bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fusion of NPM to RARA in t(5;17) Disrupts Its Ability To Interact with YY1 Transcription Factor.
Authors: Redner Robert L. , Sumandeep Sumandeep 
Published: April 2020

Abstract
The transcription factor Ying-Yang 1 (YY1) can function either as a transcriptional activator or transcriptional repressor, depending on context. It has been reported that YY1 is a myeloid transforming gene interfering with neutrophilic differentiation. Further, it has been shown that the nucleolar phosphoprotein Nucleophosmin (NPM) directly interacts with YY1 in cells. As a consequence of NPM overexpression, YY1 repression is diminished. We previously reported the expression of an NPM fusion protein in the t(5;17)(q35;q21) variant of acute promyelocytic leukemia. The fusion protein encodes the N-terminal 118 amino acids of NPM fused to the retinoic acid receptor alpha (RARA). We previously showed that NPM-RAR inhibits myeloid differentiation in both U937 and transgenic model systems. We hypothesized that NPM-RAR might contribute to abnormal myeloid differentiation by altering YY1-mediated transcription pathways. To test this hypothesis, we sought to determine whether the NPM-RAR protein interacted with YY1 in the same manner as wild-type NPM. We generated a eukaryotic expression vector encoding FLAG-tagged YY1, and used it to transform COS cells. Full-length NPM, or NPM-RAR, were subcloned into a vector encoding the maltose binding protein (MBP), in the appropriate reading frame to generate MBP-fusions. The MBP proteins were expressed in bacteria, and purified by affinity chromatography. MBP, MPB-NPM, or MBP-NPM-RAR were incubated with YY1-COS lysates, and complexes were captured on amylose resin. The resin-complexes were washed, and the presence of YY1 was detected by immunoblotting using an anti-FLAG antibody. As expected, MBP-NPM pulled down FLAG-YY1 under these conditions. However, we were unable to detect association between MBP-NPM-RAR and FLAG-YY1. To determine whether this finding might be attributable to lack of post-translational modification of NPM-RAR in the bacterial system, we co-transfected expression vectors for FLAG-YY1 and NPM-RAR into COS cells. Lysates were subjected to immunoprecipitation using anti-RARA polyclonal antisera, and the presence of co-precipitating YY1 was detected by immunoblotting using an anti-FLAG antibody. Again, we were unable to detect direct interaction between the proteins. We conclude that NPM-RAR does not directly interact with YY1. This data does not exclude the possibility that YY1 activity could be altered in t(5;17) by decreased expression of wild-type NPM.
Feasibility of Gene Expression Profiling Generated from Fine Needle Aspiration from Patients with Follicular Lymphoma and Large B-Cell Lymphoma.
Authors: Goy Andre , Stewart John , Barkoh Bedia , Remache Yvonne , Hart Susan , Neal Susan , Katz Ruth , Sneige Nour , Gilles Frederic 
Published: April 2020

Abstract
We tested the feasibility of performing gene expression profiling using amplified RNA from Fine Needle Aspiration (FNA) obtained from lymph nodes. Twenty-four samples from patients with a diagnosis of Follicular Lymphoma (FL) or Large B-Cell Lymphoma (LBCL) were obtained after informed consent. The diagnosis were performed by two pathologists and classified into two groups (10 LBCL and 14 FL) using conventional morphology and immunophenotyping. Total RNA was extracted using RNaqueous (Ambion, Austin, TX). Total RNA (100ng /per sample) was subjected to 2 cycles of standard double-stranded cDNA synthesis using Superscript Choice System (Invitrogen, Grand Island, NY) and in vitro transcription for target amplification as per GeneChip’s Eukaryotic Small Sample Target Labeling protocol (Affymetrix, Inc, Santa Clara, CA). The biotinylated cRNA from each sample was hybridized to Affymetrix HG-U133A chips. Gene expression profiling results were first analyzed by Principal Component Analysis (PCA) using a list of 146 probe sets representing 62 genes characteristic of Acivated B-Cell (ABC) or Germinal Center (GC) signature. This analysis identified 5 LBCL cases with ABC cell signature (Fig 1). Using a list of 207 probe sets representing 113 genes involved in FL transformation (K Elenitoba-Johnson, PNAS 2003), PCA analysis identified two overlapping clusters corresponding to FL and GC-DLBCL. To further improve this classification, we generated a list of 82 genes differentially expressed between FL and GC-LBCL. Using this list of genes, PCA analysis demonstrated a clear separation between FL and GC-LBCL (Fig 2). These results demonstrate that comprehensive transcription profiles can be performed in patients with NHL using RNA obtained from FNA and that one can distinguish among FL and LBCL lymphoma genetic subtypes.
Figure Figure
Motexafin Gadolinium Mobilizes Zinc and Inhibits Thioredoxin Reductase in Human Lymphoma Cell Lines.
Authors: Magda Darren , Lecane Philip , Cheryl Lepp , Miller Richard A. 
Published: April 2020

