bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of the Cone and Plate(let) Analyzer as a Point-of-Care Method for Monitoring Anti-Platelet Therapy.
Authors: Spectre Galia , Brill Alexander , Gural Alexander , Shenkman Boris , Turetzky Natalia , Savion Naphtali , Varon David
Published: April 2020

Platelet anti-aggregants play a major role in the treatment of cardiovascular disorders. Recently it has been suggested that a significant proportion of patients fail to respond to these agents according to different testing methods. We have developed a new method for evaluating the response to platelet anti-aggregants, the modified Cone and Plate(let) Analyzer (CPA) test. The method is based on the observation that pre-activation of platelets by an agonist will result in reduced platelet deposition on a polystyrene surface under arterial flow conditions (the phenomenon of platelet adhesion refractoriness). In vitro studies demonstrated that the basic platelet adhesion (surface coverage, SC 12.3±6.8%) was significantly reduced in response to pre-incubation of the sample (for 1 min) with arachidonic acid (AA, SC 2.1±1.5%), adenine diphosphate (ADP, SC 1.3±0.6%) and epinephrine (EPI, 2.9±0.9%). This effect was selectively inhibited by aspirin (for the response to AA, SC 8.1±3.8%), and by 2-Methylthio-AMP triethylammonium (2-MeSAMP), a selective inhibitor of P2Y12 (for the response to ADP, SC 4.8±2.0%), p<0.001. The potential application of this method for monitoring the effect of aspirin therapy (daily doses of 100, 300 and 500 mg), among 20 healthy volunteers was investigated in comparison to turbidimetric aggregometer. All volunteers responded to aspirin in the three doses according to the aggregometer test (reduced max. aggregation from 88±8% to 12.4±7%), with no evidence for a dose response manner in this dose range. Basic platelet adhesion as tested by the CPA (SC 9.8±2.2%), was unaffected by aspirin in the three tested doses. However, the response to pre-incubation with AA was significantly inhibited by aspirin in all patients (SC of 0.6±0.3% before aspirin therapy and 3.5±1.3%, 4.4±1.7%, and 4.1±2.0% following therapy with 100, 200 and 500 mg aspirin per day, respectively, p<0.001 for all doses), again with no dose response effect. This effect was specific for AA, since no difference in SC was observed when ADP was used as an agonist. A significant correlation between the effect of aspirin as tested by the modified CPA and by the turbidimetric aggregometer using AA as an agonist (R2=0.55) was observed. In conclusion, the CPA was found useful point-of-care method for testing the response of platelets to aggregating agonists as well as for monitoring the effect of anti-platelet drugs.
Direct and Indirect Effects of Oxymetholone on Growth and Apoptosis of Hematopoietic Progenitor Cells In Vitro.
Authors: Jo Deog-Yeon , Kim Seong-Woo , Hwang Jin-Hee , Yun Hwan-Jung , Kim Samyong
Published: April 2020

Despite a tarnished reputation, androgens remain a common treatment for aplastic anemia, especially in the Orient. Increased apoptosis of hematopoietic cells is characteristic of aplastic anemia; however, it has not been established whether androgens affect apoptosis of hematopoietic progenitor cells. In most previous studies regarding the in vitro effects of androgens on hematopoietic cells, whole bone marrow cells were used, rather than purified hematopoietic progenitor cells such as CD34+ cells. With these questions in mind, we investigated the direct and indirect effects of oxymetholone and other androgens on apoptosis and growth of normal hematopoietic progenitor cells (HPCs) in vitro. Oxymetholone did not rescue normal BM CD34+ cells and colony-forming cells (CFCs), other than mature erythroid CFCs, from apoptosis induced by growth factor deprivation. Unexpectedly, both testosterone and 5-dihydrotestosterone (5-DHT) at a concentration of 10−5 M, but not oxymetholone, increased the percentage of annexin-positive apoptotic cells (62.2 ± 5.9%, P < 0.05; 61.7 ± 6.4%, P < 0.05, respectively) compared with the controls (52.6 ± 5.6%). The addition of either stromal cell-derived factor-1 (SDF-1) or stem cell factor (SCF) partially relieved the increase in apoptosis induced by 5-DHT, and the addition of both SDF-1 and SCF completely reversed it. Oxymetholone did not rescue CFCs from interferon-gamma (IFN-g)-induced inhibition of clonal growth of BM CD34+ cells in methylcellulose cultures. Furthermore, oxymetholone did not mitigate IFN-g-induced suppression of CD34+ cell survival in the presence of growth factors. In a methylcellulose clonogenic assay, oxymetholone stimulated the clonal growth of colony-forming unit-erythroid at low concentrations, while not affecting colony-forming unit-granulocyte/macrophage or burst-forming unit-erythroid. Oxymetholone did not reverse the IFN-g-induced inhibition of colony formation by CD34+ cells. Interestingly, oxymetholone stimulated the production of SCF and thrombopoietin in normal human bone marrow stromal cells (BMSCs) through transcriptional regulation while inhibiting the production of interleukin-6. In agreement with this, oxymetholone-treated BMSCs better supported the survival and growth of HPCs. These results suggest that oxymetholone exerts most of its myelostimulatory effects via the regulation of cytokine production in BMSCs, rather than by direct action on hematopoietic progenitor cells.
Quantitative and Qualitative Protein Expression Mapping of Highly Enriched G-CSF Mobilized CD34+ Stem Cells from Peripheral Blood.
Authors: Lannert Heinrich , Franz Thomas , Eckstein Volker , Hofmann Reiner , Lenze Angela , Horsch Kerstin , Miesala Katrin , Ho Anthony D.
Published: April 2020

Background: Proteome analysis is a direct measurement of proteins in terms of their presence and relative abundance in a defined system. The overall aim of a proteomic study is characterization of the complex network of cell regulation. Different states of a cell can be compared and specific qualitative and quantitative protein changes can be identified. We focused our first proteom-investigations on G-CSF mobilized CD34+ stem cells from peripheral blood (PB).

