bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lineage Shift of a Megakaryocytic-Erythroid Leukemia Cell Line by Adhesion to Fibronectin.
Authors: Yamada Hisashi , Iwase Satsuki , Horiguchi-Yamada Junko 
Published: October 2019

Abstract
Acute megakaryocytic leukemia (AMgL) occurs in about 1% of AMLs and has poor prognosis in adults. Research of AMgL has been hampered by this rarity. To overcome this obstacle, the established immortal AMgL cell lines are useful materials for the study of megakaryocytic malignancies. A few cell lines with the characteristics of megakaryocytes have been established, but half of the cell lines are derived from blastic crisis of CMLs. In normal hematopoiesis, erythroblasts and megakaryocytes have a common precursor. And erythroid and megakaryocytic leukemias frequently share their phenotypes simultaneously. However, the mechanisms of lineage shift between the two remain unknown. We recently established an AMgL cell line named JAS-R. Megakaryocytic-erythroid JAS-R cells showed the phenotypic change by adhesion to culture dishes. Adherent JAS-R cells (named JAS-RAD) show megakaryocytic phenotype concomitant with erythropoietin secretion and MDR1 expression, while suspension cells (JAS-REN) show erythroid characters. Because the adhesion requires bivalent cation and is inhibited by RGDS tetrapeptide, it is presumably mediated by integrins. As for ligands, fibronectin was found to be the most powerful, using dishes coated with collagens (Type I and type IV), laminin, fibronectin and poly-D-lysine. Next we studied the phenotypic changes of JAS-REN cells along with the adhesion to fibronectin. JAS-REN cells were seeded on fibronectin-coated dishes and cultured for 48 hours. Then adherent and floating cells were harvested, separately. Cells were also treated with phorbol ester (TPA). Flow cytometry analysis disclosed that adherent cells had megakaryocytic phenotype with increased CD41a and CD61, and with decreased CD235a. The gene expression profile revealed that FLI1, RUNX1 and GFI1 were increased in fibronectin-adherent cells while GATA1, FOG1 and NFE2 were not changed. These characteristics of fibronectin-adherent cells resembled JAS-RAD. Similar results were observed in TPA-treated cells. Among these transcription factors, the induction of FLI1 was prominent. The promoter activity of FLI1 studied by the luciferase assay was higher in JAS-RAD than JAS-REN. These data suggest the substantial change of AMgL blast cells depending on the growth milieu in vivo. These changes might influence the result of chemotherapy
Methylation Profiling of Apoptotic Genes in Leukemia Cell Lines and Patients.
Authors: EL Ahdab Samih , Castoro Ryan , Abbas Hussein A. , Ahmed Saira , Jelinek Jaroslav , Issa Jean-Pierre 
Published: October 2019

