bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA Damage Induced by DNA Topoisomerase I- and Topoisomerase II- Inhibitors Detected by Histone H2AX Phosphorylation in Leukemic Cells.
Authors: Halicka Dorota H. , Huang Xuan , Ozkaynak Fevzi M. , Seiter Karen , Traganos Frank , Darzynkiewicz Zbigniew 
Published: April 2020

Abstract
Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (γH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of γH2AX with: (a) cell cycle phase; (b) caspase-3 activation; and (c) apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase I (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX). In response to TPT or CPT maximal increase of γH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of γH2AX was detected at 2 h in G1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of γH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in γH2AX was apparent at a concentration range between 0.4 and 1.6 μM TPT. Thus, the intensity of γH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in γH2AX immunofluorescence. On the basis of our laboratory results, the present clinical study is evaluating ex vivo the feasibility of assessing DNA damage induced by treatment with topoisomerase inhibitors in patients with acute leukemias.
Study on Telomerase Activity of Human Bone Marrow Mesenchymal Stem Cells.
Authors: Li Jingyuan , Lai Xiaoyu , He Huang 
Published: April 2020

Abstract
Human mesenchymal stem cells(hMSCs) have multiple differentiate potential, and it can differentiate into adipocytes, osteogenic cells, chondrocyte and neural cells et al. It has been reported that telomerase activity in hMSCs is negative, but it is still controversial and telomerase activity in hMSCs-derived adipocytes has not been reported. We investigate the telomerase activity in hMSCs before and after their committed differentiation into adipocytes in vitro and cryopreservation. hMSCs were isolated from normal human bone marrow fellowed by cell culture in DMEM with low glucose containing 10% FBS. The FACS was performed to examine the expression of cell surface molecules and analyze cell cycle of primary hMSCs.Then some of hMSCs were induced to differentiate into adipocytes in vitro by being treated with adipocytic medium fellowed by being stained with oil red O, and the others were cryopreserved in liguid nitrogon for three months. TRAP assay(telomerase repeat amplification protocol assay)was employed to detect telomerase activity in those hMSCs. T293 cells and α-Interferon were analyzed with each test as an additional positive control and negative control respectively. Telomerase activity was expressed as a percentage of the relative telomerase activity (RTA) of the hMSCs relative to the RTA of T293 cells. The results indicated the cells were positive for SH2, SH3, CD90 and negative for CD34, CD45, AC133. It was showed that the majority of primary hMSCs(85%) was at cell cycle of G0/G1 phase and the minority of hMSCs was at S, G2 or M phase. 80% hMSCs was orange adipocytes after they were treated with adipocytic medium for 3–4weeks. Telomerase activity was negative in hMSCs both at the beginning of culture and at the later stages during cell expansion,telomerase activity in hMSCs-passage 1–3(n=10) and hMSCs-passage 4–7(n=9) made no significant difference(1.46±0.83% vs 1.46±0.67%, p=0.99). Cryopreservation did not affect the telomerase activity in hMSCs. Telomerase activity in fresh hMSCs(n=13) and frozen hMSCs(n=6) made no significant difference(1.41±0.44% vs 1.51±1.07%, p=0.64). Telomerase activity in hMSCs-derived adipocytes(n=3) was significantly higher than in hMSCs(n=19)( 11.8±2.52% vs 1.46. ±0.67%, p&lt;0.00001). It is concluded that hMSCs are telomerase-negative, and the stage of culture or cryopreservation does not affect their telomerase activity. After being induced to differentiated into adipocytes, hMSCs telomerase activity is upregulated.
High Efficiency Transduction of Human Mesenchymal Stem Cells Using Retroviral Gene Transfer with Triple Reporter Genes.
Authors: Niu Ting , Najjar Amer M. , Robinson Simon N. , Yang Hong , Decker William K. , McMannis John D. , Ng Jingjing , Thomas Michael W. , Xing Dongxia , Champlin Richard E. , Gelovani Juri G. , Shpall Elizabeth J. 
Published: April 2020