Methods: Mononuclear cells from healthy donors were isolated by a standard Ficoll-Hypaque gradient separation method after leucapheresis from PB. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. Sample preparation, determination of protein concentrations, 2D-gel-electrophoresis with a 1D-separation isoelectric focusing (IEF) with immobilised pH gradient (IPG) strips (17cm), 2D-separation with SDS-PAGE were performed and described in Proteome Works System (BioRad), pH range from 3 until 10 and molecular weight from 5 until 200kDa. Sypro ruby stained gels were used for protein identification by peptide mass fingerprint analysis with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

Results: A.) The pattern of the G-CSF mobilized CD34+ fraction from PB cells is complex and shows >1000 protein spots (dependent on protein concentration) in silver stain using PDQuest.

B.) The most dominant 150 protein spots were characterized by MALDI-TOF analyses. A sample of identified spots and their functional groups are specified:

1. Cytosketeletal proteins: tubulin, actin, profilin, tropomyosin. 2. Signaling proteins: enolase, rho GDP dissociation inhibitor 2, glutathione S transferase, nucleophosmin. 3. Metabolism: phosphoglycerate kinase I, glyceraldehyde-3P-dehydrogenase. 4. Protein folding: heat shock proteins (HSP 60, HSP70c), molecular chaperones (GRP78).

Conclusions: Proteomics is a highly effective method to describe the gen-expression and the cell biology of stem cells on real protein transcription. Highly purified CD34+ cells from PB demonstrate a complex proteom. Our preliminary results show that cell cycle determining chaperones (folding of other protein and responsible for their functional activation or deactivation) are dominantly expressed. Thus cytoskeletal proteins dominate the protein pattern of CB stem cells. Further Proteom-comparison with other subsets of stem cells from different origins (e.g. fetal liver, cord blood, bone marrow) are currently achieved.

Figure Figure
Bonr Marrow Reconstitution from Mouse Embryonic Stem Cell-Derived Hematopoietic Cells.
Authors: Yoshimoto Momoko , Hsi Chang , Umeda Katsutsugu , Iida Midori , Heike Toshio , Nakahata Tatsutoshi
Published: April 2020

Differentiation of embryonic stem (ES) cells in vitro yield different kinds of hematopoietic progenitors including primitive and definitive hematopoietic cells. It has been reported that HOXB4 induction enable york sac (YS) cells and embryoid body-derived cells to engraft in the irradiated adult mice, however, since the characteristic of ES-derived transplantable cells is not clear, generating hematopoietic stem cells (HSCs) in vitro still remains to be resolved. We previously reported the generation of definitive HSCs from both early YS and intraembryonic paraaortic splanchnopleures (P-Sp) on AGM-S3 stromal cells derived from the aorta-gonad-mesonephros (AGM) region at 10.5 days post coitum (Matsuoka, et al; Blood 2001) Co-cultureing on AGM-S3, these YS cells and PS-Sp cells acquired the reconstituting potential of adult irradiated mice. Here we intended to make HSCs using this stromal cells. We differentiated ES cells labeled with GFP on OP9 stromal cell, which is supportive for Hematopoietic differentiation. After 4 days, we sorted Flk1+ cells, which is considered as a marker of hemangilblasts, and transferred them onto A-9, subline of AGM-S3, or OP9 stromal cell layer with cytokines. After several days incubation, we examined the emergence of CD34+ c-kit+ cells and colony forming ability of CD34+ or CD34− cells. CD34+ cells contained more CFU-Mix than CD34− cells. When compared on A-9 or OP9, cultured cells on OP9 contained more CFU activity than on A-9. We sorted and cultured CD34+ c-kit+ cells on OP9 for 7–10 days, and confirmed Ter119+, Gr-1+, or Mac-1+ cells differentiated from CD34+ c-kit+ cells by FACS analysis. Next, we cultured Flk1+ cells on A-9 or OP9 for 7–15 days and transplanted all the collected cells into 2.4Gy irradiated NOD-SCID mice. After 3 months after transplantation, FACS analysis showed no GFP+ cells in the recipient BM. However, PCR analysis detected donor derived DNAs in BM when Flk1+ cells were cocultured on A-9. We next transplanted 1×104 of CD34+ CD45+ or CD34+ CD45− cells from Flk1+ cells cocultured on A-9 or OP9 into 2.4 Gy irradiated NOD-SCID mice. PCR analysis revealed donor derived DNAs in mice transplanted with CD34+ CD45+ cells on A-9 and with CD34+ CD45− cell on OP9. These data suggested that CD34+ cells differentiated from Flk1+ cells have powerful hematipoietic activity and showed different potential cultured between on A-9 and on OP9.
A Novel hTERC Deletion Manifesting with Features of Dyskeratosis Congenita and Genetic Anticipation.
Authors: Benke Stan , Houston D. S. , Dokal Inderjeet , Vulliamy Tom
Published: April 2020