Abstract
^equal contribution Apoptosis is a morphologically and biochemically distinct form of eukaryotic cell death that occurs under a variety of physiological and pathological conditions. Apoptosis is executed via 2 main pathways that lead to the activation of caspases: the death receptor (DR) pathway and the mitochondrial pathway. Defects in apoptotic mechanisms play an important role in enabling neoplastic cells to survive by ignoring death inducing signals leading to protection from the lack of survival factors. DNA methylation is a reversible mechanism of gene inactivation that might be responsible for impaired apoptosis in leukemia cells. Methylation profiling of apoptotic genes can identify possible gene targets for epigenetic therapy using demethylating agents like 5-aza-deoxycytidine and azacytidine. It can also be used for classification and prognosis. We performed a comprehensive DNA methylation analysis of apoptosis pathway genes with CpG islands in promoter/exon 1 regions in 5 leukemic cell lines (TF1, K562, Raji, HL60 and Jurkat), 20 patients with acute myelogenous leukemia (AML), 20 patients with acute lymphoblastic leukemia (ALL), and 6 control samples (normal peripheral blood cells). We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Our analysis covered a total of 25 genes: 11 in the intrinsic pathway (BAX, BAK, HRK, CYC1, DIABLO, ATM, APAF1, MAP3K5, CHEK2,PAK1, and BNIP 3), 4 in the extrinsic pathway (TRADD,PYCARD, CARD 10 and BID), 5 genes of the Caspase family (Caspase 2, 3, 6, 7, 9) in addition to 4 of the programmed cell death genes (PDCD 2, 4, 6, and 11). By using a 15% cutoff level to call a gene methylated, methylation was observed in 7 genes in at least one cell line: BNIP3 (Raji), PYCARD (K562), HRK (Tf1, Raji and Jurkat), CARD10 (Raji and HL-60), BID (Raji), PAK1 (Raji) and Casp 6 (Raji). We then performed methylation analysis of 4 genes (BNIP3, PYCARD, PAK1 and CARD 10) in AML and ALL patient samples. Methylation frequencies over a 15% cutoff are summarized in Table 1. We conclude that DNA methylation-associated epigenetic silencing is likely to play a role in modulating apoptosis in a subset of AML and ALL patients, with PYCARD being the most frequent target.
Table 1 Genes Methylation in AML patients Methylation in ALL patients PYCARD 6/20 4/20 BNIP3 1/20 3/20 CARD10 0/20 2/20 PAK1 0/20 1/20
Acetylation of PML Plays a Key Role in Histone Deacetylase Inhibitor-Mediated Apoptosis through Enhanced PML Sumoylation.
Authors: Hayakawa Fumihiko , Kitabayashi Issay , Pandolfi Pier P. , Naoe Tomoki 
Published: October 2019

Abstract
The promyelocytic leukemia (PML) protein is a potent tumor suppressor and proapoptotic factor, and is functionally regulated by posttranslational modification such as phosphorylation, sumoylation, and ubiquitination. Histone deacetylase (HDAC) inhibitors are a promising class of targeted anticancer agents and induce apoptosis to cancer cells. In addition to their effects on histones, HDAC inhibitors increase the acetylation level of several non-histone proteins such as transcription factors, which are important for their effects to cancer cells. However, the mechanism of HDAC inhibitor-induced apoptocis is largely unknown. We report here a novel posttranscriptional modification, acetylation, of PML. By the screening using antibody array, we identified PML as a new acetylation target of Trichostatin A (TSA), a HDAC inhibitor. PML acetylation was enhanced by coexpression of p300 or treatment with TSA. We also showed that increased PML acetylation was associated with increased sumoylation of PML in vitro and in vivo. PML involvement in TSA-induced apoptosis was demonstrated by PML knocking down in Hela cells or PML overexpression in PML−/− MEF cells. Furthermore, PML with acetylation-defective mutation showed disability to mediate the apoptosis, suggesting the importance of PML acetylation for it. Our work provides new insights into the PML regulation by posttranslational modification, and new information about the therapeutic mechanism of HDAC inhibitors.
Human Tissue Factor Pathway Inhibitor-2 Induces Caspase-Mediated Apoptosis in a Human Fibrosarcoma Cell Line (HT-1080).
Authors: Kempaiah Prakasha , Kisiel Walter 
Published: October 2019

