2 overexpression or Atm deficiency, (2) transactivation-dependent apoptosis does not always play a major role in p53-dependent apoptosis and the exclusive activation of transactivation-independent pathway can fully induce apoptosis, (3) p53 activation plays a critical role in the early phase of fludarabine-induced apoptosis in vitro, and (4) Mdm2 inhibition and fludarabine synergistically induce apoptosis, which may overcome fludarabine-resistance in CLL.
Curcumin Enhances Differentiation of All-Trans Retinoic Acid (ATRA)-Sensitive and ATRA-Resistant Acute Promyelocytic (APL) Cells.
Authors: Kini Ameet R. , Nagabhushan Moolky , Tallman Martin S. , Roychowdhury Shantanu
Published: October 2019

The introduction of ATRA-based differentiation therapy has significantly enhanced outcomes in patients with APL. However, retinoic acid syndrome and ATRA-resistance remain significant concerns. It would therefore be useful to develop drugs that reduce the therapeutic doses of ATRA needed, and would be effective in ATRA-resistant cases. We have shown previously that curcumin, the yellow compound isolated from spice turmeric, suppresses the initiation and promotion stages of cancer development. In the present study we evaluated whether curcumin affects differentiation of NB4 APL cells. The NB4 cells were derived from a patient with APL, and differentiate in response to ATRA, while NB4-R1 cells are resistant to ATRA. Treatment of NB4 cells with 5 μM curcumin enhanced ATRA-mediated differentiation. Differentiation was assessed by evaluating CD11b expression, nitroblue tetrazolium (NBT) reduction and by morphologic examination. This curumin-mediated enhanced differentiation was apparent at 1 μM as well as 0.1 μM of ATRA. Curcumin alone did not cause differentiation of the NB4 cells, although higher concentrations of curcumin caused apoptosis. We then examined the effect of curcumin on the ATRA-resistant NB-R1 cells. Addition of ATRA and curcumin together induced differentiation of the NB4-R1 cells, whereas either agent alone did not cause differentiation. The differentiation was characterized by increased CD11b expression, NBT reduction and the typical morphologic changes. In addition, differentiation of the NB4-R1 cells was accompanied by restoration of the PML-oncogenic domains (PODs). These results indicate that curcumin may be another unconventional therapeutic agent in APL, following the successful use of ATRA and arsenic trioxide.
In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.
Authors: Kletzel Morris , Tallman Sarah C. , Olszewski Marie , Huang Wei
Published: October 2019

Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan.

Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control.

Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005).

24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88

The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells.

Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.
A Novel Mechanism of Action of Methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate (CDDO-Me): Direct Permeabilization of the Inner Mitochondrial Membrane To Inhibit Electron Transport and Induce Apoptosis.
Authors: Samudio Ismael , Konopleva Marina , Pelicano Helene , Huang Peng , Frolova Olga , Bornmann William , Ying Yun-ming , Evans Randall , Andreeff Michael
Published: October 2019

CDDO-Me is a synthetic oleanolic acid derivative that displays antitumorigenic and anti-inflammatory activities, and we have previously reported that this agent potently activates the intrinsic apoptotic pathway in leukemia cells. Here we demonstrate that mitochondrial dysfunction induced by CDDO-Me in leukemia cells is mediated by direct permeabilization of the inner mitochondrial membrane that results in the rapid depletion of mitochondrial glutathione (GSXm), loss of cardiolipin, and inhibition of mitochondrial respiration. More importantly, we demonstrate that in addition to activating the intrinsic apoptotic pathway, the mitochondrial effects of CDDO-Me may mediate its anti-inflammatory activity by modulating the generation of superoxide anion (O2−). Interestingly, CDDO-Me did not increase the generation of O2−, and in fact pretreatment of leukemia cells with CDDO-Me prevented the increase of this reactive oxygen species elicited by inhibition of complex I or III in the absence of de novo protein synthesis. CDDO-Me, but not other inhibitors of respiration, induced a time- and dose-dependent, cyclosporin A-independent permeability transition (mPT) of isolated mitochondria that was sensitive to sulfhydryl antioxidants but not to EDTA. PT induced by CDDO-Me and Ca2+ was accompanied by loss of GSXm suggesting that the increased permeability of the inner mitochondrial membrane facilitates the loss of this antioxidant. Finally, transmission electron microscopy revealed that CDDO-Me rapidly induced caspase-independent mitochondrial swelling and loss of inner membrane structure prior to the release of cytochrome c. Taken together, our results indicate that CDDO-Me is a novel antileukemic agent that induces apoptosis and inhibits mitochondrial electron transport via perturbations in inner mitochondrial membrane integrity.
Motexafin Gadolinium and Other Metallotexaphyrins Upregulate Heme Oxygenase-1.
Authors: Sirisawad Mint , Chen Jun , Ramos Jason , Miller Richard A. , Naumovski Louie
Published: October 2019

