bluuebunny/crossref_metadata_2025 huggingface dataset:
Immortalized Human Placenta Derived Mesenchymal Cells by Transfection with Bmi-1 Lost the Abilitiy To Differencitate into Cell Types of Osteocytic, Condoricytic and Neural Lineages.
Authors: Zhang Xiaohong , Soda Yasushi , Takahashi Kenji , Bai Yuansong , Igura Koichi , Mitsuru Ayako , Tojo Arinobu , Takahashi Tsuneo A.
Published: April 2020

Introduction: We previously reported that mesenchymal cells derived from chorionic villi of human placenta could differentiate into various cell types including osteocytic, condoricytic and neural lineages. However, these cells can slowly proliferate with limited life span. In the present study, we transduced human placenta-derived mesenchymal cells (PDMCs) with cDNA encoding Bmi-1 and telomerase reverse transferase (hTert), respectively or simultaneously by lentiviral vector, and tested transduced cells for their ability to differentiate into multi-lineages and to support expansion of CD34+ cells in cord blood as feeder cells in the presence of cytokine cocktails.

Method: The lentivairal vector expressing either Bmi-1, hTert or hrGFP was prepared as previously. The human PDMCs were isolated from chorionic villi of human placenta using the explant culture, and were cultured in DMEM (low glucose) with 10% FBS. The established cells were infected with either of the three viruses or a combination of Bmi-1 and hTert virus at the multiplicity of infection that allowed almost 100% of infection efficiency based on hrGFP fluorescence by flow cytometry. Those infected cells and uninfected cells served as control were then continuously propagated. These cells were then transferred into the specific culture media to differentiate them to osteocyte, condorocyte and neural cells as reported previously.

Results: The control and PDMC/hTert cells showed a broad and flat shape suggesting that these cells were senescence and ceased proliferation. Cells infected with Bmi-1 and Bmi-1/hTert escaped the crisis and continued proliferation. These cells have been maintained in culture for more than 1 year and continued to proliferate near to 100PD. We found that Bmi-1 over-expression could induce endogenous telomerase activity and elongate telomere length in PDMCs. The expression of p16 mRNA and protein were down-regulated in prolonged culture cells, but p14ARF was not. The differentiation potential of transduced cells was gradually lost as culture was prolonged as well as the ability to support the expansion of CB CD34+ cells. Gene expression analysis on Bmi-1- infected cells at different PD revealed that more than 100 genes were induced or repressed in several different functional categories.Extensive increase in expression of the IGFBP2 mRNA and higher concentration of IGFBP2 was observed.

Conclusion: Bmi-1 induced Telomerase activity and immorlized human placenta derived mesenchymal cells. The ability of transduced cells to differentiate into various cell types and support expansion of CB CD34+ cells were lost with propagation suggesting that subtle transformation has occurred in the Bmi-1 transduced cells.
Selective Depletion of Alloreacting CD25+ Cells from Stem Cell Allografts Can Reduce Acute Graft-Versus-Host Disease Following Matched Related Donor Transplantation.
Authors: Solomon Scott R. , Tran Thao , Carter Charles S. , Hensel Nancy , Wisch Laura , Mielke Stephan , Read Elizabeth , Ghetie Victor , Schindler John , Vitetta Ellen , Barrett John
Published: April 2020