Abstract
Motexafin gadolinium (MGd, Xcytrin®) is an anti-cancer agent that selectively localizes in tumors and promotes redox stress by oxidizing intracellular reducing species. MGd, at low concentrations, induces expression of metallothioneins (MT) and zinc transporter 1 (ZnT-1) transcripts in vitro. In the present study, we describe the effects of MGd on zinc ion homeostasis, thioredoxin reductase activity and cytotoxicity in lymphoma cell lines. Human lymphoma (Ramos, DHL-4, HF-1) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Zinc (0–100 μM) and 0–25 μM MGd were added for 4–6 hr. Medium was exchanged and thioredoxin reductase activity was assessed by measuring the rate of lipoate reduction (Biaglow, Anal. Biochem.281:77, 2000). In other experiments, cells were treated with MGd and zinc, and analyzed by flow cytometry using annexin-V, propidium iodide, and the ion-specific fluorescent probe, FluoZin-3-AM™. RNA from treated cultures was harvested and metallothionein and ZnT-1 induction assessed by Northern blotting. Treatment with MGd and zinc led to synergistic increases in free intracellular zinc levels, inhibition of lipoate reduction, MT and ZnT-1 induction, and cytotoxicity. In DHL-4, exposure to 10 μM MGd and 50 μM zinc for 3 hr led to a 2.5-fold increase in FluoZn-3 fluorescence, relative to treatment with zinc alone. In Ramos, this treatment led to 1.4, 2.6, 5.2, and 30-fold increase in FluoZn-3 fluorescence after 2, 4, 6, or 24 hr. There was a 2-fold increase in annexin-V positive Ramos cells after 6 hr, and an 11-fold increase in propidium iodide permeable cells by 24 hr. The rate of lipoate reduction decreased to 69%, 47%, and 58% of control in Ramos, DHL-4, and HF-1 cell lines after 5 hr under these conditions. MT and ZnT-1 transcript levels were elevated within 4 hr after treatment with MGd, zinc, or the combination, and remained elevated for at least 24 hr. For example, in HF-1, MT transcript levels were increased 20, 40, and 173-fold by MGd, zinc, or the combined treatment after 8 hr. These observations support the characterization of MGd as a redox cycling agent that increases the intracellular availability of free zinc. This activity leads to inhibition of thioredoxin reductase and, ultimately, induction of cell death. The proposed mechanism of action supports the use of this agent alone or in combination with various chemotherapy agents that induce redox stress.
Repeated Application of Sequence-Specific SiRNA Molecules Leads to an Effective Downmodulation of All Clinically Relevant bcr-abl Gene Variants.
Authors: Wohlbold Lara , van der Kuip Heiko , Moehring Alexandra , Granot Galit , Oren Moshe , Vornlocher Hans-Peter , Aulitzky Walter E. 
Published: April 2020

Abstract
Fusion transcripts such as bcr-abl encoding pathological oncogenic proteins represent ideal targets for a tumor-specific RNA interference (RNAi) approach. The aim of the present study was to optimize the efficacy of bcr-abl RNAi. We evaluated several synthetic siRNAs targeting the fusion sites of all common bcr-abl transcript variants (e14a2, e13a2, or e1a2). Significant knock-down of p210Bcr-abl and p190Bcr-abl fusion proteins was successfully achieved in bcr-abl expressing 32D cells and human leukemic K562 and MEG-01 cells. Repeated application of siRNA proved to be significantly more efficient compared to single treatment. The optimized protocol led to a total decrease of up to 75–90% in Bcr-abl protein levels, which was accompanied by a loss of viability of up to 90%. The target specificity of the siRNA was high, and even a single point mutation in the siRNA-sequence led to significant, albeit not complete, loss of siRNA efficacy. To expend the duration of the knock-down effect, we explored the use of plasmids driving the stable expression of short hairpin RNA (shRNA). Efficient downregulation of Bcr-abl protein was achieved in K562 cells transfected with a pSUPER-based plasmid encoding bcr-abl shRNA.
Challenging E2F-1 and c-Myb Block of Myeloid Terminal Differentiation with the Tumor Suppressor-Egr1.
Authors: Gibbs John D. , Borden Alaina A. , Liebermann Dan A. , Hoffman Barbara 
Published: April 2020