Heme oxygenase-1 (HO-1) is an inducible enzyme that is upregulated by heme and acts to degrade heme into bilirubin, carbon monoxide and iron. HO-1 is known to be upregulated in response to oxidative stress and has anti-apoptotic properties. Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that produces oxidative stress in cancer cells by reacting with various reducing metabolites and protein thiols to generate reactive oxygen species. Also, MGd is an expanded porphyrin that has a chemical structure which resembles heme. Since redox stress and heme upregulate HO-1 expression, we hypothesized that MGd would produce similar effects. We treated 8 hematopoietic tumor-derived cell lines with MGd and found that 2 of them (Ramos and HF-1) upregulated HO-1 protein. In Ramos lymphoma cells, we found that MGd induced HO-1 up to 8 fold by 24 hrs and that longer incubations did not result in appreciably greater amounts of protein. HO-1 levels returned to baseline in Ramos cells 48 hrs after cells were cultured in fresh media in the absence of MGd. HO-1 levels were elevated at baseline in HF-1 cells (compared to Ramos) and increased about 2 fold with MGd treatment. Other metallotexaphyrins, europium texaphyrin and dysprosium texaphyrin, also induced HO-1 expression. To determine if HO-1 expression was important for cell survival, we co-treated cells with MGd and tin protoporphyrin (SnPP), an inhibitor of HO-1 enzymatic activity. We found that both HF-1 and Ramos cells were sensitized to MGd-induced cell death by SnPP. These results demonstrate for the first time that metallotexaphyrins can upregulate HO-1 expression. The induction of HO-1 could be related to oxidative stress and/or metallotexaphyrins acting as heme mimetics.
Deletion of Any of BCR or ABL Gene on Derivative Chromosome 9 Is a Poor Prognostic Marker That Indicates Rapid Disease Progression in Chronic Myelogenous Leukemia.
Authors: Lee Young Kyung , Kim Young Ree , She Cha Ja , Yoon Sung-Soo , Kim Young Soo , Cho Han Ik , Kim Hee Chan , Lee Yun Song , Lee Dong Soon
Published: October 2019

To evaluate the prognostic significance of the submicroscopic deletions of ABL or BCR gene associated with t(9;22) in CML, we investigated the incidence of ABL or BCR deletion on derivative chromosome 9 using fluorescent in situ hybridization (FISH), and analyzed the survival. FISH was performed using LSI BCR/ABL, dual fusion translocation probe on bone marrow cells of 86 patients with CML. Of 86 patients, ABL deletion was detected in 13 (15.1%) patients and BCR deletion in 8 patients (9.3%). Patients with ABL deletion showed shorter event-free survival time (EFS) than those without ABL deletion (P=0.020). Patients with BCR deletion showed significantly short overall survival time (OS) (P=0.039). Patients with ABL and/or BCR deletion (14/86 patients, 16.3%) showed significantly short OS and EFS (median OS: 43.0 months, median EFS: 40.0 months), compared to the patients without any deletions of BCR or ABL gene (median OS: 94.0 months, median EFS: 90.0 months)(P=0.041 for OS, P=0.008 for EFS). Patients with BCR deletion, all except one had a concomitant ABL deletion, suggesting that BCR deletion occurs in conjunction with ABL deletion. In patients with ABL deletion only, BCR/ABL rearrangement with b2a2 mRNA type tended to be more frequent than in patients without any deletion of the two genes (P=0.073). Deletion of any of BCR or ABL gene on derivative chromosome 9 was associated with both short OS and EFS. We conclude that deletion of not only ABL gene, but also BCR gene is a poor prognostic marker that indicates rapid disease progression in CML.
Study on Clinical and Biological Characteristics of Childhood Acute Leukemia with MLL Gene Rearrangements.
Authors: He Jun , Chen Zi-xing , Xue Yong-quan , Pan Jin-lan , He Hai-long , Li Jian-qin , Wu Ya-fang , Huang Yi-ping , Zhu Ling-li
Published: October 2019