Abstract
Human tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa extracellular matrix-associated Kunitz-type serine proteinase inhibitor that regulates the plasmin and trypsin- mediated activation of zymogen matrix metalloproteinases and growth factors essential for tumor growth, invasion, and metastasis. We previously demonstrated that HT-1080 human fibrosarcoma cells do not express TFPI-2, but restoration of TFPI-2 expression in these cells by stable transfection with the human TFPI-2 cDNA markedly inhibited their growth and metastasis in-vivo. In this study, 1 μM concentrations of either recombinant human TFPI-2 or its mutated first Kunitz-type domain (R24K KD1), were offered to HT-1080 cells, incubated for 48 h, and the degree of apoptosis assessed by nuclear fragmentation, ethidium bromide/acridine orange (EB/AO) staining, fluorescence-activated cell sorting (FACS) and immunoblotting. Agarose gel electrophoresis of DNA from HT-1080 cells treated with either TFPI-2 or R24K KD1 for 48h indicated DNA fragmentation with a ladder pattern typically associated with apoptosis. EB/AO staining of TFPI-2- and R24K KD1- treated cells revealed that 40–70% of the cells were apoptotic in relation to vehicle-treated cells as judged by fluoresence microscopy. Co-administration of TFPI-2 with polymyxin B produced similar results, suggesting that apoptosis was not endotoxin-dependent. Consistent with our earlier studies showing its enhanced inhibitory activity relative to TFPI-2, R24K KD1 was able to induce apoptosis in 68% of HT-1080 cells after 48h of treatment compared to 39% for the parent TFPI-2 at an equivalent concentration. Moreover, HT-1080 cells treated with a KD1 preparation lacking the reactive site arginine residue (R24Q KD1) produced only an 18% apoptosis rate, thereby linking the observed apoptosis with serine proteinase inhibition. FACS analysis, using propidium iodide and annexin-V labeling, revealed similar apoptotic rates to that seen by EB/AO staining assays. In addition, immunoblotting experiments of vehicle and TFPI-2-treated cells indicated increased caspase-3 activation in TFPI-2-treated cells, thus providing evidence that apoptosis is caspase-mediated. We also observed up-regulation of the proapoptotic Bax protein by immunoblotting following treatment of HT-1080 cells with either TFPI-2 or R24K KD1. Taken together, our results demonstrate that treatment of HT-1080 cells with exogenous TFPI-2 or R24K KD1 activates caspase-mediated, proapoptotic signaling pathways and induces apoptosis. In addition, our data provides suggestive evidence that peritumor application of either TFPI-2 or R24K KD1 may facilitate tumor apoptosis in-vivo.
Sumoylation of TRF1 Is Essential for Its Recruitment to ALT-Associated PML Bodies.
Authors: Yu Jian , Lan Jianping , Zhu Yuanyuan , Lai Xiaoyu , Huang He 
Published: October 2019

Abstract
To achieve unlimited proliferative potential, most cancer cells activate telomerase to maintain telomeres. However, some cancer cells elongate telomeres through a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). These ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB), which comprises telomeric DNA and a number of proteins, including PML protein, the telomere binding proteins TRF1 and TRF2, replication factor A, and recombination factors Rad51, Rad52, and the Rad50/Mre11/NBS1 complex. TRF1, as the first identified telomeric binding protein, binds the duplex telomeric repeats at telomere ends. It plays an important role in telomere length control, telomeric ends shelter and cell cycle regulation. In ALT cell lines, TRF1 co-localized with PML protein at APBs, but the exact mechanism of its recruitment to APBs is not clear. Here we show that TRF1 localizes to PML bodies in about 5% of an asynchronously growing culture of U2OS cells and the percentage of cells with colocalization of TRF1 and PML bodies increases to about 40% in cells enriched in G2/M, which is consistent with the previous studies. Furthermore, our results show that TRF1 is modified by the small ubiquitin-like protein SUMO-1 in vivo and in vitro. Firstly, 293T cells were transfected with Flag-TRF1, HA-Ubc9, GFP-SUMO1 and then immunoprecipitated by using FLAG-M2 gel under denaturing conditions. Immunoblotting with GFP and Flag antibodies demonstrated that TRF1 is modified by SUMO-1 in vivo. Next, in vitro SUMO-1 conjugation assay of TRF1 was employed. The results showed that TRF1 is conjugated with SUMO-1 in the presence of purified recombinant protein SAE1/SAE2, Ubc9, SUMO1, His-TRF1 and ATP. Either SAE1/SAE2, Ubc9, SUMO-1 or ATP was omitted from the reaction abolished the sumoylation of TRF1. Previous studies have shown that sumoylation controls the recruitment of several proteins to PML bodies, so we examined whether sumoylation of TRF1 is required for its recruitment to APBs. We mutated the potential sumoylation sites of TRF1 according to the computational prediction and then transfected it into U2OS cells to examine its localization. The results showed that TRF1 mutant does not localize to PML bodies. Taken together, all these results suggest that TRF is modified by SUMO-1 and sumoylation of TRF1 is essential for its recruitment to APBs in ALT cells.
The Kinase Inhibitors Sunitinib (Sutent®) and Sorafenib (Nexavar®) Differentially Affect Reactivity of NK Cells Against Renal Cell Cancer.
Authors: Manuela Schlicke , Matthias Krusch , Baessler Tina , Kanz Lothar , Salih Helmut R. 
Published: October 2019

