bluuebunny/crossref_metadata_2025_split huggingface dataset:
Myeloblasts Convert into Fibroblastoid Stromal Cells To Create a Microenvironment for Proliferation of Leukemia Blasts in Acute and Chronic Myelogenous Leukemia Cases.
Authors: Tashiro Haruko , Noguchi Mitsuho , Shirasaki Ryosuke , Gotoh Moritaka , Kawasugi Kazuo , Shirafuji Naoki 
Published: April 2020

Abstract
Object: Cancer stem cell has been analyzed in leukemia, which behaves similarly to normal stem cells on their self-renewal and self-conversion into abnormally differentiated cells to make an exact recapitulation of the original heterogeneous leukemia cells. We observed a primary in vitro culture of non-adherent leukemia blasts prepared from various kinds of acute leukemia and chronic myelogenous leukemia cases, and biological and biochemical characteristics were analyzed.
Method: Leukemia blast-rich fractions were prepared from patients’ blood or bone marrow after gradient sedimentation method, which were cultured for a long term. When the appearance of the cultured cells converted into fibroblastoid cells, cells were divided into clones, analyzed molecularly to identify whether they were originated from leukemia clone, and their histochemical, biochemical and functional characterizations were determined.
Results: Morphological changes into fibroblastoid stromal cells were observed in AML with t (11; 19) (p23; p13.1), M4E, Ph-positive biphenotype and CML (chronic and myeloid blast phase) cases but neither in ALL nor in CML-lymphoid blast cases. The generated fibroblastoid cells had enough functions equal to those of the normal bone marrow fibroblasts on their molecular expression (CD106, fibronectin), production of cytokines (VEGF, IL-7) and giving the activity of proliferation to normal hematopoietic cells. These cells maintained their characteristics observed in the original leukemia blasts (expression of CD13, CD33 and myeloperoxidase), which cells also expressed CD 34 and 133. Leukemia blasts proliferated extensively when cultured on the expanded fibroblastoid cells derived from leukemia blasts.
Discussion: These results indicate that leukemia blasts can create their own microenvironment for proliferation.
Bone Marrow Stroma-Produced TGF-beta1 Confers Chemoresistance of Leukemic Cells.
Authors: Tabe Yoko , Xu Yuanyuan , Jin Linhua , Andreeff Michael , Konopleva Marina 
Published: April 2020

Abstract
TGF-beta1 is secreted at high levels by bone marrow stromal cells and osteoblasts, however its role in leukemia/stroma interactions is unknown. We investigated the effects of TGF-beta1 on survival and proliferation of human monoblastic U937 cells. First, the production of TGF-beta1 by bone marrow-derived mesenchymal stem cells (MSCs) and by U937 cells was analyzed by ELISA. U937 cells secreted approximately 0.1 ng/ml of TGF-beta, while TGF-beta1 level was over 20-fold higher in MSC conditioned medium (2.1±1.7 ng/ml at 72 hours). When U937 cells were co-cultured with MSC, TGFbeta concentration was 2.2±0.2 ng/ml. Hence, we selected 2 ng/ml concentration of recombinant TGF-beta to examine the effects on survival and proliferation of U937 cells exposed to chemotherapy or serum starvation. TGF-beta1 significantly reduced Ara-C or serum withdrawal-induced apoptosis of U937 cells in particular when U937 cells were co-cultured with MSCs (AnnexinV(+)%;U937 alone, control 34.5±8.4, TGF-beta1 18.4±4.5, Ara-C 88.6±3.0, Ara-C/TGF-beta1 60.4±8.0, p=0.04; U937 co-cultured with MSC, control 19.4±2.8, TGF-beta1 3.5±1.0, Ara-C 69.0±3.6, Ara-C+TGF-beta1 24.9±3.3, p=0.01). Treatment with rhTGF-beta1 also enhanced percentage of cells in G0/1 cell cycle phase hence promoting quiescence (G0/G1 phase; control 50.1±6.7, TGF-beta1 63.8±5.5, p=0.04). Most