bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fancd2 Deficiency Impairs Autophagy Via Deregulating The Ampk/Foxo3a/Akt Pathway
Authors: Sertorio Mathieu , Amarachintha Surya , Wilson Andrew , Pang Qishen 
Published: October 2019

Abstract
Fanconi anemia (FA) is a genetic disease characterized by bone narrow failure and high risk of malignancy. The disease is due to a deficiency in the FA DNA repair pathway. Impaired function of the FA pathway leads to a decrease in survival, self-renewal and function of hematopoietic stem cells (HSCs). Previous reports have shown that FOXO3a directs a protective autophagy program for HSCs. Autophagy is an intracellular degradation system that enables cell to survive during stress like nutriment deprivation or oxidative stress by recycling damage proteins and organelles like mitochondria. Our laboratory has shown that FA-deficient cells are hypersensitive to oxidative stress and that FANCD2/FOXO3a interaction directs anti-oxidative response and cell survival. The FANCD2/FOXO3a axis could play a role in the protective autophagy activity in HSCs. For these reasons, we decided to study autophagy in the context of FANCD2-deficient (FA-D2) human lymphoblast cells exposed to oxidative stress. Interestingly, we observed an impaired activation of autophagy in the FA-D2 cells, detected by LC3-II immunoblot and flow cytometry, compared to FANCD2-corrected (control) cells. We found an increased necroapoptosis as soon as 1 hour after H2O2 treatment in FA-D2 cells. Paradoxically, we observed a profound decrease in the activity of the mTORC1 complex (as determined by S6 and S6k1 phosphorylation) in FA-D2 cells. In order to explain this autophagy deregulation, we determined the activation of AKT known to up-regulate mTORC1 activity. AKT activation (monitored by phospho-ser473) was significantly decreased in FA-D2 cells compared to control cells. Consequently, FOXO3a was over-activated in FA-D2 cells after H2O2 treatment. Consistently, we found markedly increased activation of AMPK known to initiate and sustain FOXO3a activation. Since AKT controls the expression of p62/SQSTM1, a protein involved in autophagy by addressing the damaged proteins/organelles to autophagic vesicles, we next examined the level of p62 in FA-D2 cells. In contradiction with the impaired autophagy in FA-D2 cells, we observed a decrease of p62 protein compared to corrected cells. To ensure that p62 protein decrease was not due to autophagy activity, we examined p62 transcription and found that the level of p62 mRNA was significantly decreased in FA-D2 cells. Our study thus identifies a deregulated AMPK/FOXO3a/AKT pathway in FA hematopoietic cells, and reveals an impaired autophagy process in which over-activated AMPK initiates FOXO3a activation that in turn inactivates AKT leading to down-regulation of p62. Thus, impaired autophagy may play a causal role in the hypersensitivity of FA-deficient cells to oxidative stress.

Disclosures:
No relevant conflicts of interest to declare.
Analysis Of Gene Expression Profile In SUDHL6 Cells Of siRNA-Mediated BCL11A Downregulation
Authors: Wu Hong , Dongmei He , Li Ding , Li Yangqiu 
Published: October 2019

Abstract

Background
The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11A gene (BCL11A) was associated with hematopoietic malignancies. However, the precise function of this transcription factor in B-cell malignancies still remain poorly characterized. Previous work from our laboratory has shown that BCL11A gene by small interfering RNA (siRNA) resulted in the growth inhibition and apoptosis of the B cell lymphoma cell line (i.e., SUDHL6 and EB1). The aim of this study was to further elucidate the molecular mechanism of this process by analyzing the gene expression profile in SUDHL6 cells after BCL11A knockdown.

Methods
FBCL11A siRNA was transfected into SUDHL6 cells to knock down BCL11A expression. Cells were collected, and RNA isolated for transcriptional profiling using the Affymetrix HG-U133 Plus 2.0 array. The global gene expression profile of the BCL11A siRNA-treated SUDHL6 cells was identified and analyzed. Twenty-one differentially expressed genes were further validated and analyzed from the BCL11A siRNA-treated SUDHL6 cells by real-time quantitative reverse transcript-polymerase chain reaction (qRT-PCR).