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic stem cell transplant (SCT), especially in older patients. We previously showed that host-reactive donor T cells are selectively depleted (SD) from an allograft ex vivo, following a short co-culture of donor cells with irradiated T cell stimulators from the recipient and subsequent treatment with an anti-CD25 immunotoxin. We report a pilot study to test the hypothesis that GVHD could be decreased in a cohort of elderly patients receiving SD allografts from HLA-identical sibling donors. Sixteen patients, median age 65 years (range 51–73), with advanced hematologic malignancies were transplanted following reduced-intensity conditioning with fludarabine and either cyclophosphamide (n=5), melphalan (n=5), or busulfan (n=6). Cyclosporine was used as the only additional GVHD prophylaxis. SD allografts contained a median CD34 dose of 4.5x106/kg (range 3.5–7.3) and an SD CD3 dose of 1.0x108/kg (range 0.2–1.5). Fifteen patients achieved sustained engraftment. The helper T lymphocyte precursor (HTLp) frequency assay demonstrated depletion of host-reactive donor T cells in 9/11 cases tested from a mean of 1/182,089 to 1/822,354 (mean 5.5-fold depletion), while third party responses were conserved. Kaplan-Meier estimates of probability of grade II-IV and grade III-IV acute GVHD were lower than those seen in a historical control group of patients receiving cyclosporine alone for GVHD prophylaxis (35±13% vs. 57±10%, p=0.34) and (7±6% vs. 38±6%, p=0.05), respectively. Of note, the two patients who developed visceral (gut ± liver) GVHD showed ineffective allodepletion by HTLp (figure). Chronic GVHD occurred in five of 14 evaluable patients. At a median follow-up of 212 days (range 60 – 690), seven of sixteen patients remain alive and in remission. Relapse deaths occurred in four patients (refractory AML [2], therapy-related MDS [1], and CMML [1]). Non-relapse mortality in this high-risk cohort of patients included graft failure [1], GVHD [2], infection [1], and myocardial infarction [1]. In summary, CD25-directed allodepletion of stem cell allografts can reduce clinically relevant acute GVHD following matched related donor transplantation.

Figure Figure
Role of Integrins in Adhesion of Hematopoietic Progenitor Cells.
Authors: Eshghi Shawdee , Zhang Jing , Griffith Linda G. , Lodish Harvey F.
Published: April 2020

The hematopoietic stem cell niche is the set of soluble growth factors, cell-cell and cell-matrix interactions that contribute to stem cell self renewal in the bone marrow. While cytokines and cell-cell interactions have been well documented, cell-matrix interactions in the niche are less understood. Integrins are a class of highly conserved cell adhesion molecules that are important in hematopoietic development and homing. However the specific role of integrins in mediating adhesion to extracellular matrix in the hematopoietic stem cell niche is unknown. The terminal stages of erythropoiesis in the fetal liver provide a good model system with which to develop several of the assays to be used with HSCs. Using flow cytometry, murine fetal liver erythroid progenitors can be separated at four distinct stages of development based on expression of CD71 and Ter119. Further FACS and quantitative PCR analysis revealed that α4β1 integrin is significantly downregulated over the course of erythroid differentiation. Using a centrifugation assay, we determined that this change is accompanied by a loss of adhesion to fibronectin, and that adhesion to fibronectin is blocked by addition of anti-integrin antibodies. Finally, fetal liver progenitor cells adhered to comb co-polymer surfaces engineered to present peptides specifically recognized by α4β1 integrins. By determining the integrin profile expressed by hematopoietic stem cells and measuring stem cell adhesion to ECM in a similar manner, we can begin to understand how these specific interactions present developmental cues important to maintaining the stem cell phenotype in vivo, in addition to leading to design parameters for ex vivo culture systems.
Transcriptional Activation of the Cardiac Homeobox Gene CSX1/NKX2-5 in a B-Cell Chronic Lymphoproliferative Disorder.
Authors: Su Xinying , Busson Maryvonne , Delabesse Eric , Azgui Zahia , Berger Roland , Merle-Beral Hélène , Bernard Olivier A. , Nguyen-Khac Florence
Published: April 2020