The rearrangement of MLL gene is reported in 70%~80% of infant and in 5%~10% of older children (under the age of 15) with acute leukemia (AL). The biological features associated with alterations in MLL gene are hyperleukocytosis, CD10−/CD19+ phenotype and very poor prognosis. To explore the MLL rearrangement in details in our AL children patients and obtain more information on the relationship between the MLL gene abnormality and clinical outcomes. The following study has been conducted. A total of 298 patients with AL attended The Affiliated Children’s Hospital of Soochow University, including 16 cases with MLL rearrangements, were recruited in this study. Of the cohort, 11 were diagnosed as ALL, 5 were AML. 9 of 16 patients were in infant age (up to 2 year) and the rest were between the age of 2 to 13 years. Fluorescence in situ hybridization (FISH) analysis using LSI MLL dual color probe. Multiplex reverse transcriptase- polymerase chain reaction (multiplex RT-PCR) were used to discriminate 13 different fusion transcripts. These results were analyzed together with R banding karyotyping and immunolphenotyping determined by flow cytometry. We have found MLL rearrangements in 16 cases of childhood AL which were accounted for 5.4% of 298 AL patients, and 56.3% of infant AL. Among 106 cases analyzed by multiplex RT-PCR, MLL gene rearrangement was found in 11 cases, including MLL/AF4 fusion gene in 2, MLL/AF6 fusion gene in 1, MLL/AF6, MLL/ELL combined with MLL/AFX or HOX11 in one of each, MLL/AF9 in 2, MLL/AF10 in 1, MLL/ELL in 2. MLL partial tandem duplication in 1. In addition an activated HOX11 gene was found in 1 case.. In 27 cases assayed by FISH, MLL gene rearrangements have been detected in 9 cases (36.0%). In 16 patients with MLL gene rearrangements, 14 (87.5%) exhibited clonal chromosome abnormalities involved chromosome 11 in 11 cases, presenting as t(4;11) in 2, t(6;11), t(8;11), t(7;8;11), and t(9;11) in one of each, respectively, trisomy 11 in 2 and 11q- in 3 cases. Among these 16 patients, 11 were B-ALL, including Pro-B and Pre-B ALL; 5 of AML-M5, 3 of these 5 M5 patients were CD7+ and CD2+. Of these 16 patients 8 received chemotherapy and 7 of them achieved complete remission, while the other 8 patients eventually gave up treatment. Our results demonstrated that multiplex RT-PCR combined with FISH provided a more accurate and sensitive method for detection of MLL gene rearrangements, including chromosome translocation, deletion and duplication. Our findings lead to the detection of novel rearrangements at molecular genetic level. These findings regarding the MLL rearrangement provide most important information in guiding therapy and predicting prognosis in childhood AL. Besides our results also provide evidence in support of the value of 11q23/MLL in WHO classification categories.
Prospective Multicentric Molecular Study for Poor Prognosis Fusion Transcripts at Diagnosis in Adult ALL Patients - The LALA94 Experience.
Authors: Gabert Jean A. , Picard Christophe , Hayette Sandrine , Bilhou-Nabera Christelle , Cayuela Jean-Michel , Macintyre Elizabeth , Frenoy Nicole , Preudhomme Claude , DuPont Madeleine , Bastard Christian , Bories Dominique , Vaerman Jean-Paul , Davi Frédéric , Dastugue Nicole , Raynaud Sophie , Lafage Marina , Fest Thierry , Gaub Marie-Pierre , Lheritier Véronique , Thomas Xavier , Charrin Christiane , Boucheix Claude , Dombret Hervé , Fiere Denis
Published: October 2019