The gene encoding the RNA component of human telomerase (hTERC) is mutated in families with the autosomal dominant form of dyskeratosis congenita (DC). The phenomenon of genetic anticipation has recently been reported to accompany this form of DC, with disease severity increasing in offspring of affected individuals. It has been postulated that anticipation in these families relates to the adverse impact of hTERC mutations on inherited telomere length, with progressive telomere shortening seen in succeeding generations (Nat Gen2004; 36:447). We describe here a novel hTERC mutation, with affected individuals presenting in adulthood with mild mucocutaneous abnormalities, bone marrow failure and a pattern of penetrance supporting the presence of disease anticipation. The proband in the family studied presented at age 49 with squamous cell carcinoma of the tongue and a history of oral leukoplakia which he had developed at age 30. Peripheral blood on presentation was remarkable only for a mild macrocytic anemia. During treatment of his malignancy, severe and irreversible bone marrow hypoplasia was precipitated by a single cycle of cisplatinum chemotherapy. The patient’s brother at age 25 had been previously diagnosed with severe aplastic anemia; this was refractory to standard immunosuppression with cyclosporine and antithymocyte globulin. No somatic abnormailites were identified in this patient. Testing for Fanconi anemia in both siblings was negative. Direct sequencing analysis of hTERC in these patients revealed both to be heterozygous for a novel hTERC mutation (79 deletion C). Further studies among family members documented heterozygosity for the mutation in the mother of these two siblings. At age 77, she displayed none of the mucocutaneous signs associated with DC, while the only abnormality seen in her peripheral blood was an elevated mean corpuscular volume. The hTERC mutation seen in this family most likely exerts its effects through disruption of the pseudoknot domain. The findings of an individual with normal longevity, minimal phenotypic expression and affected offspring are further evidence of genetic anticipation being an important feature of autosomal dominant DC. Correlation with determination of telomere length has been initiated.
Parathyroid Hormone (1–34) Treatment Induces Hematopoietic Precursor Cell Expansion in Murine Long-Term Bone Marrow Culture.
Authors: Drize Nina , Svinareva Daria , Nifontova Irina , Chertkov Joseph
Published: April 2020

Parathyroid hormone (PTH) induces activation and increases the number of osteoblasts. An increase the stem cells number (Lin-Sca1+cKit+) was observed in mice after 4 weeks of PTH treatment, that suggest osteoblasts participation in regulation of hematopoiesis (Calvi, et al., 2003, Nature, 425, 841). Long-term bone marrow culture (LTBMC) was used for the study of PTH influence on hematopoietic progenitors of different stages of maturation. Rat PTH (1–34) (final concentration, 10−7M) was supported during whole culture period. Cell number, colony-forming units in culture (CFU-GM, CFU-C-21 days) and cobblestone area forming cells (CAFC) were measured during 10 weeks. The number of mature cells in culture did not changed during PTH treatment. The number of studied progenitors did not changed significantly during 3 and 6 weeks of PTH treatment. The number of CFU-GM, CFU-C-21 days and CAFC 14 days, CAFC 21 days, CAFC 28 days increased 7 –10 fold in suspension fraction of LTBMC after 10 weeks of PTH treatment.

Table 1. Number of different precursor cells in suspension fraction of LTBMC after PTH treatment.

To evaluate the possibility of stem cells expansion by using of PTH treated adherent cell layers (ACL) of LTBMC, the PTH-treated for 4 and 8 weeks cultures, were irradiated with 40 Gy and seeded with 2 × 106 bone marrow cells depleted of adherent cells. Following 24 hours the number of survived CAFC 8 – 28 was analyzed. On PTH-treated ACL the number of CAFC 21 increased 2,7 ± 0,4 fold as compared with fresh bone marrow or culture with non-treated ACLs. The number of CAFC 28, which characterizes the number of marrow repopulating cells, increased 2 fold only after cultivation on ACL treated with PTH for 8 weeks.

Table 2. CAFC number in bone marrow cultivated 24 hours on PTH treated ACL of LTBMC

The data suggest possibility to increase stem cell expansion ex vivo on pharmacologically manipulated microenvironment.

Time of cultivation, weeks Specificity of progenitor Number of precursors, per 100000 cells Control Control PTH treatment 3 CFU-GM 50 ± 5.2 77 ± 5.6 6 CFU-GM 33 ± 4 31 ± 5.9 10 CFU-GM 20 ± 4.8 147.5 ± 3.5 3 CFU-C-21 55 ± 5.9 76.6 ± 11.2 10 CFU-C-21 11.5 ± 1.7 102.0 ± 2.0 3 CAFC-7 21.6 20.3 6 CAFC-7 25.9 26.9 10 CAFC-7 0.9 3.0 3 CAFC-21 1.01 0.69 6 CAFC-21 0.43 0.48 10 CAFC-21 0.28 3.11 3 CAFC-28 0.32 0.55 6 CAFC-28 0.12 0.14 10 CAFC-28 0.1 0.82 Age of CAFC (days) Number of CAFC per 2 x 10^6 bone marrow cells not treated PTH (4 weeks) PTH (8 weeks) CAFC-7 424.1 804.5 647.5 CAFC-14 262.5 529.8 295.2 CAFC-21 93.5 272.7 226.2 CAFC-28 38.3 30.1 79.2
Human Marrow Megakaryocyte Differentiation: Multiparameter Correlative Analysis Identifies Von Willebrand Factor as a Sensitive and Distinctive Marker for Early (2N and 4N) Megakaryocytes.
Authors: Tomer Aaron
Published: April 2020

Human megakaryocyte differentiation and maturation were studied in fresh marrow aspirates using multiparameter flow cytometric correlative analysis. The expression of glycoprotein (GP)IIb/IIIa, GPIIIa, GPIb and CD36 correlated directly with cell size and ploidy (r ≥ 0.97) however, GPIb acquisition was relatively slow. von Willebrand factor (vWF) is robustly expressed by early (2N and 4N) megakaryocytes, enabling their complete resolution from the other marrow cells at a level superior to that achieved with GPIIb/IIIa. Expression of myeloid CD45 and IgG-FcγRII (CDw32) increased with megakaryocyte maturation and contrasted with the declining expression of HLA-DR (negative in platelets). Interleukin-6 receptor expression in megakaryocytes was higher than in other marrow cells. Using the time-of-flight technique, the diameter of the megakaryocyte population was 37±4μm (mean±1SD) compared to 14±2μm for the total marrow cells, ranging from 21±4μm for 2N cells to 56±8μm for 64N cells. Cell size directly correlated with cell DNA (r=0.98). Receptor density of GPIIb/IIIa and GPIb decreased with the transition from 2N to 4N cells, then reached maximum at 32N-cells. We conclude that the present methods are useful for studying in vivo human megakaryocytopiesis in normal and altered states. The expression of vWF is a sensitive and distinctive marker for the identification of young marrow megakaryocytes.
Oncogenic Tyrosine Kinases Regulate Proliferative and Survival Signals through Activation of Id1.
Authors: Gu Ting-Lei , Tam Winnie F. , Chen Jing , Lee Benjamin H. , Wang Andrew Z. , Cooper Michael R. , Monti Stefano , Golub Todd , Gilliland Gary D.
Published: April 2020