Abstract
Cotransplantation of ex vivo-expanded mesenchymal stem cells (MSC) together with hematopoietic stem cells hastens hematopoietic recovery in animal models and potentially humans. The in vivo mechanisms of migration, homing and long-term viability of MSC following implantation are not well understood. Reporter gene labeling would permit the tracking of transplanted MSC in vivo through noninvasive imaging techniques, namely fluorescence, bioluminescence, and nuclear imaging. Accordingly, a gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was utilized to transfer a triple fusion gene reporter system, expressing enhanced green fluorescent protein (eGFP), luciferase, and herpes simplex virus type one thymidine kinase (HSV1-tk) into MSC. Human MSC were isolated and propagated from normal bone marrow aspirates by culturing ficoll-separated fractions in alpha-MEM medium supplemented with 20% fetal bovine serum. FACS analysis confirmed the MSC phenotype, with cells expressing HLA-Class I, CD105, CD73, CD90 and CD166 antigens and not expressing HLA-Class II, CD80, CD31, CD34 and CD45 antigens. No significant phenotypic changes were observed in early and late passage numbers of MSC. MSC were transduced during their exponential growth phase (as determined by growth curve measurements), with virus-containing medium (VCM) from the PG13 packaging cells (targeting Pit-1 receptors) for 24 hours in the presence of different concentrations of the cationic polymer, polybrene, ranging from 2 μg/ml to 12μg/ml. The 2μg/ml concentration appeared to be optimal for transduction of MSC. VCM was harvested following a 24-hour incubation of the PG13 packaging cells at 32°C or 37°C. These initial experiments suggested that incubation at 32°C appeared to be optimal for virus production. Repeated VCM exposure at 24-hour intervals for 2 or 3 days did not increase the percentage of transduced cells as determined by flow cytometry (eGFP-positive). VCM harvested at 32°C added to MSC in the presence of 2μg/ml polybrene, yielded a 62% transduction efficiency following one round of exposure. U87 human glioma cells were used throughout as a positive control to verify retrovirus efficacy and demonstrated &gt;90% transduction efficiency. MSC clones highly expressing triple reporter genes are being selected and propagated in our laboratory to image transduced cells in NOD/SCID mice, thereby providing a better understanding of the potential role of these cells in transplantation processes.
Superiority of 3-D Hematopoietic Stem Cell (HSC) & Stroma Cell Co-Cultures in Supporting the Proliferation of Transplantable HSC.
Authors: Meretzki Shai , Kadouri Avinoam , Zipori Dov , Berger Ora , Rowe Jacob M. 
Published: April 2020

Abstract
Stem cells are intimately associated in-vivo with discrete niches within the 3-dimensional (3-D) bone marrow micro-environment collectively provide molecular signals that mediate their differentiation and self-renewal. A recently developed Plug-Flow Bioreactor System (PluriXTM), which closely mimics the physiological bone marrow environment by combining 3-D stromal cultures with a continuous flow system, is capable of supporting long term maintenance/expansion of transplantable human HSC.
The stroma cells, growing inside the PluriXTM bioreactor on porrosive carriers enable the propagation of high density (&gt;100-fold input) 3-D stroma cell cultures for prolonged periods of time. Within 40 days, the number of primary human stroma cells seeded in the bioreactor at a density of 5*104 cells/ml reached 1.5*106 cells/ml and the stroma cell lines reached a density of 5*107–1*108 cells/ml. Three-dimensional static stroma HSC co-cultures were found to be superior to 2-D co-cultures in supporting the maintenance of CB HSC and early hematopoietic progenitors.
When cord blood CD34+ cells were seeded on 2-D & 3-D stroma cell static cultures, the 3-D stroma cultures proved superior in supporting the growth of CD34+, CD34+38− and CD34+38−CXCR4+ cells. The 3-D primary human stroma static cultures supported these cells 4.7, 22, 7.4 fold better than the 2-D cultures, respectively.
The CD34+ cells seeded on the high-density 3-D stroma cell cultures within the PluriXTM bioreactor demonstrated significant superiority to 2-D and 3-D stroma cell static cultures and 2-D stroma cultures in supporting the growth and expansion of HSC and earlier hematopoietic progenitors.
These findings verify that a system based on 3-D stroma co-cultures is preferable for ex-vivo expansion of human HSC’s. The PluriXTM system offers wide-range potential for expansion of stem cells from cord blood and production of specific blood products.
Altered Replication Timing of IGH Alleles in Burkitt’s Lymphoma Depends on IGH Breakpoint Position.
Authors: Guikema Jeroen E.J. , de Boer Conny , Gadiot Jules , Schuuring Ed , Kluin Philip M. 
Published: April 2020