Results
FThere were 659 genes differentially expressed between the BCL11A siRNA- and negative control-transfected cells. These included 294 upregulated genes and 365 downregulated genes. The differentially expression genes are involved in various signaling pathways including metabolic pathways, focal adhesion, the MAPK signaling pathway, the cell cycle, the JAK-STAT signaling pathway, the TGF-beta signaling pathway, the WNT signaling pathway, apoptosis, and BCR signaling. qRT-PCR validation of the selected differentially expressed genes demonstrated agreement with the microarray analysis. There was a significant difference in the relative expression level of most of the selected genes differentially expressed between the BCL11A siRNA- and negative control siRNA-treated cells (P<0.05). After the transfection of BCL11A siRNA, among the apoptosis-related genes in the BCL-2 family, BCL2L11 was upregulated 7.24-fold, and BCL-2 was downregulated 3.23-fold.

Conclusions
Our results indicate that BCL11A is involved in gene networks with cancer related functions. BCL11A may play a role in gene expression events related to apoptosis.

Disclosures:
Li: This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.
Impact Of Rad50 On Telomere Recombination Mechanisms In Primary Bone Marrow and B-Cell Lymphomas Lacking Telomerase
Authors: Morrish Tammy , Behera Vivek , Budman Joshua , Dria Stephen , Morgan Michelle , Velliquette David , Bekbolysnov Dulat , Skaist Alyza , Wheelan Sarah 
Published: October 2019

Abstract
The unlimited growth that occurs in tumors requires telomere maintenance. Yet, a portion of human tumors lack telomerase, and maintain telomeres using recombination-based mechanisms. Studies in other model organisms indicate that two different pathways of recombination-based mechanisms impact telomere maintenance and rely on the DNA repair proteins Rad50 and Rad51. In the Rad50-dependent pathway telomere recombination occurs within the telomere repeats. In contrast, recombination using the Rad51-dependent pathway occurs within repetitive sequences in the subtelomeres. Using a mouse B-cell lymphoma model lacking telomerase, Eμmyc+mTR-/-, and immortalized fibroblast cells lacking the RNA component of telomerase (mTR-/-) we have examined the impact of inhibiting Rad50 and Rad51a on telomere recombination. We find inhibiting Rad50 or Rad51a in Eμmyc+mTR-/- B-cell lymphomas, and in mTR-/- immortalized fibroblasts, has a synergistic effect on DNA damage sensitivity to mitomycin but not camptothecin. Inhibiting Rad50 in telomerase deficient cells also results in telomere shortening and in some tumors, reduced growth. In contrast, when Rad50 or Rad51a is inhibited in cells with telomerase, DNA damage sensitivity from mitomycin is not observed when compared to cells expressing a control shRNA. In addition inhibiting Rad50 in cells with telomerase does not significantly impact telomere length or recombination. Next we developed a comparative genomic hybridization (aCGH) approach that detects recombination events in the subtelomeres. Using these subtelomere arrays we find B-cell lymphomas lacking telomerase exhibit a significant increase in subtelomere recombination compared to primary cells. We also examined the impact of inhibiting Rad50 on subtelomere recombination events. Our findings using aCGH suggest that inhibiting Rad50 does not impact subtelomere recombination in Eμmyc+mTR-/- B-cell lymphomas. Overall, our findings suggest that inhibiting either Rad50 or Rad51a in mTR-/- cells has a synergistic impact on the sensitivity to DNA damaging agents in contrast to cells with mTR+/+. Currently we are testing the impact of inhibiting Rad51a on subtelomere recombination. In addition these results further support that Rad50 contributes to telomere recombination mechanisms in tumors lacking telomerase and will provide insight into the mechanism of subtelomere recombination in mammalian cells.