The oncogenic tyrosine kinases, such as BCR-ABL, TEL-ABL, TEL-PDGFbR and FLT3-ITD, play a major role in the development of hematopoietic malignancy. They activate many of the same signal transduction pathways, including the RAS/MAPK pathway, STAT/JAK pathway and PI3-K/AKT pathway. Although activation of signal transduction pathway has been extensively studied, few bone fide target genes have been identified. To identify the critical target genes that are required for transformation in hematopoietic cells, specific tyrosine kinase inhibitors were applied to 32Dcl3 cells expressing various activated tyrosine kinases. Using Affymetrix oligonucleotide arrays, we have identified inhibitor of DNA binding 1 (Id1), which is involved in development, cell cycle and tumorigenesis, as a common target gene of oncogenic tyrosine kinases. To characterize the biological consequence of Id1 expression, we inhibited the expression of Id1 in two human leukemia cell lines (Molm-14 and K562) by an antisense Id1 construct. In both cell lines, we observed a growth inhibition associated with release of inhibition of p27Kip. In addition, inactivation of Id1 sensitized K562 cells to Trail-induced apoptosis. Our findings suggest that Id1 is an important target of constitutively activated tyrosine kinases, and may be a therapeutic target for leukemias involving oncogenic tyrosine kinases.
Transcription Factors Analysis in Neuronal-Like Cells Derived from Adult Human Bone Marrow.
Authors: Bulvik Shlomo , Ben-Zur Tali , Levy Yossef S. , Burshtein Alex , Melamed Eldad , Offen Daniel
Published: April 2020

Stem cells are immature cells with self-renewal capacity and ability to differentiate into multiple cell types including bone, cartilage, fat, tendon and muscle. Transplantation of stem cells or their derivatives to the adult brain have been proposed as future therapy for neurodegenerative diseases. Parkinson’s disease (PD) is a progressive neurodegenerative disease involving the deterioration of dopaminergic neurons of the substantia nigra, a part of the midbrain. Most of the currently used drugs are based on dopamine replacement which, although it reduces symptoms is limited owing to severe side effects. Alternatively, strategies of cell therapy for the treatment of PD focused on replacing damaged neurons with cells that may improve the functioning of the damaged cell population. In this study, we demonstrate that bone marrow stromal cells isolated from human iliac crest can be induced to differentiate into neural-like cells. Human bone marrow stromal cells differentiation was induced with N2 supplements, retinoic acid, butylated hydroxyanisole, isobutylmethylxanthine and dibutyryl-cyclic-AMP. A few hours after differentiation the cells changed their phenotype to neural-like cells, spindle shaped, with cell bodies and long branches. Moreover, immunostaining demonstrated the expression of specific neuronal markers such as nestin, neurofilaments and neuron specific enolase. To define the transcription factors associated with the differentiation, we used the TranSignal Protein/DNA kit (Panomics Inc). This new array-based technology allows functional analysis of dozens of eukaryotic transcription factors. Nuclear proteins were purified and incubated with mix labeled DNA binding oligonucleotides corresponding to the consensus sequences of 96 transcription factors. The DNA probes were then dissociated from the complexes and used to hybridize the TranSignal Array. We found that the differentiated human bone marrow stromal cells induced the expression of several neuron-specific transcription factors such as GAG (glycosaminoglycan), FKHR (human forkhead in rhabdomyosarcoma) and DR-2. The activation of these transcription factors in a time course manner was evident also by electrophoretic mobility shift assay. In conclusion, we show that the induced differentiation of neuronal like cells from bone marrow is associated with activation of specific neuronal transcription factors. Our study advances the possible use of autologous transplantation as a new strategy for cell therapy in neurodegenerative diseases.
Transdifferentiation of Bone Marrow-Derived Cells Co-Cultured with Fetal Liver Cells into Hepatic-Like Cells without Fusion.
Authors: Yamada Yasuhiro , Yonemura Yuji , Nishimoto Eishi , Mitsuya Hiroaki
Published: April 2020

Several research groups have reported that bone marrow cells (BMCs) transdifferentiate into hepatocytes in rodents. However, it is yet to be studied what factors effectively trigger and sustain the transdifferentiation of BMCs to hepatocytes. In the present study, we investigated whether murine BMCs in the presence of fetal liver cells (FLCs) could differentiate into hepatic-like cells in vitro without fusion. Fractionated BMCs from C57Bl/6-TgN(ACTbEGFP)10bs mice and FLCs from B6.129S7-Gt(ROSA)26Sor mice were co-cultured at 1x105 cells and 1x106 cells in 10% FCS-containing medium supplemented with hepatocyte growth factor on laminin-coated dishes. Hepatocyte-specific markers among BMCs were detected as assessed by immunocytochemistry for albumin and reverse transcription-polymerase chain reaction (RT-PCR) for alpha-fetoprotein (AFP), albumin, and cytokeratin-19 mRNAs. We also found that Sca-1+ BMCs containing both hematopoietic stem cells and AFP-expressing cells could differentiate into hepatic-like cells and such cells were seen adherent to dish together with FLCs in the early phase of culture. Moreover, the AFP-expressing cells were found in a Sca-1+ cKit- cell fraction, which also differentiated into CD45− GFP+ albumin+ cells and proved to be positive for GFP but negative for LacZ as assessed by RT-PCR and immunocytochemistry. These results suggest that albumin+ cells developed through transdifferentiation from BMCs but not through spontaneous cell fusion between BMCs and FLCs.
The Dynamics of Chromatin Modification during RA Induced Promyelocyte Differentiation.
Authors: Harris Robert G. , Krause Diane S.
Published: April 2020