From 1994 to 2000, 984 adults aged from 15 to 55 years with newly diagnosed Acute Lymphoblastic Leukemia (ALL) were eligible for randomization in the multicentric LALA-94 clinical protocol. The t(9;22), t(1;19) and t(4;11) translocations corresponding to BCR-ABL, E2A-PBX1 and MLL-AF4 fusion gene transcripts respectively, were considered as independent poor prognostic factors. Standardized RT-PCR analysis of these fusion gene transcripts were performed by 17 laboratories in order to provide data before the second randomization (J35) on 787 patients. In this multicentric study, validated data were available for therapeutic stratification for 91% of these analysed patients. No false positive RT-PCR was reported. Secondarily to retrospective BCR-ABL FISH, few false BCR-ABL negative RT-PCR were identified, leading to the design of new BIOMED-1 primers for b3-a3 junctions detection. Moreover, the LALA-94 study allowed to define new guidelines for molecular analysis at diagnosis.

Like in other studies, the BCR-ABL transcript was found to be the most frequent molecular abnormality in B-ALL (24%) whereas MLL-AF4 and E2A-PBX1 were detected in 5% and 3.5% of B-ALL, respectively. Epidemiological and clinical data of MLL-AF4 and E2A-PBX1 were concordant with previous publications. Interestingly, because of the large number of reviewed patients, the different BCR-ABL subtypes (M-BCR and m-BCR) were statistically characterized by few clinical data.

M-BCR subgroups had a higher age than m-BCR (p= 0.016) and occurs especially during the second semester (p= 0.034). Moreover, the comparison of clinical data at diagnosis of M-BCR variants showed that median age of b3a2 was statistically younger than b2a2 (p= 0.04) and that b3a2 occurs more frequently in man (p= 0.02). For the first time, these data suggest that these BCR-ABL breakpoints: m-BCR and M-BCR and also b2a2 and b2a3, are secondary to different physio-oncologic mechanisms even if therapeutic regimens including the same targeted therapy (tyrosine kinase inhibitor) for all BCR-ABL variants is the rule today.
FOXP3 Expression in B and T Cell Lymphomas.
Authors: Roncador Giovanna , Garcia Juan Fernando , Garcia Jose Francisco , Maestre Lorena , Lucas Elena , Cigudosa Juan Cruz , Sanchez Lydia , Ohshima Koichi , Nakamura Shigeo , Banham Alison , Piris Miguel Angel
Published: October 2019

Foxp3, which encodes a forkhead/winged helix transcription factor designated Scurfin, is a key regulatory gene required for the development and function of regulatory CD4+CD25+ T cells (Treg), a subpopulation of T-cells specialized in maintaining the balance between immunity and tolerance.

Humans with defects in the FOXP3 gene, develop strong activation of the immune system, leading to multiorgan autoimmune disease, allergies, inflammatory bowel disease and severe infections, collectively known as the IPEX syndrome (immune deregulation, polyendocrinopathy, enteropathy, X-linked inheritance syndrome)

Because of the importance of FOXP3 in the development and function of Treg cells, and its potential use as a specific Treg marker, we have developed several monoclonal antibodies against FOXP3, for use on paraffin-embedded tissue sections and evaluated its expression in a large series (150 cases) of B- and T-cell lymphomas. In reactive lymphoid tissue, strong nuclear FOXP3 expression was observed in approximately 5% of interfollicular T-cells. FOXP3 expression in tumour cells was confined to most of Adult T-cell Leukaemia/Lymphoma (ATLL) cases (68%), with some variability in protein expression. In other lymphoma types, FOXP3 expression was only detected in the reactive T-cell background, and the number of FOXP3-positive reactive T-cells was variable, ranging from almost a complete absence (Burkitt’s lymphoma) to abundant infiltrate (common in follicular lymphoma).

