bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bone Marrow Small Molecule Radioprotectors.
Authors: Epperly Michael W. , Franicola Darcy , Dixon Tracy , Zhang Xichen , Komanduri Paavani , Greenberger Benjamin , Wipf Peter , Koide Kazunori , Greenberger Joel S. 
Published: October 2019

Abstract
Development of small molecule radioprotectors is a major national priority. Two groups of compounds have particular promise. The first group targets the mitochondria based upon previous data with transgene MnSOD which when expressed in the mitochondria prevents apoptosis and increases radioprotection. These agents contain the antioxidant tempol or nitric oxide synthetase inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) attached to a hemi-gramicidin linker which targets the mitochondria. The second group consists of the dietary agent resveratrol and acetylated variants. Mouse hematopoietic progenitor 32Dcl3 cells were incubated for 1 hr in 10 μM tempol, AMT, or gramicidin linked tempol XJB-5-125 (tempol), XJB-7-75 (tempol) or JP4-039 (AMT). In separate experiments, 32Dcl3 cells were incubated for 1 hr in resveratrol or acetylated resveratrol. The cells were then irradiated to doses ranging from 0 to 8 Gy, plated in 0.8% methylcellulose, and incubated in a 5% CO2 incubator for 7 days. Colonies of greater than 50 cells were counted with the data analyzed using linear quadratic or single-hit, multi-target models. 32Dcl3 cells incubated in 10 μm tempol before irradiation resulted in no change in radiation sensitivity while incubation in XJB-5-125 or XJB-7-75 had decreased radiosensitivity. XJB-5-125 had an increased Do of 1.91 ± 0.67 Gy compared to 1.32 ± 0.09 Gy for 32Dcl3 cells incubated in tempol and 1.35 ± 0.27 Gy for control 32Dcl3 cells (p = 0.045 or 0.040, respectively). Incubation in XJB-5-75 resulted in an increased shoulder on the survival curve with an ñ of 19.4 ± 2.6 compared to 8.7 + 1.6 for cells incubated in tempol or 6.9 +1.8 for control 32Dcl3 cells (p = 0.025 or 0.022). Incubation in JP4-039 resulted in an increased Do of 2.2 ± 0.1 Gy compared to 1.24 ± 0.15 or 1.13 ± 0.06 for cells incubated in AMT or control 32Dcl3 cells only, respectively (p = 0.0115 or 0.0098, respectively). Incubation of 32Dcl3 cells in resveratrol or acetylated resveratrol before irradiation resulted in an increased shoulder on the survival curve of 33.2 ± 5.7 or 57.5 ± 9.9, respectively, compared to 6.9 ± 1.8 for 32Dcl3 cells (p = 0.0122 or 0.0072, respectively). These compounds were tested in mice receiving an LD50/30 irradiation dose. C57BL/6NHsd mice were injected intraperitoneally with 10 mg/kg of XJB-5-125, XJB-7-75or JP4-039 or 25 mg/kg of resveratrol or acetylated resveratrol and irradiated 10 mins later along with control mice to 9.5 Gy whole body irradiation. The mice injected with XJB-5-125, XJB-7-75, JP4-039 or acetylated-resveratrol had increased survival compared to control irradiated mice (p ≤ 0.0004). Therefore, four new small molecules have been identified which demonstrate significant radioprotective properties both in vitro and in vivo.
Mobilised Peripheral Blood Could Be a Suitable Source of Mesenchymal Stem Cells?.
Authors: Almici Camillo , Verardi Rosanna , Braga Simona , Neva Arabella , Russo Domenico , Rossi Giuseppe , Marini Mirella 
Published: October 2019