Abstract
Sunitinib (Sutent®) and Sorafenib (Nexavar®) are multikinase inhibitors licensed for the treatment of patients with advanced renal cell cancer (RCC) because of their direct and indirect anti-tumor and anti-angiogenic effects. Sunitinib and Sorafenib target, among others, vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptors (PDGFR), the stem cell factor receptor (cKIT) and Fms-like tyrosine kinase-3 (Flt3). Beyond that, Sorafenib additionally inhibits c-RAF and B-RAF. NK cells play an important role in the immune surveillance of tumors. Their reactivity is governed by different surface receptors leading to activation of various kinases, phosphatases and transcription factors. Up to now, nothing is known regarding the influence of Sunitinib and Sorafenib on NK cells. Here we report that Sunitinib and Sorafenib differentially affect NK cell cytotoxicity. Analysis of peripheral blood mononuclear cells from 10 independent donors revealed that Sorafenib but not Sunitinib significantly (p<0.05, Mann-Whitney U-test) reduced cell surface CD107a expression (about 50%) on NK cells as a surrogate marker for degranulation in response to tumor targets. In line, pharmacological levels of Sorafenib significantly (p<0.05, Mann-Whitney U-test) diminished cytotoxicity of primary NK cells against the RCC cell lines A498, ACHN and CAKI-2 (from 46% to 14%, from 52% to 22% and from 63% to 42%, respectively) in chromium release assays. Again, no inhibition of cytotoxicity was observed in the presence of pharmacological concentrations of Sunitinib. Since activation of MAP kinases leading to ERK phosphorylation regulates the movement of lytic granula towards target cells, which is responsible for NK cell cytotoxicity we investigated the influence of Sunitinib and Sorafenib on ERK phosphorylation. While Sunitinib had no effect, Sorafenib, alike the specific c-RAF inhibitor ZM336372 or the specific MEK inhibitor PD98059, markedly reduced ERK phosphorylation as determined by Western Blot. Our results indicate that NK cell anti-tumor reactivity is impaired by Sorafenib but not by Sunitinib due to the additional inhibitory effect of Sorafenib on the ERK pathway, which has been demonstrated to dictate NK cell cytotoxicity. We therefore conclude that Sunitinib is exquisitely suitable for combination with immunotherapeutic approaches in RCC patients.
Morphine Stimulates the Phosphorylation and Nuclear Translocation of EGFR in Lung Cancer Cells.
Authors: Nguyen Julia , Johnson Katherine H.N. , Pasrija Teena , Kratzke Robert A. , Hyams Aaron R. , Stinson Brittany A. , Gupta Kalpna 
Published: October 2019