Abstract
The proliferation and differentiation of hematopoietic cells are tightly regulated to maintain cellular homeostasis. Understanding the mechanism of this regulation may identify potential therapeutic targets against leukemia. Previous works have shown that several negative regulators of differentiation, namely oncogenes c-Myb, E2F-1 and c-Myc were capable of blocking the IL6-mediated myeloid terminal differentiation program of M1 myeloblastic leukemia cells. On the other hand, a positive regulator of differentiation, Egr-1 has been shown to activate the macrophage differentiation program of M1 cells in the absence of IL-6. Interestingly, recent work showed that Egr-1 could override the block of differentiation imparted by deregulated c-Myc in the presence of IL-6, and could reverse the leukemic phenotype associated with deregulated c-Myc. From such observations, we asked whether exogenous expression of Egr-1 in M1 cells could override the earlier block of differentiation imparted by the oncogenes c-Myb or E2F-1. The established M1Myb-Egr cells and M1E2F-Egr cells were analyzed. The M1E2F-Egr cells underwent growth arrest followed by macrophage differentiation and subsequently apoptosis. In addition, exogenous Egr-1 only partially abrogated c-Myb block of differentiation. The M1Myb-Egr cells failed to undergo growth arrest, however, were able to enter intermediate-late stage macrophage differentiation with concomitant phagocytic functionality. These data demonstrate that E2F and c-Myb each block myeloid differentiation via different mechanisms. Egr-1 can completely override the E2F block but cannot abrogate the c-Myb block to allow M1 cells to terminally differentiate or growth arrest. Furthermore, Egr-1 appears to behave as a tumor suppressor, and therefore may serve as a possible target against various forms of leukemia.
Detection of Human Telomerase Reverse Transcriptase (hTERT) Protein Expression in Bone Marrow Cells of Hematological Disorder by Flow Cytometry.
Authors: Handa Hiroshi , Murakami Hirokazu , Matsushima Takafumi 
Published: April 2020

Abstract
Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. Recently, it has been reported that telomerase activity level was correlated with the progression and prognosis of hematopoietic malignancies, thus, it is important to analyze telomerase activity. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and unable to isolate specific cell sub-populations. We developed immuno-fluorescence flow cytometry based assay to detect hTERT expression using a monoclonal antibody. This method allows sub-population of cells to be separated according to hTERT expression more easily, combining antibody against cell surface antigen. Expression level of hTERT in T cell leukemia cell line Jurkat and myeloma cell line RPMI8226, KMS12PE, KMS28PE, were very high and well correlated in between this method and real time quantitative PCR. Human mature granulocytes supposed to have no telomerase activity did not show any hTERT expression in this flow-cytometric assay. Immuno-histochemistry demonstrated the specific nuclear expression of hTERT. Bone marrow samples were obtained from 8 MDS patients (5 RA, 1 RAEB, 2 RAEB-t) and analyzed hTERT isolating MDS blast cells. In the blast population, cells expressing hTERT were 33.1% (21.4–40.3) in RA patient sample, while those in RAEB and RAEB-t patients sample were significantly higher at 75.1% (68.2–81.9). When CD34 positive hematopoietic stem cells were analyzed, the cells expressing hTERT were higher in RAEB and RAEB-t than in RA. Mature granulocyte population in bone marrow cells did not show any positivity. Our result suggests that hTERT expression and telomerase activity in the MDS blasts or CD34 positive stem cells were up-regulated during the disease progression and the high expression level of hTERT and telomerase activity were not due to the blast expansion. This technique allows us to easily analyze hTERT expression in hematopoietic malignancy even though the malignant cells were very small in bone marrow sample, e.g. MDS, MM or MGUS of which telomerase activity were supposed to be correlated with the disease progression and prognosis.
Evaluation of the Ras/B-Raf/SHP-2 Axis in B Cell Malignancies.
Authors: Mitsiades Constantine S. , Mitsiades Nicholas , Fanourakis Galinos , Goudopoulou Athina , McMullan Ciaran J. , Voutsinas Gerasimos , Munshi Nikhil C. , Anderson Kenneth C. 
Published: April 2020