Epigenetic histone modifications have recently been shown to be important in gene regulation and to be correlated with gene expression profiles. In particular, particular modifications at Lysine 4 and Lysine 9 of Histone H3 may be highly indicative of both active and silenced chromatin. Promoters of highly active genes are generally tri-methylated at Lysine 4 and acetylated at Lysine 9 whereas silenced chromatin is unmethylated or mono methylated at Lysine 4 and di or tri-methylated at Lysine 9. NB4 is a human acute promyelocytic leukemia (t15:17) cell line that can be differentiated down the neutrophil lineage by induction with supraphysiological levels of retinoic acid (RA). We have examined post-translational modifications at Lysine 4 and Lysine 9 of Histone H3 at specific promoters using chromatin immunoprecipitation (ChIP) during differentiation of NB4 cells. We have performed quantitative (q) RT-PCR for the Myeloperoxidase (MPO) and Defensin-alpha (Def-a) genes after 0,12, 24, 48 and 72 hours of RA induction of NB4 cells. Concurrently we performed ChIP followed by qPCR for Di- and Tri-methylated Lysine 4 of Histone H3 and acetyl- and di-methylated Lysine 9 of Histone H3 at the promoters of these genes. MPO mRNA is expressed at time 0, and decreases progressively starting at 24 hours such that by 48 hours mRNA levels are negligible. This correlates nicely with a sharp decrease in tri-methyl Lysine 4 and acetyl Lysine 9 levels by 12 hours, with a smaller decrease again at 24 hours on the MPO promoter. By 72 hours a concurrent increase in dimethyl Lysine 9 levels is seen, indicating chromatin silencing. Conversely, def-a is initially not expressed in NB4 cells. After 24 hours of induction with RA, an increase in mRNA levels is seen. This correlates well with a sharp increase of acetyl Lysine 9 in the promoter and a concurrent decrease in dimethyl lysine 9 levels. In conclusion we have shown that during RA induced differentiation of promyelocytic leukemia cells, chromatin modifications slightly precede changes in mRNA expression levels and recapitulate other studies linking Lysine 4 methylation with active transcription and Lysine 9 methylation with gene silencing.
Antagonistic Roles of the ERK and p38 Pathways in Chemically Induced Erythroid Differentiation of Murine Erythroleukemia Cells.
Authors: Blank Volker , Boudia Mansouria Merad , Todorova Margarita , Moore Amy P.
Published: April 2020

Murine erythroleukemia (MEL) cells provide a valuable model to study the molecular events leading to erythroid differentiation. Maturing erythroid cells synthesize large quantitities of hemoglobin, a process requiring the coordinated synthesis of heme and globin. Here, we investigated the role of the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways in chemically induced differentiating MEL cells. We showed that treatment of DMSO- or HMBA-induced MEL cells with the MEK1/2 inhibitor UO126, blocking the ERK pathway, results in the increase of beta-globin and 5-aminolevulinate synthase-2 (ALAS-2) transcript levels. We found that addition of the p38 inhibitor SB203580 has the opposite effect, leading to decreased beta-globin and ALAS-2 mRNA levels. The regulation on the transcript level correlated with increased or decreased hemoglobin levels in the presence of MEK1/2 and p38 inhibitors, respectively. Our results suggest that the ERK and p38 pathways play antagonistic roles in DMSO- and HMBA induced erythroid differentiation in MEL cells. This data also provide a novel link between MAPK signaling and the regulation of intracellular heme levels, as ERK and p38 MAPK signaling cascades appear to differentially regulate one of the key enzymes of the heme biosynthesis pathway.
Expression and Functional Differences of CBFA2T (ETO, MTG) Gene Family Members in Hematopoiesis.
Authors: Mortazavi Fariborz , Kadkol ShriHari , Bruno Annette , Baraoidan Kristine , Ackerman Steven , Wickrema Amittha , Saunthararajah Yogenthiran
Published: April 2020

The CBFA2T (ETO, MTG) family has three similar family members - CBFA2T1-T3. CBFA2T1 (ETO, MTG8) and CBFA2T3 (ETO2, MTG16) are targeted by chromosomal translocations in acute myeloid leukemia. To better understand the usual hematopoietic function of this gene family, we examined the expression of CBFA2T RNA using RQ-PCR in cell-lines and human CD34+ hematopoietic cells during macrophage and erythroid differentiation. RQ-PCR on extracted RNA was performed with an icyclerQ instrument (Bio-Rad) using the Quantitect SYBR Green RT-PCR kit (Qiagen) and in vitro transcribed RNA to construct standard curves. CBFA2T3 was the most highly expressed family member in human CD34+ cells, the erythro-leukemia line K562, the myeloid line MPD, the T cell line Jurkatt and the B-cell line LCL-11. However, CBFA2T3 expression decreased by >50% during both macrophage and erythroid differentiation of human CD34+ cells. In contrast, CBFA2T1 expression was almost undetectable in human CD34+ cells and all cell lines except K562 but increased more than 20 fold during erythroid (but not macrophage) differentiation of human CD34+ cells. Extrinsic over-expression of CBFA2T1, but not CBFA2T2, significantly increased glycophorin-A and hemoglobin A expression in K562 cells, consistent with a regulatory role for CBFA2T1 in erythroid differentiation. CBFA2T2 (MTGR1) was moderately expressed in human CD34+ cells and all the cell lines and demonstrated a 2.5 fold increase in expression with macrophage differentiation but essentially no change with erythroid differentiation of human CD34+ cells. These findings suggest that despite their similarity, the CBFA2T family members have distinctive regulatory roles in hematopoietic differentiation.
In Vivo Induction of Cytokine mRNA and Protein in Mice by the Radioprotectant Steroid 5-Androstenediol.
Authors: Grace Marcy B. , Chang Cheng-Min , Singh Vijay K. , McLeland Christopher , Inal Cynthia E. , Jackson William E. , Whitnall Mark H.
Published: April 2020