Disclosures:
No relevant conflicts of interest to declare.
Optimization Of Stepwise Hematopoietic Differentiation Of Human iPSCs
Authors: Schuening Friedrich , Srinivasakumar Narasimhachar , Zaboikin Michail , Zaboikina Tatiana 
Published: October 2019

Abstract
Since their discovery in 2006, induced pluripotent stem cells (iPSCs) with their ES cell-like self-renewal and differentiation capability, are set to revolutionize the field of regenerative medicine. There is tremendous interest in the field of hematology for derivation of hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPCs) by in vitro differentiation of IPSCs. IPSCs can be differentiated into HSC/HPCs by coculture on feeder cells, such as OP9, or by using stepwise differentiation protocols on defined media. Neither approach produces high yields of HSCs or HPCs. With an intention to improve this, we systematically investigated various parameters for in vitro differentiation of iPSCs into HPCs.
iPSCs were derived from human adult dermal fibroblasts by transduction with the Yamanaka retroviral vectors (encoding human Klf4, Oct3/4, Sox2 and cMyc) or by electroporation with the Yamanaka Epstein–Barr virus-based episomal plasmid vectors (encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA). One iPSC clone of each variety was then subjected to a stepwise differentiation protocol described by Niwa and coworkers [PLoSOne. (2011); 6(7):e22261] followed by hematopoietic colony forming (CFU) assays in MethoCult (STEMCELL Technologies, Vancouver, Canada). The original protocol calls for the use of Stemline II serum-free medium (Sigma, St. Louis, MO) supplemented with various growth factors/cytokines. We investigated the use of APEL medium described by Ng and coworkers [Nature Protocols. (2008); 3(5): 768] as a possible substitute for Stemline II. We also tested the effect of varying the number of colonies seeded in 6-well plates and the efficiency of hematopoietic differentiation after seeding iPSCs as single cells. The results, based on the number of hematopoietic colonies obtained in MethoCult following differentiation, showed that the APEL medium (>100 CFU/100,000 cells) was a superior substitute to the Stemline II medium (<10 CFU/100,000). When IPSCs were seeded as single cells, at initial densities of 10, 100 or 1,000 cells/cm2 in the presence of Y-27632 Rock inhibitor, only the cells at starting density higher than 1,000 per cm2survived but did not yield hematopoietic CFUs in MethoCult. When seeded as colony fragments, lower density of seeding in 6-well plates (< 20 colonies/well) was superior to higher density (>50 colonies/well) for obtaining HPCs. Other parameters that can affect differentiation, such as bone-morphopoietic protein (BMP) and O2 concentration, are being investigated.
Figure A. Cellular markers detected at different time points of stepwise hematopoietic differentiation. B. CFUs in MethoCult.

Disclosures:
No relevant conflicts of interest to declare.
LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation
Authors: Jin Yue , Guo Yidi , Liang Dongxue , Li Yue , Li Zhe , Hu Xin 
Published: October 2019

Abstract
GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated.
In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter. In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival.
Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes.

Disclosures:
No relevant conflicts of interest to declare.
STUDY ON ANTIANGIOGENENIC EFFECT OF ACANTHOPANAX SENTICOSUS IN HL60 CELL LINES
Authors: Fangyuan Chen , Minyue Zhang , Jiayi Cai , Lijing Shen 
Published: October 2019

Abstract

Introduction
Many studies have been confirmed that neovascularization, the formation of new blood vessels from existing vasculature, plays an essential role in growth, development and metastasis of acute leukemia. At present, antiangiogenic therapy of leukemia become the new hot spot. Acanthopanax senticosus(Chianese name Ci Wu Jia ,CWJ) is a kind of Chinese herb, which contain natural flavonoid compounds and have been proven to inhibit leukemia cell proliferation. But there is no detailed report about the relationship with the inhibition of leukemia cells and the inhibition of angiogenesis effect. In this study, we should demonstrate the inhibition of leukemia cell growth and antiangiogenic mechanism through HL60 cell lines, further confirm the inhibitory effect on leukemia and antiangiogenic effect of Acanthopanax senticosus,

Methods
HL60 cells were treated with different concentrations of Acanthopanax senticosus (25°¢50°¢75°¢100°¢200µg/ml). Cell proliferation were detected using Cell Counting Kit-8. Kinds of transcription factors in Dll4/Notch (Delta-like 4 is the only ligand of Notch expressing in endothelium) and VEGF(R) signaling pathway were evaluated using quantitative real-time PCR (qRT-PCR) and Western blotting.