Abstract
The Ras/Raf/MEK/MAPK pathway is a major regulator of tumor cell proliferation. Ras mutations have been known in many malignancies for several years, whereas more recent reports have shown that activating mutations of the BRAF gene are present in a large percentage of human malignant melanomas and thyroid carcinomas and in a substantial proportion of colon carcinomas (Nature2002;417:949–54). The vast majority of these mutations represent a T1796A change resulting in a V599E substitution within the activation segment of B-Raf. This mutant B-Raf kinase is constitutively active and results in inappropriate stimulation of downstream MAPK/ERKs and tumor cell proliferation. The PTPN11 gene encodes SHP-2 (Src homology 2 domain-containing protein tyrosine Phosphatase), a nonreceptor tyrosine protein tyrosine phosphatase (PTPase) that modulates Ras signaling. Germline mutations in PTPN11 are responsible for Noonan syndrome (NS), whereas somatic mutations have been detected in juvenile myelomonocytic leukemia (JMML). We investigated the presence of mutations in the Ras/B-Raf/SHP-2 axis in 25 lines from B-cell malignancies, including 19 multiple myeloma (MM) cell lines. DNA was isolated and B-Raf (exons 11 and 15), PTPN11 (3 and 13) and K-, N- and H-Ras sequences were amplified by PCR using specific flanking primers. The PCR products were purified and sequenced in automatic sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems). Ras gene mutations were found in 11/25 (44%) of the studied cell lines. None of the previously described activating B-Raf mutations, including the most prevalent V599E, were found in our panel. Only one MM cell line demonstrated a C1332A substitution, which corresponds to a D444E change, which is predicted to have a minimal, if any, impact on kinase activity. A single PTPN11 G178C substitution, corresponding to a G60R change (previously described in NS and AML), was detected in another MM line. These data confirm the previously known role of Ras mutations in B-cell malignancies and identify a rare occurrence of a PTPN11 mutation in our panel. On the other hand, activating mutations of the B-Raf kinase are unlikely to play a significant part in the pathogenesis of B cell malignancies, in contrast to their prominent role in several types of solid tumors. In total, our data suggest that the Ras/Raf/MEK/MAPK can be overactive in these malignancies due to activating genetic events upstream of B-Raf, such as the previously described activating Ras mutations.
KSHV-GPCR and CXCR2 Transforming Capacity and Angiogenic Responses Are Mediated through a JAK2-STAT3 Dependent Pathway.
Authors: Burger Meike , Hartmann Tanja , Burger Jan A. , Schraufstatter Ingrid U. 
Published: April 2020

Abstract
The Kaposi’s sarcoma herpesvirus encodes a G-protein-coupled chemokine receptor termed KSHV-GPCR. Expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi’s sarcoma. Previously, we have shown that CXCR2, the closest human homologue, is similarly able to transform cells if continously stimulated or constitutively activated by amino acid exchange D138V of the DRY sequence. Here, we demonstrate that STAT3 activation is a prerequisite for transformation in KSHV-GPCR and CXCR2 transfected NIH 3T3 cells. In KSHV-GPCR and D138V transfected cells, STAT-3 is constitutively phosphorylated on Tyr705. In CXCR2 transfected NIH 3T3 cells and human microvascular endothelial cells (HMEC), which express the CXCR2 constitutively, STAT3 is phosphorylated upon stimulation with IL-8 (CXCL8). Focus formation in NIH 3T3 cells transfected with the KSHV-GPCR, CXCR2, or the D138V mutant, was blocked by the specific JAK2 inhibitor AG490. Typical functions of the CXCR2 including actin stress fiber formation, haptotaxis, and the angiogenic response in HMEC shown by tube formation in Matrigel were blocked by AG490. These data suggest that the transforming capacity and migratory responses that are involved in tumor development, metastasis and angiogenesis in KSHV or CXCR2-expressing cells is at least partially mediated through a JAK2-STAT3 dependent pathway.
ABCChips as New Diagnostic Tool to Monitoring Multidrug Resistance in Human Tumor Cells to Chemotherapeutics Agents by Expression Profiling of ATP-Binding Cassette Transporter Genes.
Authors: Gillet Jean-Pierre , Efferth Thomas , Steinbach Damiel , de Longueville Françoise , Bertholet Vincent , Remacle José , Sayer Herbert G. 
Published: April 2020