Abstract
Monoallelic expression of immunoglobulin genes is epigenetically regulated and maintained. Asynchronous replication of the IGH alleles has been implicated in allelic exclusion. Usually, the functional IGH allele replicates early in the S-phase of the cell cycle whereas the non-functional allele replicates late. Previously, the intronic enhancer region (Eμ) and the 3′ Cα enhancer region have been designated as putative replication initiation sites. Activity of these replication origins are likely to be involved in regulation of asynchronous replication. By use of interphase and DNA fiber FISH we have performed a detailed analysis of the configuration of the t(8;14) chromosomal translocation in Burkitt’s lymphoma patients and cell lines and showed that the breakpoints in all studied cases were perfectly reciprocal without loss of IGH genomic material. An important implication is that in patients and cell lines harboring a breakpoint in a downstream switch region (Sγ or Sα) the Eμ and the 3′ Cα enhancer region are physically separated from each other, whereas in patients and cell lines with the IGH breakpoint located in the JH-region both enhancer regions remain on the der(14) chromosome. We therefore studied the IGH replication timing in Burkitt’s lymphoma cell lines harboring different IGH breakpoints by use of BrdU-FISH and interphase FISH on sorted cell cycle fractions. In two cell lines with JH-region breakpoints (Jiyoye and DG-75) the t(8;14) was invariably targeted to the late replicating IGH allele. In contrast, in all three cell lines with switch-region breakpoints (CA-46, Namalwa, BL-65) asynchronous replication of the IGH alleles was lost. As the position of the breakpoint in the MYC locus at 8q24 differed substantially between these cell lines (DG-75 and CA-46: intron 1 of the c-myc gene; Jiyoye, Namalwa, and BL-65: 100 kb to &gt;500 kb centromeric from c-myc) it is unlikely that the position of the 8q24 breakpoint is of crucial influence on IGH replication timing. We speculate that the Eμ and the 3′ Cα enhancer region regulate the IGH replication timing in a cis-acting manner as physical separation of both enhancer regions results in loss of asynchronous replication.
Evaluation of Focal Adhesion Kinase Gene Expression in Leukemia and Cancer Cell Lines Using Combined Molecular Cytogenetic Investigations and Quantitative Real Time RT-PCR.
Authors: Campos Lydia , Tchirkov Andrei , Cornillon Jerome , Mihaescu Mirella , Giollant Michel , Piselli Simone , Vago Philippe , Cottier Michele 
Published: April 2020

Abstract
FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase protein implicated in integrin-mediated signal pathways. The human gene fak is located on chromosome 8q near c-myc. Cells that overexpress FAK show increased migration and increased cell survival with apoptosis inhibition. The aim of the present study was to investigate the genomic or post-genomic origin of this overexpression in seven tumor cell lines with different levels of FAK expression: K562, an erythroblastic cell line; U937, a monoblastic cell line; KG1a, a poorly differenciated myeloblastic cell line; HL 60, a promyelocytic cell line; Jurkat, a lymphoblastic cell line; A549, a bronchial adenocarcinoma cell line; M4Beu, a lymph node metastasis melanoma cell line. Total or partial chromosome 8 qualitative or quantitative abnormalities and the presence of double minute chromosomes bearing coding genomic DNA fragments were investigated using both conventional cytogenetic (standard karyotype after R- and G- banding) and molecular cytogenetic such as multicolor karyotype (M-FISH), FISH and comparative genomic hybridization (CGH). The mRNA expression was investigated using quantitative real time RT-PCR and the protein level using immunocytochemistry and western blot (immunoblot) analysis. Except for the Jurkat cell line, karyotypes revealed complex modifications. FISH and M-FISH allowed to precisely identify the chromosomal origin of the genomic material on each chromosome and CGH allowed to characterize relative loss and gain of coding genomic DNA and to identify their chromosomal origin. Summarized results are shown in the following table:
No detectable RT-PCR product was observed for HL60 and U937 cell lines. The preliminary results show that there was usually a good correlation between PCR data and western blot analysis. However, FAK overexpression for KG1a cell line most probably has a genomic origin, while in K562 and A549 cell lines the origin is most probably at the transcription or post-transcription level without any abnormality of chromosome 8. Dysregulation of fak gene is observed in hematopoietic as well as solid tulor cell lines, and may contribute to leukemogenesis.&lt;/CENTER&gt;
Cell line FISH 8q24-qter CGH Chr. 8 RT-PCR RT-Q-PCR Western blot RT-Q-PCR results in copy number ratio K562 Normal Normal + 130.1 ++++ KG1a Amplified enh(8)(q11qter) + 37.5 ++++ Jurkat Normal Normal + 8.7 + (low) U937 Normal enh(8)(pterqter) 0 0 0 HL60 Amplified amp(8)(q23-24) 0 0 0 M4Beu Normal Normal + 43.5 0 A549 Normal Normal + 46 ++++
High Expression of Micro-RNA BIC / miR155 in All Subtypes of Hodgkin Lymphoma.
Authors: van den Berg Anke , Kluiver Joost , de Jong Debora , Koekoek Theresia , Blokzijl Tjasso , Poppema Sibrand , Kroesen Bart-Jan 
Published: April 2020