Abstract
Mesenchymal stem cells (MSC) are multipotent cells that are considered one of the most promising product for cellular therapy in regenerative medicine. MSC have been obtained and expanded from bone marrow and umbilical cord blood in adequate amounts for clinical applications. Under the right conditions, MSC could migrate from bone marrow into the peripheral circulation; however MSC have not been routinely isolated from peripheral blood, and studies are rare and not conclusive. The aim of the present study was to evaluate mobilised peripheral blood (MPB), obtained from patients undergoing apheresis collection of circulating hematopoietic progenitor cells, as a potential source of MSC for clinical applications. MPB samples (500–900 × 106 cells, N = 17) were separated by negative lineage-depletion immunoselection (RosetteSep). Selected cells were seeded in multi-well plates at low density in MesenCult Basal Medium without and with different combinations of growth factors (EGF, PDGF-BB, b-FGF). On reaching confluence, adherent cells were detached by 0.25% trypsin-EDTA treatment and replated for at least two passages. At each passage, surface antigen expression was analyzed by flowcytometry (CD45, CD34, CD105, CD44, CD73, CD166, CD31, HLA-DR and VE-caderine). Following immunoselection 9.5–17.1 × 106 cells were recovered from MPB samples. Cultured cells reached confluency in 3–4 weeks on first passage and in two weeks thereafter. Immunophenotyping showed negativity for CD45 antigen. The absence of growth factors in culture medium conditioned MSC growth capability, while the addition of PDGF-BB+EGF or b-FGF was able to boost the number of CD45−/CD73+/CD90+ cells in culture (see figure). However expansion remains still sub-optimal, having been reached in 8/17 samples. In conclusion, we demonstrate that MSC can be obtained from MPB, but expansion requires longer time period and appears more difficult compared to bone marrow. Therefore, further studies need to be conducted to find better culture conditions and optimal growth factor combinations to support MPB-derived MSC expansion.
Figure Figure
Implication of AML1/RUNX1 Function in the Homeostasis and Leukemic Transformation of Hematopoietic Stem Cells.
Authors: Ichikawa Motoshi , Takeshita Masataka , Goyama Susumu , Asai Takashi , Nitta Eriko , Nakagawa Masahiro , Kawazu Masahito , Chiba Shigeru , Ogawa Seishi , Kurokawa Mineo 
Published: October 2019

Abstract
Transcription factor AML1/RUNX1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction and c-Kit-positive cells in the G0 cell cycle status were increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. Thus, the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia. To identify mechanisms through which functional loss of AML1 exerts leukemogenic potential, we focused on the AML1-Evi-1 chimeric protein, which is generated by the t(3;21) chromosomal translocation and disturbs the normal function of AML1. We introduced AML1-Evi-1 and its mutants into murine bone marrow cells, and evaluated hematopoietic cell transformation by colony replating assays. The transforming activity of AML1-Evi-1 was impaired when any of the major functional domains of AML1-Evi-1 was lost. Moreover, overexpression of Evi-1 could not transform AML1-deleted bone marrow cells, suggesting that fusion of AML1 and Evi-1, rather than AML1 suppression and Evi-1 overexpression, is essential for AML1-Evi-1 leukemogenesis. Intriguingly, among the hematopoietic progenitor cell fractions, AML1-Evi-1 could transform only the uncommitted, immature hematopoietic cells, which contrasts with MLL-ENL, a chimeric protein generated in t(11;19) leukemia. AML1-Evi-1 transformed cells show a surface marker profile different from that of the cells transformed by AML1-MTG8/ETO, another leukemic gene product that also perturbs AML1 function. These results provide a valuable clue to a distinct mechanism determined by the Evi-1 moiety in the AML1-Evi-1 leukemogenesis and to a role of AML1 loss in the self-renewal of leukemic stem cells.
GATA and BCLxl Downregulation in Erythropoiesis during In Vitro Lineage Specific Differentiation of MDS Hematopoietic Progenitor Cells Is Not Induced by Activated Notch Pathway.
Authors: Hopfer Olaf J. , Komor Martina , Koehler Ina S.N. , Freitag Claudia , Hoelzer Dieter , Thiel Eckhard , Hofmann Wolf-Karsten 
Published: October 2019