Abstract
A major problem in the treatment of tumors represents the development of resistance to chemotherapy. Many mechanisms are responsible for the failure of treatment, the main one being the activation of the ABC transporters. In the present investigation, we developed a low density DNA microarray which contains 38 ABC transporter genes. This tool has been validated with three different characterized multidrug-resistant sublines (CEM/ADR5000, HL60/AR, MCF7-CH1000) and their corresponding drug-sensitive parental cell lines (CCRF-CEM, HL60, MCF7). The multidrug-resistant cell lines used are known to overexpress either the MDR1, MRP1, or BCRP genes. Interestingly, we found not only the overexpression of these genes but also of other ABC transporter genes by using low density microarrays. These results were corroborated by quantitative real time RT-PCR. The microarray allowed the determination of the expression profile of ABC transporters in a single hybridization experiment and may, hence harbor the potential as diagnostic tool to detect drug resistance in the clinic.
Induction of Apoptosis and Cell Cycle Arrest in Acute Myeloid Leukemia Cells by Histone Deacetylase Inhibitor, Suberoyl Hydroxamic Acid (SAHA): Lack of Cross-Resistance to Cytosine Arabinoside and Etoposide.
Authors: Dreyer Sabine , Decker Thomas , Wagner Michaela , Peschel Christian , Licht Thomas 
Published: April 2020

Abstract
Inhibitors of histone deacetylases (HDAC) such as SAHA are being introduced into the clinical treatment of hematopoietic neoplasms. These compounds can induce apoptosis or cell cycle arrest by modification of the chromatin structure of malignant cells, thereby modulating the expression of target genes. We investigated the effects of HDAC inhibitors, SAHA and trichostatin A, in twelve acute myeloid leukemia (AML) cell lines. Cytotoxicity was determined with the use of a tetrazolium-based colorimetric assay. The 50% inhibition concentrations (IC50) of SAHA were in the micromolar, of trichostatin A in the nanomolar range. Three cell lines were 3–5 times more resistant to both HDAC-inhibitors than the other leukemias, namely the myelomonocytic leukemia ML-2, and the erythroleukemias HEL and K562. All investigated AML cells were, however, more sensitive to SAHA than three patient samples of normal CD34+ progenitor cells mobilized into peripheral blood. A close association between the IC50 of TSA and SAHA was noted within the panel of AML lines (r=0.78). In contrast, chemosensitivity to HDAC-inhibitors was not significantly correlated with IC50 for etoposide, cytosine arabinoside, or staurosporine, respectively. To distinguish between growth arrest and induction of apoptosis by SAHA, we analyzed the cell cycle status by staining with propidium iodide, and exposure of phosphatidylserine by an Annexin V assay. Cell cycle arrest in G1 phase was observed in four AML cell lines with an increase of G1 cells by 20–49% in comparison with untreated cells. One cell line, KG-1a, displayed G2 arrest. SAHA induced apoptosis in eight cell lines with one line displaying both apoptosis and G1 cell cycle arrest simultaneously. KASUMI-1 cells bearing the AML1/ETO gene fusion, a target for HDAC’s, underwent apoptosis upon exposure to SAHA. Constitutive expression of the cell cycle inhibitor p27Kip1, determined by Western blotting, was associated with increased numbers of G1 cells following treatment with SAHA (p=0.036, one-tailed Mann-Whitney test). Conversely, constitutive expression of cyclins A, B1, D3 and E, as well as p53 and p16INK4 was not predictive for induction of cell cycle arrest. Expression of P-glycoprotein (ABCB1) as assessed by flow cytometry was not correlated with the IC50 for SAHA. The anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP were analyzed by Western blotting. No association with resistance to SAHA-induced apoptosis was noticable. In summary, AML cells from permanent lines were more sensitive to SAHA than normal hematopoietic progenitor cells. The potential role of p27Kip1 in modulation of the cytotoxicity of HDAC inhibitors requires further study. Lack of cross-resistance with drugs used in clinical treatment suggests that HDAC inhibitors may be useful for treatment of chemoresistant AML.