In conclusion, the availability of a FOXP3 monoclonal antibody, not only provides an important tool for the study of the development and function of Treg cells, but also represents a useful marker for the identification of ATLL cases in formalin-fixed paraffin-embedded tissue sections.

The presence or absence of Treg cells in the tumour environment could also play a role in the immune surveillance of tumours, thus implying a potential additional value for the detection of this cell population in tumour samples.
Quantitative Comparison of BCR/ABL Transcript Levels in Bone Marrow and Peripheral Blood Specimens from Patients with Minimal Residual Disease.
Authors: Faulkner Nicole E. , Sirko-Osadsa D. Alexa , Rosenblum-Vos Lynne S. , Rohlfs Elizabeth M. , Browne Patrick , Hallam Stephanie E.
Published: October 2019

Chronic Myeloid Leukemia (CML) is diagnosed by the presence of the Philadelphia chromosome (Ph+). The number of cells containing the bcr/abl chromosomal translocation diminishes dramatically after current standard of care treatment. Patient progress is monitored quantitatively and long-term prognosis is favorable if the Ph+ cells are no longer detected by cytogenetic analysis or FISH (sensitivity of 20% and 2% respectively). Molecular monitoring of cDNA BCR/ABL transcripts is widely used to assess levels of minimal residual disease (MRD). In our laboratory, quantitative RT-PCR allows detection of BCR/ABL transcript down to 1 x 10−5 or one BCR/ABL transcript in 100,000 reference gene transcripts. Serial values from bone marrow (BM) and blood specimens are tracked over time and monitored for increases that may indicate unfavorable changes in disease status. Though the literature indicates that the levels of BCR/ABL transcript in BM and blood samples are comparable at the time of diagnosis, this may not be true in patients with MRD. To explore this question we compared BCR/ABL transcript levels in paired BM and blood samples that were either collected together or separately but within a short time frame. In 36% of these cases both specimen types had detectable low-level BCR/ABL transcript. The levels of transcript in bone marrow were consistently higher than the levels in the associated blood specimen, though there was a wide range of variability (3-50 fold). Additionally, when blood levels of transcript were near the lower limit of detection, we were more likely to observe a larger difference relative to bone marrow transcript levels than when blood levels of transcript were higher. These results indicate that while both BM and blood specimens are valuable for serial molecular monitoring, they may not be equivocal when sampled from patients with MRD. If a patient has been monitored using blood specimens, periodic concurrent testing of blood and bone marrow specimens can provide clinical utility.
In Vivo Effects of Cyclosporine A, Mycophenolate Mofetile and Rapamycin on Adoptively Transferred CD4+CD25+ Regulatory T Cells in Prevention of Experimental Acute Graft-Versus-Host Disease.
Authors: Zeiser Robert , Nguyen Vu , Beilhack Andreas , Baker Jeanette , Schulz Stephan , Contag Christopher H. , Negrin Robert S.
Published: October 2019