Abstract
We recently demonstrated that the noncoding BIC gene was highly expressed in Hodgkin Reed-Sternberg (HRS) cells of the vast majority of both classical and nodular lymphocyte predominant (NLP) Hodgkin lymphoma (HL). The BIC gene belongs to the family of noncoding mRNA-like molecules and was suggested to function through its noncoding RNA transcript. The human BIC gene shows a high degree of sequence identity to the Mouse and Gallus gallus BIC genes, which was most pronounced in a region containing a ~70 nucleotide stem-loop structure. Recently published data revealed expression of miR155 micro-RNA in murine tissue. The miR155 sequence was identical to the first 22 nucleotides of the ~70 nucleotide stem-loop structure of the BIC gene indicating that BIC is a primary micro-RNA transcript. Micro-RNAs (miRNA) are endogenous small RNAs (20–22 nucleotides) that inhibit protein translation by paring to the messages of protein-coding mRNAs. Based on recent data, it is clear that deregulation of miRNA expression, processing or stability may lead to deregulation of important cellular processes and thereby contribute to the development of cancer.
In this study we analyzed the expression of miR155 in HL and NHL samples and studied the regulation of BIC/miR155 expression. Northern blot analysis revealed that HL cell lines, which all have a high level of BIC transcripts, also have high levels of the functionally mature miR155. NHL samples with low levels of BIC transcripts also have low levels of miR155. These data provide experimental support for processing of primary BIC transcripts to the functionally mature miR155 fragments and indicate that miR155 expression is specific for HL. To analyze whether BIC expression could be induced in NHL cell lines we studied several stimuli in the wild type Burkitt lymphoma derived cell line Ramos and a NF-κB deficient variant cell line. Real-time RT-PCR analysis revealed a high level of BIC and of the 70 nucleotide miR155 precursor in anti-IgM stimulated Ramos cells. Anti-IgM stimulation of the NF-κB deficient Ramos cell line revealed only low levels of BIC and pre-miR155. These data demonstrate that transactivation of NF-κB is required for continuous upregulation of BIC in Ramos cells. Stimulation of Ramos cells with PMA/calcium-ionophore, which directly activates PKC, significantly upregulated the level of BIC and pre-miR155. This suggests that activation of PKC and its downstream signaling components, including NF-κB are involved in the regulation of BIC expression. Thus, whereas HRS cells typically do not express surface immunoglobulin, upregulation of BIC in these cells may be induced by constitutively activated NF-κB.
Microarray Analysis of the Short Isoform of Ikaros Overexpressed in Patients with Hematologic Malignancies.
Authors: Kataoka Itaru , Ishimaru Fumihiko , Tanimoto Mitsune 
Published: April 2020