Transcription deregulation resulting from genomic rearrangement is frequently observed in human lymphoid malignancies. For example, chromosomal aberrations, such as translocations or inversions, involving the immunoglobulin (Ig), T cell receptor (TCR) and the BCL11B loci, are expected to juxtapose the strong regulatory elements of these genes to the affected genes on the other chromosomes, resulting in such activation. Recently, ectopic activation of two orphan homeobox genes located ~2Mb apart on chromosome 5q35, HOX11L2/TLX3 and CSX1/NKX2-5 have been described, as a result of their fusion to BCL11B on chromosome 14q32 in acute lymphoblastic leukemia of T-lineage. Fusion of TLX3 to TCRδ has also been reported. We describe a novel translocation t(5;14)(q35;q11) identified in a case of B-cell chronic lymphoproliferative disorder. FISH and molecular studies located the chromosome 14 breakpoint within the TCRδgene, at the Dδ3 segment, and the chromosome 5 breakpoint 2.5kb downstream (telomeric) of the NKX2-5 gene. As a result of this translocation, both NKX2-5 and TLX3 were transcribed, whereas only NKX2-5 was detected at the protein level. To investigate the expression of these two genes in B-cell chronic proliferations, we screened 20 B-cell chronic lymphocytic leukemias (B-CLL) and 21 atypical B-CLL samples by RT-PCR and RQ-PCR. No sample demonstrated NKX2-5 expression and only one patient showed TLX3 expression. This patient had an evolutive B-CLL, with complex chromosomal abnormalities, without involvement of the band 5q35. In conclusion, this study identifies a new translocation involving a NK-like homeobox gene in a mature B lymphoproliferative disorder, underscoring precedent identification of NKX2-5 activation in two human lymphoid leukemia cell lines. It also demonstrates that deregulation of homeobox encoding genes is not a specific feature of acute leukemic proliferations, but is also observed in chronic malignancies.
Lymphoma Associated Chromosomal Abnormalities Can Be Easily Detected by FISH on Tissue Imprints. an Underused Diagnostic Alternative.
Authors: Buño Ismael , Nava Paola , Alvarez-Doval Angela , Simon Ainhoa , Gema Gonzalez-Pardo , Alvarez-Rodriguez Federico , Diez-Martin Jose L. , Menarguez Javier
Published: April 2020

Detection of specific chromosomal abnormalities is essential in the diagnosis of several lympholiferative disorders. However, conventional cytogenetic studies are not frequently carried out from biopsies as it is in bone marrow (BM) specimens. On the contrary, FISH is usually performed on paraffin embedded tissue, an alternative with potential technical nuances both in its application and its interpretation. In our experience, FISH on tissue imprints is the ideal alternative to overcome these problems. In the present study, 46 tissue imprints and 17 BM smears from 43 patients with lymphomas were selected to investigate the presence of t(14;18)(q32;q21), t(11;14)(q13;q32), t(8;14)(q24;q32) and t(3;var)(q27;var). Representativity of the samples was assured prior to FISH by rapid May-Grümwald staining (Diff-Quick, QCA, Spain). Twenty imprints from reactive palatine tonsils and adenoids were used as negative controls. FISH was performed with specific dual-color dual-fusion FISH (D-FISH) probes for the first 3 translocations and a dual-color break-apart FISH probe for t(3;var)(q27;var) following the instructions of the probes supplier (Vysis, Inc). All except one sample (a BM smear stored at room temperature over 9 years), rendered satisfactory FISH hybridizations. In any case, all 43 patients could be successfully studied by FISH (the case referred above in which FISH failed in a first attempt was successfully analyzed using a different sample). The results supported the suspected diagnosis of follicular lymphoma in 22 patients, mantle cell lymphoma in 12 patients, large B-cell lymphoma in 5 patients, marginal zone lymphoma in 2 patients and B chronic lymphocytic leukemia in 2 patients. In one case, the observation of t(11;14) by FISH allowed to reclassify the case from follicular to mantle cell lymphoma and also to demonstrate clonal evolution by studying sequential tissue imprints stored for more than 6 years. Negative results also aided in the assignment of patients to proper diagnostic categories. FISH performed on tissue imprints and BM smears constitutes an optimal strategy for retrospective and prospective investigation of chromosomal abnormalities in lymphomas. Tissue imprints and BM smears conventionally stored at room temperature even for long periods of time can be safely used and sent by ordinary mail without special considerations for this purpose. Based on our experience we recommend to perform and routinely store tissue imprints whenever fresh tissue is available.
Heterogeneous Chromosomal Mechanisms Generating the 5′RUNX1/3′CBFA2T1 Gene in Acute Myeloid Leukemia.
Authors: Specchia Giorgina , Albano Francesco , Anelli Luisa , Zagaria Antonella , Liso Arcangelo , La Starza Roberta , Mancini Marco , Cuneo Antonio , Saglio Giuseppe , Fazi Paola , Testoni Nicoletta , Vicari Laura , Liso Vincenzo , Rocchi Mariano
Published: April 2020