Results
Acanthopanax senticosus inhibited the growth of HL60 cells, and the time and concentration dependence(Fig.1). We extracted RNA and protein from these cells at 12hr, 24hr and 48hr respectively, found that Acanthopanax senticosus remarkably results in VEGF, VEGFR2(VEGF Receptor 2), DLL4 down-regulation based on the time and the concentration dependence, and mild inhibit VEGFR1(VEGF Receptor 1) and Notch1 factors gene expression(Fig. 2). Western blotting also showed a significant inhibition protein of VEGFR2, DLL4 and Notch1, mild inhibited the expression of VEGF and VEGFR1 protein, and with time and concentration dependence (Fig. 3).

Summary
Acanthopanax senticosus can inhibited proliferation of HL60 cells in vitro and anti-angiogenesis effect mainly via inhibition of VEGFR2-mediated signaling. It has an instantaneous effect on Dll4/ Notch signaling pathway. The data have elucidated the potential roles of several key signaling pathways in angiogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Identification of secreted proteins that can promote erythroid progenitor expansion
Authors: Xu Bing , Wang Xiangmeng , Li Peng , Xiao Yiren , Dong Huijuan , Zhou Yong , Li Yanyan 
Published: October 2019

Abstract
Erythroid blasts are progenitors that can differentiate to mature red blood cells (RBC). Identification of these growth factors and understanding their downstream pathways can help us to optimize RBC production ex vivo. We derived a stromal cell line (PL16) from mouse embryonic fetal livers that promotes proliferation of mouse and human erythroid blasts in vitro. Among specifically highly expressed secreted proteins in PL16, we identified a new growth factor (FA) that is capable of stimulating human erythroid blast expansion in vitro significantly. Furthermore, erythroid blasts from our in vitro expansion system can differentiate into functional mature red blood cells.

Disclosures:
No relevant conflicts of interest to declare.
Data From The Russian Cohort Of The International Disease Registry For Paroxysmal Nocturnal Hemoglobinuria (PNH)
Authors: Lisukov Igor , Kulagin Alexander , Maschan Alexey , Shilova Elena , Abdulkadyrov Kudrat , Zaritskiy Andrey , Ivanova Valentina , Bogdanova Yulia , Volkova Svetlana , Gavrilenko Andrey , Kaporskaya Tatyana S , Meresiy Sergey , Schneider Tatyana , Yakupova Svetlana , Anchukova Ludmila , Bakirov Bulat , Kosacheva Natalia , Krinitsina Tatiana , Ustyantseva Viktoria , Shatokhin Yuriy , Rumyantsev Alexander , Afanasyev Boris 
Published: October 2019

Abstract

Introduction
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disease that can lead to life-threatening complications including thrombotic events (TE), chronic kidney disease (CKD) and pulmonary hypertension. An international PNH Registry was implemented in 2008 to enhance understanding of the natural history of PNH, to describe treatment outcomes, and to evaluate the long term safety of eculizumab in treated patients.

Methods
This Registry is a non-interventional, prospective, multicenter, observational study. All patients with a diagnosis of PNH (confirmed in accordance with international diagnostic guidelines) or a detected PNH clone are enrolled irrespective of age or therapy. Data on patient demographics, medical histories, disease characteristics and treatment are collected at enrollment, every 6 months thereafter and/or at discontinuation. Descriptive statistics are used to describe the data; n, median and range (min–max) for continuous variables and percentages for categorical parameters.