Abstract
We analyzed expression of the transcription factor Ikaros in human hematologic malignancies and found overexpression of the short isoform of Ikaros, Ik-6, in patients with blast crisis of chronic myeloid leukemia and pre-B cell acute lymphoblastic leukemia. We have made several stable transfectants which overexpress Ik-6 and analyzed the consequence of Ik-6 overexpression.
First, overexpression of Ik-6 in FDH-1, which was established from a patient with early pre-B acute lymphoblastic leukemia, and Ramos conferred resistance to dexamethasone- and anti-IgM-induced apoptosis, respectively. Upon treatment with dexamethasone, FDH-1 cells demonstrated massive apoptosis measured by staining with Annexin V. In contrast to mock transfectants, Ik-6 transfectants showed less marked apoptosis (mock 86.4% vs Ik-6 34.4–73.6%, p=0.01). We also used Ramos as a model of anti-IgM-induced apoptosis, and flow cytometric analysis was performed with Annexin-V staining. In contrast to mock transfectants, Ik-6 transfectants showed less pronounced apoptosis (mock 40.4% vs Ik-6 23.1–27.4%, p=0.03). Next, overexpression of Ik-6 in Nalm6 lead to impaired cell growth and cell cycle progression. Cell growth and cell cycle were analyzed and compared with mock transfectants and Ik-6 transfectants using MTT assay and DNA content analysis with PI staining, respectively. MTT assay demonstrated that, after three days of growth, Ik-6 transfectants grew more slowly than mock transfectants (Ik-6/mock ratio 0.62–0.67; p=0.01). DNA content analysis showed that, after three days of growth, more Ik-6 transfectants underwent G0–G1 arrest than mock transfectants (Ik-6/mock ratio 1.15–1.21; p=0.01), and less Ik-6 transfectants entered into S phase than mock transfectants (Ik-6/mock ratio 0.84–0.90; p=0.05).
Finally, overexpression of Ik-6 promoted all-trans retinoic acid (ATRA)-induced granulocytic differentiation, and, on the other hand, inhibited vitamin D3 (VD3)-induced monocytic differentiation of HL-60. Upon induction of granulocytic differentiation by ATRA, mock transfectants showed 74.5% positivity of myeloperoxidase (MPO) staining. In contrast, Ik-6 transfectants showed 51.5–56.8% of positivity of MPO staining (p=0.04). NBT reduction assay demonstrated 43.8% positivity with mock transfectants and 62.3–67.3% positivity with Ik-6 transfectants (p=0.001). Upon induction of monocytic differentiation by VD3, mock transfectants showed 51.0% positivity of nonspecific esterase (NSE) staining. In contrast, Ik-6 transfectants showed 26.8–36.0% of positivity of NSE staining (p=0.03).
To understand the underlying mechanisms, we report the results of microarray analysis of Ik-6 transfectants.
Distinct Biological Impact of Dephosphorylation vs Downregulation of p210Bcr-Abl: Implications for Imatinib Mesylate Response and Resistance.
Authors: Kurzrock Razelle , Talpaz Moshe , Li Lan , Estrov Zeev 
Published: April 2020

Abstract
Imatinib mesylate acts by suppressing phosphorylation of its kinase target - Bcr-Abl. We hypothesized that loss of p210Bcr-Abl (the kinase target) may lead to imatinib mesylate resistance. Two model systems were studied: K562 cells (CML blast crisis line) and MO7E/MBA-1 cells (with MBA-1 cells representing MO7E cells stably transfected with BCR-ABL). Relative resistance to imatinib mesylate developed when p210Bcr-Abl expression was abolished. Furthermore, K562 cells were significantly more growth suppressed after exposure to imatinib mesylate (p210Bcr-Abl is present but dephosphorylated), than after downregulation of Bcr-Abl expression. Signaling pathways which were functional in the absence of Bcr-Abl expression - NF-KB and MAP kinase activation or the growth factor pathway -- were disrupted when p210Bcr-Abl was present but dephosphorylated, suggesting that an intact, but enzymatically inactive Bcr-Abl, may interfere with critical growth/signaling pathways. Downregulation of p210Bcr-Abl may be a mechanism by which imatinib mesylate-resistance emerges. Samples from 3 of 15 patients with imatinib mesylate-resistant CML blast crisis had undetectable levels of p210Bcr-Abl. We conclude that retention of a dephosphorylated p210Bcr-Abl has a biologic impact distinct from that of downregulation/loss of p210Bcr-Abl and, in a subset of patients, loss of the target of the kinase inhibitor may lead to imatinib mesylate resistance.