Translocation t(8;21)(q22;q22) is a common karyotypic abnormality detected in about 15% of Acute Myeloid Leukemia (AML) cases. The rearrangement results in fusion of the RUNX1 (also known as AML1) and CBFA2T1 (also known as ETO) genes generating a 5′RUNX1/3′CBFA2T1 transcriptionally active fusion gene on derivative chromosome 8. In 1 to 8.5% of AML cases insertions events generating a 5′RUNX1/3′CBFA2T1 fusion gene have been reported, whereas the occurrence of inversions accompanying the t(8;21) has never been observed. We report a screening of 82 AML cases bearing the RUNX1/CBFA2T1 rearrangement detected by RT-PCR; all cases were tested by Fluorescence In Situ Hybridization (FISH) with BAC and PAC clones specific for CBFA2T1 and RUNX1 genes. This analysis allowed us to reveal five cases with ins(21;8), one with ins(8;21), and two with a pericentric chromosome 8 inversion followed by a t(8;21) translocation. A detailed molecular cytogenetic characterization of breakpoints has been performed in all cases.

FISH co-hybridization experiments with CBFA2T1 and RUNX1 probes revealed the presence of a functional fusion gene on the der(21) instead of the der(8) chromosome in five cases with ins(21;8); a single fusion signal on the der(8) chromosome was detected in the case with ins(8;21). The use of the same clones in FISH studies showed the presence of a single unexpected fusion signal on the 8p derivative chromosome in addition to faint CBFA2T1 and RUNX1 signals on the long arm of der(8) and der(21) chromosomes, respectively. These results suggested that a pericentric chromosome 8 inversion involving CBFA2T1 gene occurred and that the chromosome 21 was rearranged with the 8p derivative chromosome.

Appropriate chromosome 21 and 8 BAC clones were employed to precisely define the size of inserted regions in cases with insertions and the breakpoint on the 8p derivative chromosome in cases showing pericentric chromosome 8 inversion. The insertion size turned out to be very heterogeneous, ranging from a minimum of 2.4 Mb to a maximum of 44 Mb. In both cases with chromosome 8 inversion, the CBFA2T1 gene represents the breakpoint at the chromosome 8 long arm whereas the 8p breakpoint showed different mapping positions in 8p21.3 and 8p21.1, respectively.

Our results illustrate that (1) heterogeneous mechanisms can lead to the generation of the 5′RUNX1/3′CBFA2T1 chimeric gene; (2) molecular cytogenetic techniques may identify cryptic chromosomal changes, not detected by conventional cytogenetic analysis; (3) the crucial role of the 5′RUNX1/3′CBFA2T1 fusion gene in leukemogenesis does not depend on its location.
Discovery and Characterization of a Novel Large Non-Coding RNA Transcript, the Mouse Alpha Gene.
Authors: Lin Rui , Liu Cheng , Edgington Thomas S.
Published: April 2020

Recently, a new class of RNAs has been identified that, like mRNAs, are usually transcribed by RNA polymerase II, but lack significant translated open reading frames. They are non-coding RNAs (ncRNAs) that exert roles directly as RNAs, and function as genetic regulators, or riboregulators. In the present study, we discovered a novel very large ncRNA. From differential gene profiling of CD31 positive tumor microvascular endothelial cells from a murine colon carcinoma, we amplified an ~380 bp nucleotide sequence, DP100. This RNA was present in tumor cells as well as tumor vascular endothelium. The full-length DP100 transcript is an ~7 Kb RNA lacking an open reading frame set for more than 66 amino acids. Also the hypothetically predicted proteins lacked significant similarity to known proteins, consistent with identity of DP100 as an unusually large non-coding RNA transcript. Further bioinformatic search demonstrated that the full-length DP100 sequence is highly homologous to the human alpha gene, which encodes an 8.5 Kb, non-coding RNA transcript, and is located in a region implicated in chromosomal abnormalities of human tumors. The full-length DP100, here designated the mouse alpha gene, is evolutionally highly conserved among vertebrates, suggesting its functional significance. The role for this ncRNA in cellular behavior is under investigation.
PML/RARα Mediates Resistance to Apoptosis and Down-Regulation of the NF-κB Pathway in Acute Promyelocytic Leukemia.
Authors: Garcia-Casado Zaida , Cervera Jose , Pajuelo Juan C. , Valencia Ana , Garcia Carlos , Martin Guillermo , Bolufer Pascual , Barragan Eva , Ballester Sandra , Oltra Juan S. , Martinez Nerea , Marty Maria L. , Piris Miguel A. , Sanz Miguel A.
Published: April 2020