importantly, inhibition of TGF-beta1 by neutralizing antibody (TGF-NA) significantly diminished the pro-survival effects of rhTGF-beta1 in serum-deprived U937 cells co-cultured with MSC (AnnexinV(+)%; U937 alone, control 27.9±9.0, TGF-beta1 20.1±3.3, TGF-NA 21.8±2.2, TGF-beta1+ TGF-NA 18.9±4.1; U937 co-cultured with MSC, control 22.2±1.9, TGF-beta1 10.9±2.5, TGF-NA 29.6±5.1, TGF-beta1+ TGF-NA 20.2±0.9, p&lt;0.001). Dissection of molecular mechanisms demonstrated that TGF-beta1 induced anti-apoptotic Bcl-2 protein expression in U937 cells. Furthermore, concomitant exposure of U937 cells to rhTGF-beta1 in MSC co-cultures results in activation of AKT signaling and pro-survival phosphorylation of Bcl-2, implicating their role in anti-apoptotic effects of TGF-beta1. Taken together, these findings demonstrate that TGF-beta1 secreted by BM stroma cells promotes survival of U937 cells and confers chemoresistance of leukemic cells within the bone marrow microenvironment. Since small molecule inhibitors of TGF-beta signaling are undergoing final steps of pre-clinical development, this approach may represent a viable strategy to enhance efficacy of chemotherapy in AML.
Patulin: A Small Molecule Mycotoxin Possessing Potent Selective In Vitro Cytotoxicity Against Malignant T-Cells.
Authors: Rollins Margo R. , Hessel Alex , Rosenthal Hilary , Waller Ned 
Published: April 2020

Abstract
Background: Peripheral T-cell lymphomas have no established standard therapy, and clinical outcomes are inferior to those seen in patients with diffuse large B-ell lymphoma. Mycotoxins are structurally diverse secondary metabolites of fungi with immunosuppressive activity. Mycotoxins are believed to kill T-cells by covalently bonding to intermediates in the p38 map kinase pathway. We hypothesized that mycotoxins may have selective cytotoxicity against malignant T-cells.
Method: The cytotoxicity of Gliotoxin and Patulin were tested against normal peripheral blood mononuclear cells and malignant hematopoietic cell lines. Growth inhibition was assessed using an MTT assay and cytotoxicity was measured using flow cytometry following staining with 7AAD and annexin. Five leukemia/lymphoma cell lines: K562, KG-1, HL60, Jurkat, HL60, and MJ were grown in log phase culture. Malignant and normal mononuclear cells were incubated with serial dilutions of either Patulin or Gliotoxin for 24 hours. The concentration of drug producing half maximal cytotoxicity (ED50) was determined for each compound and each target cell population. Multiple replicates of this experiment were run 2 to 4 times and average values for experiments can found in the graphs below.
Results: Gliotoxin and Patulin were cytotoxic to normal and malignant hematopoietic cells at micromolar concentrations in the flow cytometry and MTT assays. Normal T-cells were more sensitive than tumor cells to the cytotoxic and growth inhibitory effects of Gliotoxin (Figure 1). In contrast, Patulin was selectively cytotoxic against malignant T-cell lines, with an ED50 of 0.5 μM for Jurkat and 5 μM for MJ compared to and ED50 of &gt; 50 μM with normal T-cells. Low micromolar concentrations of Patulin produced more than 50% cell death in the Jurkat T-cell line with less than 20% killing of normal T-cells. CD11c+ type I dendritic cells were equally sensitive to both Patulin and Gliotoxin.
Conclusion: Gliotoxin and Patulin are cytotoxic against normal and malignant hematopoietic cells at micromolar concentrations. The selective cytotoxicity of Patulin on malignant T-cell lymphoma and leukemia cell lines suggests potential therapeutic activity in T-cell malignancies. The action of Patulin on intermediaries in the p38 map kinase pathway in normal and malignant T-cells will be discussed.