Abstract
Notch signals have recently been shown to inhibit erythroid and megakaryocytic differentiation of hematopoietic progenitor cells. In myelodysplastic syndrome (MDS) its role in dyserythropoiesis has not been fully elucidated. Therefore we asked whether dysregulation of Notch pathway elements might be associated with impaired GATA1 and BCLxl expression and ineffective erythropoiesis being a hallmark of MDS hematopoiesis. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO and TPO. Cell harvest was at days 0, 4, 7 and 11. Expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). RNA expression of GATA1 and of BCLxl was steadily upregulated, particularly during late normal erythropoiesis. During normal megakaryopoiesis expression of both genes was up to 50 times lower as compared to normal erythropoiesis. In contrast, during MDS erythropoiesis a loss of typical late upregulation of GATA1 and BCLxl was observed. DLK1 expression during erythropoiesis showed increased expression particularly in high risk MDS vs. normal controls. Expression of HES1 was increasing during the course of normal erythropoietic and megakaryopoietic differentiation but not in lineage specific cells from MDS patients. In conclusion our data show that the central erythropoietic transcription factor GATA1 and the associated antiapoptotic molecule BCLxl are markedly downregulated during MDS erythropoiesis which may contribute to the ineffective erythropoiesis seen in this disease. Increased DLK1 expression in differentiated stem cells from high risk MDS patients was seen. However, an upregulation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 could not be detected.
The Expression of MT1-MMP in Human Hematopoietic Cell Lines and Its Influences on the Biological Behaviors of HL60.
Authors: Jiang Juxiang , Ma Zhenni , Dong Ningzheng , Bai Xia , Ruan Changgeng 
Published: October 2019

Abstract
Membrane type matrix metalloproteinase-1(MT1-MMP) is the identified member of characterized MMPs. Recent researches suggest that MT1-MMP participates in the progress of hematology malignancies, but the underlying mechanisms have not been clearly defined. To explore the effects of MT1-MMP in hematology malignancies, the expression of MT1-MMP as well as MMP-2 and TIMP-2 in 13 malignant hematopoietic cell lines was investigated and its eukaryotic expression vector was constructed and tranfected to HL60 with low expression of MT1-MMP. mRNA expressions of MT1-MMPs, MMP-2 and TIMP-2 in hematopoietic cell lines with different lineages were semi-quantified by RT-PCR. The gelatinolytic activity of MMP-2 was visualized on gelatin zymograms and the expression of MT1-MMP protein was detected by flow cytometry. The eukaryotic expression vector pcDNA3.1/V5-His-TOPO-MT1-MMP was constructed and identified by enzyme digestion, PCR and sequencing, the vector was transfected into HL60 with liposomes and the transfection efficiency was analyzed by RT-PCR, real-time PCR and flow cytometry. HL60/pcDNA3.1 cells were adopted as controls. Cell proliferation was examined by MMT, and expressions of MMP-2, Bcl-2 and Bax were analyzed by real-time PCR. Invasive potential and cell cycle of the transfected cells were studied with transwell plate and flow cytometry. The expression patterns of the MT1-MMP/MMP-2/TIMP-2 were varied in 13 hematopoietic cell lines. No obvious correlation between the origin of the cells and the expression level of the detected genes was observed (P>0.1), while MT1-MMP significantly correlated to MMP-2 (R=0.64, P=0.02). The expression vector of MT1-MMP was successfully constructed. Significantly enhanced proliferative ability was observed in HL60/MT1-MMP than in control (P<0.01). No obvious difference was observed as to the expression of MMP-2, Bcl-2 and Bax between the two groups. At 48h after transfection, significantly increased invasiveness was noted in HL60/MT1-MMP (15.42±2.38%)than in control(5.16±0.78%)(P<0.01). At 24h after transfection, higher ratio of G2 and S phase cells was found in the experiment group than in control (67.5±3.57% vs 57.9±2.52%) (P<0.05), whereas at 48h and 72h after transfection, no obvious difference of cell cycle was observed between the two groups. The association between expression of MT1-MMP and MMP-2 in malignant hematopoietic cell lines may suggest the close relation of their functions. Over-expressing MT1-MMP in leukemic cell lines exhibit enhanced proliferative and invasive potential. No direct relationship can be identified between MMP-2 and enhanced invasiveness. Directive involvement of MT1-MMP may be absent in the regulation of apoptosis induced by the Bcl-2/Bax pathway.
Two Novel Translocations That Disrupt the RUNX1 Gene in Acute Myeloid Leukemia.
Authors: Dai Haiping , Xue Yongquan , Pan Jinlan , Wu Yafang , Wang Yong , Shen Juan , Zhang Jun 
Published: October 2019