We previously showed 5-androstenediol (5-AED) injected sc enhanced survival in mice exposed to whole-body gamma-irradiation, ameliorated radiation-induced neutropenia in mice, dogs, and monkeys, and induced increases in bone marrow GM-CFC in irradiated mice. Radiation-induced decreases in platelets, natural killer cells, red blood cells, and monocytes were also blunted by the steroid. Administration of 5-AED caused functional activation of circulating granulocytes (phagocytic ability), monocytes (oxidative burst), and natural killer cells (surface CD11b expression). To test our hypothesis that 5-AED acts via initiation of a cytokine cascade in hematopoietic tissue, we performed real time quantitative PCR on spleen samples taken from mice and correlated the results with serum ELISA. PCR was performed for mRNAs of GM-CSF, IL-6, IL-10, IL-12, and IFNg. Spleens and serum were taken at 3 time points: 4 h after 5-AED or vehicle (PEG-400) injection, and 4 or 24 h after irradiation (7.5 Gy whole-body gamma) or sham-irradiation. Each group contained 5 mice. Results were analyzed by 2-way ANOVA for PCR results 4 and 24 h after irradiation, and by a t-test 4 h after injection. Radiation alone induced increases in mRNAs for all cytokines at both 4 and 24 h, except for IFNg, which was not elevated at 4 h. 5-AED alone did not induce a significant increase for any cytokine, but the increases bordered on significance for IL-12 at 4 h after irradiation or sham-irradiation (28 h after injection, p = 0.07), and for GM-CSF 24 h after irradiation or sham-irradiation (48 h after injection, p = 0.08). There was a significant positive interaction between radiation and 5-AED for IL-6, 4 h after irradiation (p = 0.006). The interaction effect bordered on significance for IL-12 4 h after irradiation (p = 0.07) and for GM-CSF 24 h after irradiation (p = 0.10). ELISA for serum cytokine protein was measured for G-CSF, IL-6, IL-10, and IL-12. Results were analyzed by the Mann-Whitney test for exact p-values. IL-6 was not detected in serum. G-CSF was detected 4 and 24 h after irradiation or sham-irradiation only in 5-AED-treated mice. IL-10 and IL-12 were detected only 24 h after irradiation, and the values in 5-AED-treated mice were significantly higher than those in vehicle-treated mice (p = 0.02). The results support our hypothesis that the previously observed increases in numbers of circulating innate immune cells and platelets, and functional activation of granulocytes, monocytes, and NK cells, result from a cytokine cascade induced by 5-AED.
Decreased Total Body Irradiation (TBI)-Induced Apoptosis in Murine Hematopoietic Side Population Cells Compared to Non-Side Population Cells.
Authors: Shen Hongmei , Yun Yun , Epperly Michael W. , Greenberger Joel S.
Published: April 2020

The side population (SP) cells of the murine bone marrow (BM) contain primitive hematopoietic stem cells (HSCs). To understand the effect of irradiation on these cells, C57BL/6NHsd mice were irradiated to 10 Gy total body dose and sacrificed 24 hours later. The bone marrow was removed and stained with Hoechst dye, and Annexin V as an early marker of apoptosis. The SP cells were analyzed by flow cytometry. Following irradiation, there was no change in the frequency of SP cells in total nucleated BM cells between non-irradiated (0.081 ± 0.033%) and irradiated mice (0.093 ± 0.027%). In contrast, there was a decrease in total nucleated BM and SP cells. SP cells decreased by 60.1% from 44873 ± 22099 SP cells per harvest to 17550 ± 4805 SP cells per harvest 24 hours after 10 Gy. The number of lymphocytes in the irradiated BM decreased from 25.7% to 4.5% after irradiation. Since the majority of the SP cells are located in the lymphocyte sorting region, there was a 6-fold decrease in the percent of SP cells found in the live lymphocyte region following irradiation (0.469 ± 0.185% compared to 2.923 ± 1.913%).

Annexin V was used to stain for the appearance of phosphatidylserine (PS) on the cell surface as an early marker for apoptosis. The appearance of Annexin V positive cells did not increase significantly with irradiation in SP compared to non-irradiated SP cells (26 ± 8% and 25 ± 12%, respectively). In contrast, the percent of apoptotic cells in the non-SP fraction was significantly increased following irradiation compared to the non-irradiated cells (35 ± 8% and 25 ± 6%, respectively) (p=0.02). Our data indicate that irradiation was not inducing apoptosis in SP cells but in non-SP cells. It might suggest that SP cells could be more resistant to radiation damage. Future investigations will be performed to understand the functional activity of SP cells from lethally irradiated mouse BM and the molecular mechanism of those SP cells in resistance to irradiation damage.
Characterization of Non-Expanded Mesenchymal Progenitors Cells from Normal Adult Human Bone Marrow.
Authors: Nathalie Boiret , Chantal Rapatel , Richard Veyrat-Masson , Laurent Guillouard , Jacques Guerin Jean , Pascale Pigeon , Stephane Descamps , Stephane Boisgard , Marc Berger G.
Published: April 2020

Adult Bone Marrow (BM) mesenchymal stem /progenitor cells (MS/PC) are a potentially useful tool for cell therapy and tissue repair. Most of the studies related to their characterization have been carried out on cells selected by adherence and expanded in vitro. We have developed a simple means of directly identifying mesenchymal subsets from fresh BM.