Results
As of May 1, 2013, the Registry has enrolled 248 patients from Russia, over 50% of whom have a history of aplastic anemia or other bone marrow disorder (BMD) (Table 1). Disease characteristics for the overall population and by clone size or LDH level are presented in table1.A total of 25 patients have received eculizumab and have available follow-up data after starting treatment; median (range) follow-up time 4.4 (0.3–8.3) months. Among the 11 patients treated with eculizumab and with available LDH levels, the median LDH ratio was 5.7 X ULN before treatment and 1.0 X ULN at last follow-up assessment.

Conclusion
Russian patients included in the International PNH Registry show broad ranges of age, clone size, and degrees of hemolysis. History of TE, impaired renal function, and signs of chronic hemolysis are present among these patients regardless of PNH clone size. History of TE was recorded more frequently in patients with PNH clone sizes ≥20%, and was also more frequent among patients with LDH levels ≥1.5 x ULN. Among patients treated with eculizumab there was a marked decrease in hemolysis (as measured by LDH levels).

Disclosures:
Lisukov: Alexion: Honoraria. Kulagin:Alexion: Honoraria. Shilova:Alexion: Honoraria. Afanasyev:Alexion: Honoraria.
Analysis Of Hematological Improvement With Iron Chelation Therapy Using Deferasirox In Subject Ts With Therapy-Refractory Severe Aplastic Anemia
Authors: Jomen Wataru , Kuroda Hiroyuki , Yoshida Masahiro , Yamada Michiko , Abe Tomoyuki , Sakurai Tamaki , Fujii Shigeyuki , Maeda Masahiro , Fujita Miri , Matsuno Teppei , Sato Masanori , Kobune Masayoshi , Kato Junji 
Published: October 2019

Abstract

Introduction
It has been shown that iron chelation therapy (ICT) improves survival and quality life in subjects with transfusion-dependent hematological disorder including aplastic anemia (AA). In most cases of aplastic anemia, hematopoiesis is impaired by immunological disturbance, so the number of CD34+ stem/progenitor cells decreases drastically in severe AA. Little is known, however, about the hematological response to deferasirox therapy in those with severe AA, especially refractory cases with immunosuppressive therapy (IST).

Methods
Nine subject 5 men and 4 women aged 20 to 83 (median age: 59.4 years) with transfusional iron overload who were administered the oral iron chelator, deferasirox (DFX) were evaluated from April 2010 to March 2013.at our hospital. Of these, one had non-severe AA, and 8 severe AA classified by severity criteria (Hematology 2011). These 8 were administered immunosuppressive therapy (IST) with rabbit antithymocyte globulin (rATG) in combined with cyclosporine A (CsA), but no hematological improvement was seen.After informed consent was obtained, all 9 were administered iron chelation therapy when serum ferritin (SF) exceeded 1,000 ng/mL or they required over 20 RBC in transfusions (or 100 mL/kg of RBC). Hematological improvements was assessed using International Working Group 2006 criteria.

Results
The initial median DFX dose was 12.0 mg/kg per day and median treatment duration was 13.4 months. Two discontinued treatment. Hematologic improvements was observed in 50% (4/8) of those with severe AA and all 4 no longer required blood transfusions, and while 3 of the remaining 4 no longer required platelet transfusion. Median time to transfusion independence was 4.3 months while that to transfusion independence was 7.2 months In these 4 subject ts, median serum ferritin was 1,708 ng/ml immediately before ICT and decreased to less than 500 ng/ml after ICT. Bone marrow (BM) biopsied was in subjects with hematological improvement, showed that BM cellularity had slightly but significantly recovered in all cases.

Conclusions
ICT using DFX improved hematopoiesis in subjects with severe AA even after IST. This finding suggested that DFX induction should be considered as a potential treatment in IST-refractory severe AA. Thus, DFX appears to support the efficacy of IST due to the chelation of cellular excess iron in BM cells.

Disclosures:
No relevant conflicts of interest to declare.