Abstract
Translocations involving 21q22 are commonly observed in both de novo and therapy-related acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). They often result in the disruption of RUNX1 and give rise to fusion genes consisting RUNX1 and different partner genes which contribute to leukemogenesis. At present, at least 21 such translocations have been reported in the literature. We report here two novel translocations involving the RUNX1 gene: t(1;21)(q12;q22) in a 53-year-old female with AML-M5b and t(11;21)(q13;q22) in a 65-year-old male with AML-M2. The abnormalities revealed by R-banding karyotypic analysis were confirmed by interphase and metaphase fluorescence in situ hybridization (FISH), chromosome painting (CP), and M-FISH.
Hidden Abnormalities and Novel Classification of t(15;17) APL Based on Genomic Alterations.
Authors: Akagi Tadayuki , Ogawa Seishi , Yamamoto Go , Sanada Masashi , Kawamata Norihiko , Miller Carl W. , Liang Der-Cherng , Shih Lee-Yung , Koeffler H. Phillip 
Published: October 2019

Abstract
Acute promyelocytic leukemia (APL) is a hematopoietic malignant disease characterized by the chromosomal translocation t(15;17), resulting in the fusion of PML-RARA genes. We examined whether genomic alterations could be found other than t(15;17) using a sensitive technique in order to subcategorized this disease on the basis of genomic status. Thirty-three t(15;17) APL samples were analyzed with high-density single-nucleotide polymorphism microarray (50K SNP-chip) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). Our analysis revealed that 10q (2 cases), 11p (3 cases) and 19q (1 case) regions were identified as loss of heterozygosity with normal copy number [we call this somatic uniparental disomy (UPD)] that could not be detected by karyotypic analysis. Nineteen samples (58%, Group A) did not have an obvious alteration. Fourteen samples (42%) showed either one or more genomic abnormalities: 6/14 of these samples (18%, Group B) had trisomy 8 either with or without duplication, deletion, and UPD; and 8/14 of these samples (24%, Group C) had abnormalities without trisomy 8. Interestingly, FLT3-ITD mutation (7/33 cases) was found only in Group A. These results suggest that the pathway of development of APL differs in each group; FLT3-ITD, trisomy 8 (probably c-Myc gene), and unknown factor(s) are involved in group A, B, and C, respectively. Here, we showed for the first time hidden abnormalities and novel disease-related genomic regions in t(15;17) APL. Our technique may become a routine, rapid, robust technique for subclassification of APL and to screen for novel therapeutic targets.
Bio-Cell Chip Method for Stable DNA Storage and Simple Extraction.
Authors: Kim Miyoung , Lee Dong Soon , Kim Tae Young , Oh Bora , Min Hyun Jung , Yoon Sung-soo 
Published: October 2019

Abstract
We have previously developed the bio-cell chip, on which cells are arrayed and immobilized on a glass slide, using multi-well device. Development of bio-cell chip was originally targeted for the application in fluorescent in situ hybridization and immunohistochemistry. Here, we suggest simple and convenient method of DNA extraction from bio-cell chip, which can handle hundreds of specimens. One uL of cell suspension was loaded on wells of the bio-cell chip and after drying, 2uL of BR-A buffer (GenScript BloodReady™Multiplex PCR System) was dropped onto the wells of bio-cell chip. After incubation for 10 minutes, each mixture was transferred to a microtube with additional buffer to a total volume of 20uL. One uL of mixture was taken and 20uL of mixture for PCR reaction was prepared; 1U BioTherm™DNA Polymerase (Genecraft, GERMANY), 1uL (10pM/ul) of each oligonucleotide primer for target sequence, 2.5mM dNTP, and 1.0uL of DNA mixture. PCR for GST M1 and hGAPDH was carried out using the MJ Research DNA thermal cycler. With this bio-cell chip extraction method, simplification of experimental procedure not only lessened the loss of specimen but also saved the time. Only 10 min of reaction time and a very small volume of samples was required without any further purification step. Amplification of DNA was possible, reflecting safety in DNA stability. Requirement for the diagnostic specimen transport is intricate; strict regulations on infectious materials and preparation of specimen package, cell fixatives such as methanol. Bio-cell chip enables transport of hundreds of samples at a thin plate without transportation media, and also simple extraction of DNA from bio-cell chip with high throughput efficiency, with excellent stability comparable to those with fresh samples.
Figure Figure
A Novel Method of Microarray Quantitave Analysis of P16 CPG Island Methylation in Leukemia Cells from Patients with Hematological Malignancies.
Authors: Shou Beiming , Chen Baoan , Zhou Dongrui , Ding Jiahua , Gao Chong , Sun Yunyu , Wang Jun , Chen Jian , Zhao Gang , Ma Yan , Song Huihui , Bao Wen , Pei Xiaoping , MA Xudong , Liu Delong 
Published: October 2019