First, we adapted our tools on MS/PC enriched by short time adherence (D1-3) before any cell division. Acquisition and analysis of the CD45-CD14- cells was performed on a EPICS Elite flow cytometer (Coulter, Miami, FL, USA). We have already shown that all CFU-F were included in adherent CD45-CD14- subset. When there was a sufficient number of collected adherent cells, the sub populations, defined by the expression of CD73, CD105, CDw90, or CD49a on CD45-CD14- cells, were sorted and plated in CFU-F assay. Then, we adapted this procedure to un-manipulated fresh BM mononuclear cell (MNC).

Adherent CD45-CD14- cells expressing the CD73 or the CD49a antigen contained all the CFU-F even though the CD105+ and CDw90+ subsets comprised less than half the total. In fresh un-manipulated BM MNC, CD73 and CD49a were also highly discriminative and allowed up to a 3 log enrichment of CFU-F when compared to BM MNC. The expression of CD105 and CDw90 appeared non-specific on fresh mesenchymal progenitors. Usual culture conditions up-regulated most of the tested antigens, in particular CD73 and CD49a with a significant increase in mean of fluorescence.

Antigen expression on CD45-CD14- subset

Conclusion: The CD45-CD14-/CD73+ and CD45-CD14-/CD49a+ phenotypes identified subsets containing all mesenchymal progenitors and sufficiently enriched to detect them in fresh BM, enabling evaluation of mesenchymal content of BM collections for cell therapy. Futhermore, this study argues for the existence of differences in antigen expression between un-manipulated mesenchymal cells (MC) and in vitro expanded MC and for the feasibility of distinguishing cell subsets in MC populations.

CD73+ CD105+ CDw90+ CD49a+ Phenotypic analysis and plating afficiency of adherent (D1-3) and un-manipulated CD45-CD14- cells. The proportion of each subset was expressed as a percentage of CD45-CD14- cells (a). The proportion of CFU-F obtained from the positive subset was reported in percentage of the total CFU-F obtained from the two subsets (positive and negative for the same antigen) (b). % (a) adherent 18.3 21.1 17.4 21.1 un-manipulated 5.2 17.4 4.5 4.4 CFU-F % (b) adherent 100.0 45.4 49.0 95.2 un-manipulated 100.0 50.5 65.6 99.7
Karyotypic Evaluation of Expanded Rat MAPCs.
Authors: Rundlett Stephen E. , Boozer Sherry L. , Suntharalingam Piratheepan , Verfaillie Catherine M. , Deans Robert
Published: April 2020

Summary

The clinical utility of an ex-vivo expanded stem cell population is dependent on both safety and efficacy. As an initial step toward ensuring that expanded adult stem cell populations are safe, we harvested bone marrows from four different strains of rat and cultured mononuclear cells under low serum media shown previously to allow for propagation of Multipotent Adult Progenitor Cells (MAPCs). We then determined the karyotype and tri-lineage potential for these expanding populations at numerous population doublings. We observe that an average of 6.7% of cells within these cultures are tetraploid and 2.7% of cells show other karyotypic alterations. In spite of this low level of karyotypic instability, no persistent chromosomal abnormalities are present in these cultures after more than 100 population doublings. As measured by qPCR and indirect immunofluoroescence for lineage specific marker genes, these cells have tri-lineage potential and are therefore considered to be MAPCs. These data support and extend previous conclusions that MAPC cultures with a nearly normal karyotype can be routinely established and expanded from rat bone marrow providing important standards for performance and safety in pre-clinical disease models.
Recurrent Chromosomal Rearrangements and a Homozygous Deletion Spanning 1-Mb Region at 10p11.2 in Adult T-Cell Leukemia/Lymphoma.
Authors: Hidaka Tomonori , Nishida Kazuhiro , Shimizu Daisuke , Sasaki Hidenori , Yamashita Kiyoshi , Kubuki Yoko , Nomura Kenichi , Taki Tomohiko , Tsubouchi Hirohito , Morishita Kazuhiro , Taniwaki Masafumi
Published: April 2020