Acute promyelocytic leukemia (APL) is genetically characterized by the translocation t(15;17) that results in the fusion gene PML/RARα. The chimeric protein renders hematopoietic progenitor cells resistant to FAS-, TNF- and IFN-induced apoptosis and caspase-3 activation, supporting a role for apoptosis impairment in the pathogenesis of APL. To better investigate apoptosis deregulation in APL, we analyzed the expression profile of 53 newly diagnosed APL patients (27M/26F; median age: 41 yr. (range: 9–80); median WBC count x 109/L: 2.67 (range: 0.5–128); median platelet count x 109/L: 19 (range: 4.4–135); FAB subtype: 39 M3/14 M3v) using the OncoChip®, a cDNA microarray especially designed for analyzing genes involved in cancer which contains 6,386 genes. After array processing, a total of 371 and 249 known genes were found to be ≥2 fold down- or up-regulated, respectively. To verify the results of microarray analysis, six differentially expressed genes (JAG1, JUN, CDKN1C, FAS, TRAIL, TRAF6 and MMP9) were tested by real-time quantitative PCR analysis (Q-RT-PCR). As initially hypothesized, numerous genes involved in apoptotic pathways were deregulated. In particular, we found a significant down-regulation of genes involved in the activation of NF-κB and of genes related to TNF-mediated apoptosis (FAS, TRAIL, TNFSF13B), with 46 and 24 genes deregulated, respectively. Expression changes in other genes implicated in apoptosis were also identified, being of special interest those affecting to the BCL2 family. Thus, anti-apoptotic genes BCL2 and BCL11A were up-regulated while pro-apoptotic members of the family were down-regulated in the analyzed series. Genes involved in the regulation of p53-dependent apoptosis (such as APAF1, p53DINP1, ATM), as well as numerous genes involved in diverse mechanisms of DNA repair, were also inhibited. Finally, the protein kinase C-δ (PKCδ) and several interferon- and interleukin-related genes were also infraexpressed in APL. These findings suggest that inactivation of apoptotic pathways is a common event in the pathogenesis of APL and may have important implications in the design of therapeutic protocols. It was of particular interest the unexpected inhibition of the NF-κB pathway. It could be in accordance with the hypothesis that NF-κB is an inductor of apoptosis under some circumstances. The role of NF-κB in promoting or repressing cell death in APL should be further investigated.
Serial Analysis of Gene Expression Revealed Consistent Downregulation of More Than 100 Genes in Hodgkin Lymphoma.
Authors: Kluiver Joost , Schwering Ines , de Jong Debora , Küppers Ralf , Poppema Sibrand , van den Berg Anke
Published: April 2020

Hodgkin Lymphoma (HL) is unique in its cellular composition, consisting of a minority of tumor cells in an inflammatory background. The tumor cells (Reed-Sternberg cells, RS cells) are thought to be derived from germinal center B cells (centroblasts, CB) that have escaped apoptosis during antigen selection. Global gene expression studies in HL have been hampered by the fact that RS cells only represent less than 1% of the cells in the tumor tissue. Only a few large-scale gene expression studies are available, mostly using cell lines derived from patients with classical HL. To get more insight into the gene expression profile of RS cells we applied the Serial Analysis of Gene Expression Technique (SAGE) on several HL cell lines. SAGE was performed on the cell lines L428 and L1236 (classical cell lines, cHL), DEV (Nodular Lymphocyte Predominant cell line, NLP) and on CB as normal counterparts.