CD4+CD25+ regulatory T (Treg) cells reduce GVHD without abrogating GVT effects in murine models and hold promise for clinical application. However, in contrast to most clinical situations, Treg have been investigated in models without immunosuppressive drugs. Therefore, we aimed to assess the impact of Cyclosporine A (CSA), Mycophenolate mofetile (MMF) and Rapamycin (RAPA) on Treg function both in vitro and in a lethal acute GVHD model. Gfp+ Treg reisolated from mixed leucocyte reactions (MLR) containing CSA but not RAPA showed significantly reduced ability to suppress CD4+CD25− T-cell proliferation in secondary MLRs (p=0.025) in a CFSE based assay. The CSA effect could be reversed to 85±3.4% by the addition of IL-2 (50 IU/ml) to the primary culture. In vivo bioluminescence imaging after major mismatch bone marrow transplantation demonstrated reduced early proliferation of donor derived luciferase-labeled conventional T-cells (Tcluc+) in animals treated with Treg. The addition of RAPA and MMF did not interfere with this suppressive effect. Conversely, combining Treg with CSA led to a significantly increased Tcluc+ proliferation as measured in photons/second/mouse (p=0.002). Treatment with Treg/CSA was associated with increased GVHD risk and lowest survival of the Treg/drug combinations. Immunofluorescence microscopy of secondary lymphoid organs revealed less donor derived Treg (CD4+FOXP3+H-2q+) per high power field in animals receiving CSA as compared to RAPA or MMF. Our data indicate that CSA, but not RAPA or MMF interferes with Treg function possibly through an IL-2 dependent mechanism. In vivo, the combination of Treg with RAPA or MMF allowed for adequate GVHD suppression and may therefore provide guidance for clinical trials.
Discrepancies in p21ras, p53, and Mdm2 Protein Expression Predict Worse Prognosis in Acute Myeloid Leukemia.
Authors: Schulz Elizabeth , Lorand-Metze Irene , Koch Rainer , Costa Solange Campelo , Costa Fernando Ferreira , Saad Sara Terezinha Olalla
Published: October 2019

Tumor progress is a multistep process in which clones of cells become abnormal by accumulating growth alterations until they are transformed and/or immortalized. In order to verify a potential role for p53, mdm2, and Ras genes in acute leukemogenesis, we investigated the expression and mutational status of these genes in a cohort of 35 adult patients with acute myeloid leukaemia (AML) at diagnosis with a minimum follow up of 4,5 years. The expression of p21ras, p53, and Mdm2 proteins was studied in bone marrow cytospins stained by APAAP (alkaline phosphatase anti-alkaline phosphatase procedure) using monoclonal antibodies Y13-219, PAb 1801 and 2A10, respectively. N-, K-, H-ras and p53 gene mutations were assessed by PCR-SSCP (polymerase chain reaction/single strand conformation polymorphism) and DNA sequencing to exclude the presence of mutations. We found 2 N-Ras gene and 2 p53 gene mutations in 4 patients. Stratification of AML patients by different cytoplasmic expression patterns (p21ras/p53, p53/mdm2, or p21ras/mdm2, and p21ras/mdm2/p53) revealed that patients with phenotypes p53+/mdm2+ (n=2) had significant best prognosis, patients p53—/mdm2— (n=16) an intermediate prognosis and patients with discrepant phenotypes p53—/mdm2+ or p53+/mdm2— (n=6) a worse prognosis (p=0.003 by Log Rank and p=0.008 by Wilcoxon). Patients with phenotypes p21ras+/mdm2+ (n=2) showed significant best prognosis, patients p53—/mdm2— (n=16) an intermediate prognosis and patients with discrepant phenotypes p53—/mdm2+ or p53+/mdm2— (n=8) a worse prognosis (p=0.07 by Log Rank and p=0.02 by Wilcoxon). Patients with phenotypes p21ras+/p53+/mdm2+(n=2) had a tendency to exhibit best prognosis, patients p21ras—/p53—/mdm2— (n=13) a tendency to intermediate prognosis and patients with discrepant phenotypes p21ras/p53/mdm2 (n=8) had a tendency to worse prognosis (p=0.06 by Log Rank and p=0.06 by Wilcoxon). However, stratification of AML patients by different expression values 0(—), 1(+) or 2(++) for each isolated variable p21ras, p53 and mdm2 using the same survival analysis demonstrated that there were no statistically significant difference among these groups. We found that discrepancies in cytoplasmic expression of p53/mdm2, p21ras/mdm2 and p21ras/p53/mdm2 confer worse long term prognosis to these categories of AML patients. These findings suggest that the signalization pathways p53/mdm2, ras/mdm2 and ras/p53/mdm2 may be active in AML and that an intact p53/Mdm2 axis is important in the prognosis of these patients.