Glioitoxin Dose Response Curves Glioitoxin Dose Response Curves Patulin Dose Response Curves Patulin Dose Response Curves
The FRA12E Fragile Site Is Localized within the SMRT Gene at 12q24.
Authors: Coignet Lionel , Dolcce Jean , Ghoshal Pushpankur , Nganga Alain , Johnson Timothy , Foreman Heather , Moran-Guiati Joseph , Houde Christiane 
Published: April 2020

Abstract
The Silencing Mediator of retinoid and Thyroid hormone (SMRT) is a transcription co-repressor whose association with nuclear receptors both in solution and bound to DNA response elements is destabilized by ligand. SMRT and a related co-repressor N-CoR are recruiting a transcriptional repression complex, that contains sin3A/B protein and histone deacetylases (HDAC1/2) to nuclear receptors. It is now well established that SMRT can interact with non-receptor proteins such as BCL6, for its repression activities. The ability of the “silencing complex” to deacetylate histones results in a condensed chromatin state that is inhibitory to transcription. We have previously shown that SMRT was localized at chromosome 12q24. In addition, down-regulation of the SMRT protein due to 12q24 rearrangements was recurrently associated with NHL transformation, supporting a tumor suppressor function for SMRT (Cancer Res, 65(11):4554–4561, 2005). We further investigated the reasons why the 12q24 region and SMRT was targeted by chromosomal rearrangements. Genetic breakage is one mechanism by which functional loss of tumor suppressor gene activity may occur. Chromosomal locations in which genetic breakage may be induced are known as fragile sites. Fragile sites have been shown to be involved in some malignancies in which the fragile site lies within known genes, such as the FHIT gene (chromosome 3p) in lung cancer, and where small deletions are consistently observed on chromosome 3. Two fragile sites exist on the long arm of chromosome 12. FRA12B is located at 12q24.13 and FRA12E has been located at 12q24.2–3. Interestingly the FRA12E region corresponds to the site of SMRT (12q24.2). To assess whether the FRA12E fragile site is localized within the SMRT gene, we studied normal lymphocytes cultured in the presence of aphidicolin (an inducer of fragile sites) and metaphase chromosomes were subsequently prepared following classical cytogenetics protocols. These preparations were then subjected to fluorescence in situ hybridization (FISH) using a set of SMRT-specific RPCI BAC clones. Using this approach, we were able to demonstrate that the FRA12E fragile site is localized within the SMRT gene. We further characterize several lymphoblastoid cell lines that were either carrier or not of the FRA12E fragile site. This provides us with a mechanistic explanation for recurrent 12q24 rearrangements seen in transformed NHLs. The FRA12E-carrier lymphoblastoid cell lines will provide us the opportunity to characterize the structure of this fragile site.
Duplication and Amplification of AML1 in a Biphenotypic Acute Leukemia: Mechanism in the Formation of Double Minute Chromosome?.
Authors: Benattar Laurence K. , Bouffard Laurence , Coignet Lionel J. 
Published: April 2020

Abstract
Amplification of AML1 has been reported in pediatric acute lymphoblastic leukemia (ALL): it occurs mainly older in older children (5 to 14 years-old) as well as in CD10 positive B ALL and a few cases of acute myeloid leukemia. The presence of double minute chromosomes (DMs) may be one of the mechanisms for oncogene up-regulation. It is a rather rare event in hematological malignancies and is generally associated with a poor prognosis. Until now, DMs have been described mainly in AML and involve mainly amplification of c-MYC and MLL.
We report here the case of a 14-year-old boy with CD10 negative biphenotypic acute leukemia (B + M).
Bone marrow cytogenetic analysis revealed a (47, XY) clone with one normal chromosome 21 and two small markers consisting of chromosome 21 material as the sole abnormality. The two markers were consistent with different forms: small acrocentric, small metacentric and appear to be a mixture of those forms until resembling double minute.