In total, about 100.000 tags were sequenced (Table 1). After normalization and exclusion of tags present only once, 3.635 genes were retained and used for further analyses. A five-fold difference in tag frequency between the HL cell lines and CB was used to select for up- or downregulated genes. Although many genes were upregulated in every individual HL cell line compared to CB, only few genes were consistently upregulated. In contrast, comparison of the downregulated genes revealed that 125 were downregulated in all three HL cell lines compared to CB (Table 2). Tags corresponding to Fascin, TARC/CCL17 and BIC, known to be highly expressed in HL indeed were observed in the SAGE libraries. Among the genes downregulated in L428 a loss of B lineage-specific genes was observed confirming the previous findings in L1236. Interestingly our SAGE results suggest that the NLP cell line DEV has an intermediate loss of B-lineage specific genes compared to CB. To gain more insight in the biology, differentially expressed genes were grouped according to gene ontology’s (based on biological process or molecular function) and pathways. We have now selected more than 70 genes that will be subjected to quantitative RT-PCR analysis to confirm our SAGE results and to study their expression in tumor cell enriched HL samples.

In conclusion: Downregulation of gene expression seems to be important in the pathogenesis of HL since we observed a consistent downregulation of more than 100 genes in HL compared to CB.

Table 1. Overview of Hodgkin lymphoma SAGE libraries

Table 2. Number of up- and downregulated genes in Hodgkin lymphoma compared to centroblasts

Library DEV L428 L1236 CB HL type NLP cHL cHL Centroblasts Tags 16316 20990 30623 29787 Genes 4703 5602 10561 11095 L428 L1236 cHL DEV cHL & NLP HL L428 and L1236 are derived from classical Hodgkin lyphoma (cHL) cases, DEV is derived from a case of Nodular Predominant Hodgkin lymhoma (NLP HL) Upregulated 396 116 14 330 7 Downregulated 329 169 141 309 125
Hypermethylation of the p15/INK4B Gene in Fanconi Anemia.
Authors: Hamanoue Satoshi , Yabe Miharu , Yabe Hiromasa , Yamashita Takayuki
Published: April 2020

Fanconi anemia (FA) is an inherited bone marrow failure syndrome with multiple complementation groups, characterized by genomic instability and predisposition to MDS and AML. Recent evidence indicates that multiple FA proteins are involved in DNA repair. Thus, increased genetic damage and secondary dysregulation of cell proliferation, differentiation and apoptosis are thought to play important roles in the development of bone marrow failure and subsequent progression to MDS/AML. However, little is known about molecular abnormalities responsible for these hematological disorders. Numerous studies indicated that epigenetic silencing of p15/INK4B, an inhibitor of cyclin-dependent kinases, plays an important role in the pathogenesis of MDS and AML. In the present study, we examined methylation status of 5′ CpG islands of the p15 gene in bone marrow mononuclear cells of FA patients, using methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Bone marrow samples were analyzed in 10 patients and serially studied in 4 of them. Hypermethylation of the p15 promoter region was detected in 5 patients (50%). This group included 3 patients with MDS: FA28-1 with refractory anemia (RA), FA87 with RAEB (RA with excess of blasts), and FA88 with later development of RA and progression to RAEB; whereas myelodysplasia was not observed in 2 patients (FA89, FA90). In two cases (FA88, FA90), p15 hypermethylation became negative during their courses, perhaps because of decreased myeloid cells. On the other hand, none of 5 patients without p15 hypermethylation had MDS. These results suggest that p15 hypermethylation is associated with development of MDS and occurs in the early phase of clonal evolution in the disease. Methylation status of p15 may be a useful prognostic factor of FA.

Patient Age at onset (year old) Time from onset (month) Cytopenia MDS Cytogenetic abnormalities p15 methylation MSP b p15 methylation COBRA c a siblings, b MSP: methylation specific PCR, c COBRA: combined bisulfite restriction analysis, d ND: not determined FA28-1a 5 128 severe RA − − + 133 severe RA − + ++ FA87 8 252 severe RAEB + + +++ FA88 5 31 moderate − − + +++ 45 severe RA + − − 58 severe RAEB + + + FA89 5 49 mild − − + + 56 severe − − + + FA90 2 2 mild − − + ++ 31 severe − − − − FA28-2a 5 51 mild − − − NDd FA28-3a 3 12 mild − − − NDd FA47 3 15 mild − − − NDd FA68 5 46 moderate − − − NDd FA91 5 129 mild − − − NDd
Regulation of the GATA3 Promoter by Human T-Cell Lymphotropic Virus Type I Tax Protein.
Authors: Gilli Simone C.O. , Salles Tereza S.I. , Saad Sara
Published: April 2020