Cytogenetic analyses and their correlation with clinical features are still limited in mature T-cell malignancies. The cytogenetics of adult T-cell leukemia/lymphoma (ATLL) is complicated by many structural and numerical abnormalities of chromosomes. Thus, it is occasionally difficult to define karyotype of ATLL only by chromosomal banding method. Using multicolor spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH), we performed molecular-cytogenetic studies on 38 patients with adult T-cell leukemia/lymphoma (ATLL) and 7 established cell lines. In SKY analysis, chromosomal bands involved in rearrangements were identified based on the inverted images of DAPI staining. Chromosome no.7 (25 cases) and no.14 (30) were frequently involved in structural rearrangements including translocations, inversions, insertions, isochromosomes, and deletions. Breakages frequently occurred at 10p11-13 (21 cases, 46.6%), and 14q11.2 (16, 35.6%), 14q32 (12, 26.7%), 7q22 (10, 22.2%), 9q22 (10, 22.2%), 13q14 (9, 20.0%), 22p11 (8, 17.7%), 8p11 (7, 15.5%), 18p11 (7, 15.5%), 21q22 (7, 15.5%) and 19q13 (6, 13.3%). Recurrent partners associated with 10p11.2-13 rearrangements were 21q22 (3 cases), 13q14 (2) and 14q32.1 (2). Partner chromosomal breakpoints involving 14q11.2 rearrangements were 14q32 (3 cases), 8p11.2-21 (3), and 11q13.3 (2), while those involved in 14q32 rearrangements were 14q11 (3) and 10p11-13 (2). Using FISH with 37 bacterial artificial chromosome clones, we analyzed breakpoints at 10p11.2-13 in two cell lines KOB4 and KK1 and one patient (HH) by walking a segment ranging from 10p13 to 10p11.2. Among 37 BAC clones used as FISH probes, 9 were assigned to 10p15, 3 to 10p14-13, and the remaining 25 to 10p11.2-12. Large genomic deletions were detected at 10p11.2 on der(10) in two cell lines. In patient HH, a homozygous deletion was identified at 10p11.2, spanning the region of 1 Mb in size. Chromosome 14q32 translocation was associated with shorter survival (median survival time; 4.2 months vs. 8.0 months), although statistically not significant. As for 10p11.2-13 and 14q11.2 rearrangements, there was no significant difference of survival between patients with and without respective abnormalities. In conclusion, SKY and FISH analysis identified recurrent chromosomal rearrangements and a homozygous deletion at 10p11.2 in ATLL. Our molecular analysis is now focusing on this genomic region to clone genes associated with development of ATLL.
Expression of the Chk2 Gene Is Downregulated in Hodgkin’s Lymphoma Cell Lines Via Epigenetic Mechanisms.
Authors: Kato Naoko , Kondo Takeshi , Tsukada Junichi , Tanaka Yoshiya , Minami Yasuhiro , Tanaka Junji , Imamura Masahiro
Published: April 2020

Chk2, the mammalian homologue of the yeast Rad53 and Cds1 genes, encodes a nuclear serine/threonine kinase that plays a crucial role in the DNA damage response and helps guard the integrity of the genome by regulating cell-cycle checkpoints, DNA repair and apoptosis. Furthermore Chk2 is regarded as a tumor suppressor gene. Alterations in tumor suppressors that are involved in the DNA damage response have been reported to be frequently involved in the pathogenesis of lymphoid malignancies. We investigated the expression levels of the genes encoding Chk2 in nine cell lines from lymphoid malignancies, including three Hodgkin’s lymphoma (HL) cell lines. We found that all three HL cell lines exhibited a drastic reduction in Chk2 mRNA and protein expression compared to the other cell lines without any apparent mutation of the Chk2 gene. Accumulating evidence demonstrates the importance of posttranslational modification of histone proteins in addition to DNA methylation, as epigenetic mechanisms involved in the organization of chromosomal domains and gene regulation. It is now generally appreciated that hyperacetylated histones H3 and H4 are associated with activated genomic regions, while hypoacetylation of histones H3, H4 and methylation on H3-lysine 9 results in gene repression and silencing. Therefore we tested the possibility that epigenetic mechanisms are involved in this aberrant expression of the Chk2 gene in these cells using the histone deacetylase (HDAC) inhibitors trichostatin A (TsA) and sodium butyrate (SB). Expression of Chk2 in HL cells was restored following treatment with the TsA and SB, or 5Aza-dC. Chromatin-immunoprecipitation (Chip) assays revealed that treatment of HL cells with TsA, SB or 5Aza-dC resulted in increased levels of acetylated histones H3 and H4, and decreased levels of dimethylated H3 lysine 9 at the Chk2 promoter. These results indicate that expression of the Chk2 gene is down-regulated in HL cells via epigenetic mechanisms. Furthermore we examined whether the down regulation of Chk2 gene in HL cells may be involved in apoptosis resistance to irradiation (IR). We found that upregulation of Chk2 in HL cells following treatment by SB resulted in increased susceptibility of the cells to IR. Therefore, our findings suggested that altered epigenetic regulation of Chk2 gene in HL cells results in the down-regulation of Chk2 kinase and abrogates DNA damage response signaling in the cells
Different Subtypes of Difffuse Large B-Cell Lymphomas Identified Using Microarrays and Phenotyping.
Authors: Poulsen Christian Bjorn , Borup Rehannah , Nielsen Finn Cilius , Borregaard Niels , Hansen Mads , Gronbaek Kirsten , Ralfkiaer Elisabeth
Published: April 2020

The mRNA expression profile in diagnostic, pretreatment samples from 52 primary nodal diffuse large B-cell lymphomas (DLBCL) were investigated using a commercially available oligonucleotide array (the Affymetrix Genechip HU133A) that contains >22.000 probe sets representing 18.400 transcripts. Hierarchical clustering indicated that 3 subtypes could be distinguishes. These included a GCB type (n=20) that showed an expression profile as similar to germinal center B-cells, an ABC type (n=25) that showed a profile as in vitro activated B-lymphocytes, and an intermediate group, type-3 (n=5) that did not fit into either of these categories. Two samples could not be classified due to inconsistency in clustering. Correlation with the genotype and phenotype showed that markers of germinal center B-cells (CD10, Bcl-6, t(14;18)) were prevalent but not exclusive to the GCB type. By contrast, MUM1 (a marker of terminal B-cell differentiation) was only expressed in the ABC type and in type-3. These results are similar to results obtained using a custom-made, spotted array - the Lymphochip (Alizadeh et al. Nature 2000; Wright et al, PNAS 2003). Comparison with the clinical features indicated that patients with GCB types of lesions showed a better initial response to CHOP or CHOP-like regimens than the remaining patients. However there was no significant difference in over-all survival. It is concluded that gene profiling with Affymetrix Genechips can be used to distinguish between GCB and ABC types of DLBCL and that these subtypes are likely to represent different, biological entities. Affymetrix Genechips should constitute a useful platform for prospective investigations, which are needed to elucidate whether gene profiling in DLBCL can be of use in choosing which treatment should be offered to patients.