Fluorescence in situ hybridization (FISH) was performed using the TEL-AML1 ES probe. Metaphase FISH showed one normal signal, one tandem signal and a third signal with an apparent variable structure. In interphase nuclei, the hybridization showed from 2 to &gt;6 signals without neither tandem duplication nor clustered signals in an area within the interphase nuclei as previously reported in B-ALL.
In our case, we hypothesize that the different forms of the “derivative/deleted” chromosome 21 lead to form DMs containing the AML1 gene. The mechanism seems to be multistep chromosomal rearrangements with formation of an iso-chromosome 21 and deletion of part of chromosome 21.
To our knowledge, it is the first case of biphenotypic AL reported with AML1 amplification and a probable double minute mechanism.
Flow Cytometric Detection of Human Telomerase Reverse Transcriptase Expression in CD34 Positive Precursor Fraction in Myelodysplastic Syndrome Bone Marrow.
Authors: Handa Hiroshi , Matsushima Takafumi , Tsukamoto Norifumi , Karasawa Masamitsu , Irisawa Hiroyuki , Saito Takayuki , Yokohama Akihiko , Yamane Arito , Murakami Hirokazu 
Published: April 2020

Abstract
Telomerase activity has been found in most common cancers indicating that telomerase detection may be a useful marker in cancer diagnosis. For detection of telomerase activity and the expression of associated genes in cells, TRAP assay and RT-PCR are customarily used. Immunohistochemical detection of hTERT is useful to detect telomerase-positive cells in a background of non- cancerous cells. We developed a method for the detection of intra-nuclear hTERT protein, in a sub-population of hematopoietic cells, using concurrent staining cell surface antigen and multi color flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas neutrophils of normal subjects showed 0% positivity, it is consistent with telomerase activity assessed by TRAP assay (r=0.71, p&lt;0.0001) and previous observations. Then we applied this method to analyze hTERT expression in myelodysplastic syndrome (MDS). Forty MDS patients samples were obtained, 36 patients were diagnosed as low risk MDS (RA), 14 patients were diagnosed as high risk MDS (RAEB or RAEB-t) according to FAB classification. All samples were acquired after informed consent was obtained from the patients.
Expression of hTERT protein was higher in CD34-positive blast-gated cells than CD34-negative blast-gated cells. The percentage of the CD34+ cells expressing hTERT ranged from 9.66% to 90.91% in low risk MDS patients, whereas from 50.46% to 97.68% in high risk MDS. The expression level was higher in the high risk group compared to that in the low risk group in MDS (p=0.0054, p=0.0084). This observation implied that telomerase up-regulation and hTERT expression were important for disease progression and could be a marker of more advanced disease. In subsets of MDS and AML bone marrow specimens obtained from these patients, we examined the hTERT expression in CD34+/CD38 high cells and CD34+/CD38 low cells containing stem cell fraction. Of interest, some of the patients showed higher expression of hTERT in CD34+/CD38 low cells than in CD34+/CD38 high cells. This observation is inconsistent with previous reports describing normal bone marrow hematopoietic cell findings. We speculated that this phenomenon could be a marker of MDS abnormality and that telomerase up-regulation may be initiated in the more primitive precursor fraction containing hematopoietic stem cells during the disease progression. Telomerase studies may be useful for definition of the risks associated with disease severity. Multi-parameter nature of flow cytometry and its ability to identify cellular sub-populations will facilitate a fuller understanding of the mechanisms of activation of telomerase.
Changes in DNA Methylation of Repetitive Elements during the Progression of Chronic Myelogenous Leukemia.
Authors: Choi Si Ho , Byun Hyang-Min , Jiang Jia Yi , Garcia-Manero Guillermo , Yang Allen S. 