The HTLV-I nonstructural protein Tax plays a crucial role in cellular transformation. It activates the transcription factors of various cellular genes and interacts with cellular proteins. There is limited data available on the interaction between specific T cell transcription factor GATA3 and Tax. Implications for the significance of GATA3 in T-cell development and function, (Th2) differentiation, and a role of GATA3 during the immune response have been reported. We had previously demonstrated decreased GATA3 mRNA expression in HTLVI infected individuals and in patients with ATLL and HAM/TSP [Gilli et al., 2000]. To determine the effect of the Tax protein on GATA3 gene expression, we investigated the interaction between this protein and its promoter and repressor regions by DNA-protein interaction and “in vitro” transcription assays. Results demonstrated an interaction between Tax and the GATA3 promoter via the transcription factor Sp1 and a role for Tax in the negative regulation of GATA3 expression, through its interaction with the repressor ZEB. This interaction may be involved in the pathophysiology of adult T cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-I-associated myelopathy.
Frequency of Genetic Deletions of the Glutathione-S Transferase (Gst) Gene in Mexican Patients with De Novo Acute Leukemia: A Preliminary Report from the “Instituto Tecnologico De Monterrey-Hematology Research Group”.
Authors: Escoboza Jose R. Borbolla , Garza-Madrid Marcos E. , Alvarado Martha , Baez Enrique , Lopez-Hernández Manuel A. , DeDiego Jose E. , Baltazar Severiano , Leon-Castanon Maria I. , Rojas-Martinez Julio C. , Ramirez-Assad Cecilia , Trueba Elvira , Villarreal Gerardo , Villela Luis M. , Cruz-Rico Jorge , Tejeda Monica , Duque Jorge , Gonzalez-Avante Mauricio C. , Pompa Teresa , Anaya Irene , Ochoa Leonel , Gonzalez Guadalupe , de la Luz Mendizabal Maria , Jimenez Rosa M. , Hernandez Roberto , Mejia Dolores , Romero Eduardo , Gil-Rondero Carlos , Juarez Jorge L. , De Leon Rosa E. , Tovar Yara E. , Penniecook Jason A.
Published: April 2020

Although there have been extraordinary advances in leukemia therapy, there is still a large subset of cases in which complete remission or prolonged leukemia-free survival cannot be achieved. Some of this variability in results is thought to be related to the differences in the effect of chemotherapy drugs on different patients. In many cases, these differences may be associated with the presence of genetic alterations that produce defective drug-metabolizing enzymes, drug transporters or drug targets. One of these is the glutathione-S transferase (GST) gene, which is known to catalyze the conjugation of toxic compounds, such as aliphatic aromatic heterocyclic radicals and epoxides, among others, to glutathione. The enzymes encoded by this gene detoxify polycyclic aromatic hydrocarbons and conjugated isothiocyanates.

Objective : Our aim is to evaluate the frequency of deletions in GSTT1 and GSTM1 of the GST gene in Mexican patients with de novo acute leukemia.

Methods : Starting in July 2003 and up until now, have included 75 samples from as many patients diagnosed with de novo acute leukemia, regardless of age, sex, or leukemia type. After obtaining informed consent, we drew blood, purified DNA and performed PCR to look for deletions in the GST gene (variants GSTT1 and GSTM1).

Results: We found GSTM1 positiveness in 54 samples (72%), of those, 24 (44.5%) were ALL and 30 (55.5%) AML. We had 18 samples (24%) positive for the GSTT1 deletion; of those, 10 (55.5%) were from patients with ALL and 8 (44.4%) from AML patients.

Conclusions ; As far as we can tell this is the first report of the frequency of these genetic deletions in Mexican acute leukemia patients. Recently a Mexican group studying the possible association of GSTT1 to lung cancer found that among Mexican controls, the frequency of this deletion was around 4.5%. This result would appear to indicate, as has been done by some other authors, that regardless of the possibility of becoming a prognostic factor, at least some polymorphism of the GST gene could be related to the genesis of this disease.
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

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

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

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

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

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

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

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

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.