Abstract
p16 is a tumor suppressor gene frequently affected by hypermethylation of promoter CpG island in malignant disorders. Phenylhexyl isothiocyanate (PHI) has been shown to induce p16 hypomethylation in leukemia cells. However, the methylation status is usually studied qualitatively or semi-quantitatively at the best. In this study, we developed a novel method to quantify p16 methylation in clinical specimen from patients with hematological malignancies as well as in U266, a multiple myeloma cell line. To establish a standard curve of p16 promoter methylation, methylated and demethylated PCR products were subcloned into plasmids respectively. The clones were selected out as completely methylated and demethylated clones, respectively. The PCR products of the two clones were mixed at different ratios and analyzed on a mciroarray chip to produce a standard curve of methylation. p16 promoter methylation was determined in U266 cells. The baseline p16 methylation was 78.2% in U266 cells. The methylation was decreased to 61.7% and 54.8%, respectively in the presence of PHI of 5 and 10 uM, respectively. We further analyzed p16 methylation status in 7 patients’ specimen, two from blood, 5 from bone marrow. Two patients had ALL, two had AML, one CLL, one MM, and one CMML. 3 patients have p16 gene hypermethylation at 17.7%, 47.5% and 84.3%, respectively. Hypomethylation occurred in two of the three patients’ cells after PHI treatment in vitro (84.3% to 64.8%, and 47.5% to 25.4%). In conclusion, a quantitative microarray method was established for quantitation of p16 methylation status. This will be used for laboratory screening of hypomethylation drugs, such as PHI, and for clinical application to monitor therapeutic efficacy of hypomethylating drugs, such as azacitidine, and to prognostify patients based on p16 methylation level.
RUNX1/EVI1 That Blocks Myeloid Differentiation Inhibits C/EBPα Function.
Authors: Tokita Katsuya , Maki Kazuhiro , Mitani Kinuko 
Published: October 2019

Abstract
RUNX1/EVI1 chimeric transcription factor produced by t(3;21) causes leukemic transformation in hematopoietic stem cell tumors such as chronic myelogenous leukemia (CML) in blastic crisis and myelodysplastic syndrome (MDS) in leukemic transformation, possibly through differentiation block of malignant myeloid progenitors. We have recently reported that Runx1/EVI1 knock-in heterozygous mice show defective hematopoiesis in the fetal liver similar to Runx1 knock-out mice, but possess dysplastic hematopoietic progenitors with high self-renewal capacity. Notably, Runx1/EVI1 knock-in chimeric mice developed acute megakaryoblastic leukemia. The molecular characterization of RUNX1/EVI1 points two major functions; one is dominant-suppressive function over wild-type RUNX1 and the other is EVI1’s own function; blockade of TGFb-mediated signal, inhibition of JNK and stimulation of AP-1 activity. C/EBPa is a key transcriptional regulator that induces the granulocytic differentiation of myeloid progenitors and several lines of evidence suggest that disturbance in C/EBPa signaling is one of the major molecular events in myeloid malignancies. In this study, we investigated whether RUNX1/EVI1 affects the expression and function of C/EBPa. We introduced RUNX1/EVI1 cDNA into LG-3 cells that differentiate along the myeloid lineage upon granulocyte colony-stimulating factor exposure, and confirmed that RUNX1/EVI1 suppressed the differentiation. To further investigate the molecular mechanisms of RUNX1/EVI1-mediated differentiation block, we analyzed RUNX1/EVI1’s effect on the functions of C/EBPa. RUNX1/EVI1 was found to associate with C/EBPa. By using the reporter assay with the CEBPA promoter, we observed a dominant-negative effect of RUNX1/EVI1 over C/EBPa-mediated transcriptional activation via the CtBP-binding site in the EVI1 portion. In the gel-shift assay, RUNX1/EVI1 down-regulated DNA-binding activity of C/EBPa. Therefore, recruitment of histone deacetylase via CtBP and disruption of DNA binding could be likely scenarios for the RUNX1/EVI1-induced dominant repression on C/EBPa. Importantly, co-expression of C/EBPa restored differentiation ability of the RUNX1/EVI1-expressing LG-3 cells. All these data argue that inhibition of C/EBPa function may be causatively related to the RUNX1/EVI1’s leukemogenic potential.