Published: April 2020

Abstract
Epigenetic changes are a common finding in cancer, and promoter hypermethylation of tumor suppressor genes leads to aberrant gene silencing. In contrast to gene promoter hypermethylation, global hypomethylation is also a well-described phenomenon in cancer. Measurement of total 5-methylcytosine in the DNA show decreases in cancer despite increases in gene specific methylation. This discrepancy can be accounted for by the decrease of DNA methylation found in DNA repetitive elements, which make up approximately over half of the human genome. Decreases in repetitive element DNA methylation are a sensitive surrogate marker of global methylation, and have been used to measure DNA methylation changes induced by chemotherapy, dietary folate and environmental exposure. Decreases in LINE1 methylation have been described in numerous human cancers. In CML decrease in LINE1 methylation has been associated with progression to blast crisis. We investigated the DNA methylation changes of DNA repetitive elements in CML during the progression of CML from chronic phase to accelerated phase and finally blast crisis. We used a previously described assay (Yang et al. 2004) employing Bisulfite-PCR Pyrosequencing to quantitatively study the DNA methylation changes associated with the LINE1, Alu, D4Z4, and NBL-2 repetitive elements (See table).
LINE1 and Alu hypomethylation was significantly associated with the progression of CML. Surprisingly, we found that NBL-2 and D4Z4 hypermethylation significantly increased during the progression of CML. Interestingly, these two repetitive elements were found to be specifically hypomethylated in immunodeficiency, centromeric instability and facial abnormalities (ICF) syndrome which is caused by a germline mutation of DNA methyltransferase 3b (DNMT3b) (Kondo T et al, 2000). This result suggests that DNMT3b may be involved in the progression of CML.
DNA methylation (Mean ± S.E.M) Repetitive Elements Normal Chronic Phase Accelerated Phase Blast Crisis LINE1 32.4 ± 1.3 29.3 ± 0.6 27.8 ± 0.8 28.3 ± 0.8 Alu 79.4 ± 1.1 77.9 ± 0.6 77.6 ± 0.9 77.5 ± 0.8 D4Z4 47.9 ± 2.9 49.0 ± 1.1 52.9 ± 1.4 57.5 ± 2.0 NBL-2 71.7 ± 2.2 71.7 ± 0.7 75.9 ± 1.3 77.2 ± 1.6
Hydroxyurea with Azacitidine or Decitabine in Combination Is Antagonistic on DNA Methylation Inhibition.
Authors: Choi Si Ho , Byun Hyang-Min , Kwan Jennifer , Yang Allen S. 
Published: April 2020

Abstract
5-azacytidine (azacitidine, Vidaza) and 5-aza-2′deoxycytidine (decitabine, Dacogen) are cytidine analogs currently used to treat myelodysplastic syndrome and as an investigational agent to treat acute myelogenous leukemia, sickle cell disease and other cancers. Decitabine is phosphorylated to a triphosphate form and incorporated into DNA where it can covalently trap DNA methyltransferase. Azacitidine is also phosphorylated to a triphosphate form, but requires reduction from a ribonucleotide to a deoxyribonucleotide by ribonucleotide reductase (RR) before incorporation into DNA. Hydroxyurea (Hydrea) is a drug routinely used to treat patients with leukemia and sickle cell disease. Hydroxyurea inhibits cell proliferation by inhibiting RR. Inhibition of RR leads to a depletion of nucleotide pools and cell cycle arrest in S-phase. Therefore, we hypothesized that the combination of hydroxyurea with azacitidine would be antagonistic to the ability of azacitidine to inhibit DNA methylation, and synergistic with decitabine in inhibiting DNA methylation. Hydroxyurea inhibits RR, which could block the conversion of azacitidine from a ribonucleotide to a deoxyribonucleotide, and therefore prevent its incorporation into DNA. In contrast, decitabine could increase its incorporation into DNA by taking advantage of a depletion of other deoxynucleotides induced RR inhibition by hydroxyurea. In order to test this hypothesis, we treated HL-60 (leukemia) and T24 (bladder cancer) cell lines with hydroxyurea in combination with either decitabine or azacitidine. DNA methylation was quantitated using Bisulfite-PCR Pyrosequencing of LINE-1 DNA repetitive elements, the MAGE-A1 gene and the p16 gene promoter region. We clearly showed that hydroxyurea at concentrations above 0.5 mM completely blocked the ability of azacitidine to inhibit DNA methylation (IC50=0.36 ± 0.04 mM). Surprisingly, we found that hydroxyurea at concentrations above 0.5mM also completely inhibited the effect of decitabine on DNA methylation (IC50=0.24 ± 0.06 mM). Further investigation showed that inhibition of the ability to block DNA methylation correlated with inhibition of the cell cycle. Using flow cytometry we clearly show that the antagonistic effect of hydroxyurea on DNA methylation inhibitors correlates with the arresting of cell division in S-phase. Aphidicolin, which inhibits DNA polymerase, also blocked the cell cycle and the ability of azacitidine and decitabine to inhibit DNA methylation. Thus, azacitidine and decitabine are S-phase specific drugs and are antagonized when used in combination with drugs that arrest the cell cycle. However, we also show that sequential treatment of hydroxyurea followed by azacitidine or decitabine could be used to avoid this antagonism. Therefore, due to the antagonistic effect of hydroxyurea with the mechanistic inhibitors of DNA methylation, azacitidine and decitabine, we recommend sequential combination treatment strategy.
Expression of LAT and PAG Adaptor Molecules in Blasts of Childhood Acute Lymphoblastic Leukaemias and in Normal B- and T-Precursors.
Authors: Burjanivova Tatiana , Vaskova Martina , Kalina Tomas , Stary Jan , Trka Jan , Zuna Jan 
Published: April 2020

Abstract
Despite the progress in leukaemia research, genetic background of some acute lymphoblastic leukaemias (ALL) is unknown. In the aetiopathogenesis of ALL the transmembrane adaptor proteins (including LAT and PAG) could play an important role as these proteins are key mediators of signalization cascades in lymphocytes.
We analysed expression of PAG and LAT at the mRNA level by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and at the protein level by flow cytometry. In our study we assessed the expression levels in 10 patients with T-ALL and in 10 patients with B-cell precursor ALL (BCP-ALL). All analysed samples had a high percentage of malignant blasts (70–97%, median 91%). Sorted and unsorted bone marrow/peripheral blood samples of healthy volunteer donors were used as controls. To determine the PAG and LAT expression levels in normal immature thymocytes we analysed also sorted cells from the normal thymuses taken from children undergoing heart surgery. We used various differentiation markers of both B- (CD10, CD19, and CD20) and T-lineage (CD1a, CD3, CD4, CD7, CD8) for sorting to compare the leukaemic samples with the most adequate control in terms of maturation status of the cells. Informed consents were obtained from all volunteer donors or their guardians.
Our data show that the expression of PAG at both protein and mRNA level during the physiological B-cell maturation is decreasing. On the contrary, PAG expression during the T-cell development and also the levels of LAT during the B- and T-cell maturation are increasing, however, the levels of LAT mRNA in B-precursors are very weak.
In childhood BCP-ALL the expression of PAG on protein and mRNA level reflects the situation in corresponding non-malignant precursors with the exception of the TEL/AML1 positive leukaemias that show tendency to the overexpression of PAG. In the T lineage ALL we found a significantly lower expression of PAG and higher expression of LAT mRNA ((p&lt;0.0001 and p=0.03, respectively, for mature T-ALL) compared to sorted thymocytes with corresponding immunophenotype. The LAT protein in BCP-ALL blasts is expressed very weakly or there is no expression at all. Also the levels of LAT mRNA are even lower than in the physiological BCP counterparts.
We present the first study assessing changes in the expression of two important adaptor molecules at both mRNA and protein levels during the physiological maturation of lymphocyte precursors. Moreover, we demonstrate changes in the pattern of expression in childhood ALL and suggest that the aberrant transmission or processing of signal through the immunoreceptors of lymphoid precursors might play an important role in the aetiopathogenesis of the disease.