Abstract
Mu opioid receptor (MOR) stimulates MAPK/ERK phosphorylation, by transactivating epidermal growth factor receptor (EGFR) in astrocytes (Belcheva MM et al J Biol Chem 2001, 276, 33847–53) and by transactivating vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells (Chen C et al Curr Neurovasc Res 2006, 3, 171–80). EGFR and VEGFR2 are two critical targets for lung cancer therapy. Since opioids used to treat pain in cancer act via MOR, it is critical to understand the role of MOR in growth and survival promoting signaling in lung cancer. Therefore, we examined if MOR co-activates, EGFR mediated signaling in non-small cell human lung cancer cells, HL2009, HL1299 and HL460; and VEGFR2 in murine blood outgrowth endothelial cells (BOEC) derived from wild type (wt) and MOR knockout (KO) mice. We observed that HL2009 cells showed a 3–5 fold higher expression of MOR (by RT-PCR) as well as constitutive activation of phospho-EGFR (by Western) as compared to HL1299 and HL460. Morphine (100 nM) stimulated the phosphorylation of EGFR, MAPK/ERK and Akt in a time-dependent manner similar to that induced by 10 nM EGF, in HL2009 but not in HL460 cells. Morphine as well as EGF-induced phosphorylation peaked after 10 and 30 min of stimulation and it was accompanied by the translocation of phospho-EGFR to the nucleus in HL2009 but not in HL460 cells. Since, nuclear expression of phospho-EGFR is associated with poor prognosis in some human cancers, it is possible that MOR activation upon opioid exposure may impart resistance to lung cancer therapy, by stimulating the translocation of phospho-EGFR to the nucleus. On the other hand, morphine also stimulated the phosphorylation of VEGFR2 and MAPK/ERK in BOEC from wt mice but not in BOEC from MOR KO mice. Thus, opioid analgesics may stimulate VEGFR2 via MOR and stimulate angiogenesis. Acquired resistance to EGFR therapy in non small cell lung cancer is associated with increased VEGF activity. Therefore, simultaneous co-activation of EGFR and VEGFR2 via MOR in lung cancer may promote cancer progression and resistance to VEGF and/or EGFR based therapies. We speculate that MOR-specific antagonists in combination with VEGF/EGFR based therapies may prove to be beneficial in treating lung cancer.
Poor Survival of CD34 Positive and CD13/33 Positive Patients in Pediatric T-Cell Acute Lymphoblastic Leukemia Is Not Explained by Expression of Multidrug Resistance Genes.
Authors: van Grotel Martine , van Noesel Max M. , Van Den Heuvel M.M. , Pieters Rob , Meijerink Jules P.P. 
Published: October 2019

Abstract
For childhood T-ALL, the significance of early differentiation markers CD34 and CD13/33 in relation to outcome is unclear. In this study of 72 pediatric T-ALL patients, CD34 and CD13/33 expression was related to a poor 5 yrs survival (p=0.0013 for CD34+ and p=0.035 for CD33/CD13+). Expression of CD34 was associated with elevated MDR1 (p=0.01) and MRP1 (p=0.019) levels and CD13/33+ expression with MRP1 levels (p=0.028). However, none of these MDR genes independently predicted for poor outcome. We conclude that T-ALL with CD34 and CD13/33 expression has a relatively poor survival that is not explained by expression of MDR genes.
Immunoglobulin and T-Cell Receptor (Ig/TCR) Gene Rearrangements in Chinese Adult Acute Lymphoblastic Leukemia Patients.
Authors: Yao Li , Chen Zi-xing , Cen Jian-nong , He Jun , Qi Xiao-fei , Shen Hong-jie 
Published: October 2019

Abstract
To determine the feasibility of Ig/TCR gene rearrangements as patient-specific targets for the detection of minimal residual disease in Chinese ALL patients, forty adult patients with B-lineage and T cell acute lymphoblastic leukemia(ALL) were enrolled in this study. The frequency and pattern of IGH, IGK, TCRB, TCRG and TCRD gene rearrangement were analyzed by BIOMED-2 multiplex PCR protocol. All DNA samples were obtained from the bone marrow of these patients. IGH, IGK, TCRB, TCRG and TCRD rearrangements were found in 86%,14%,27%,77%,59% of adult patients with B-lineage ALL, respectively. While in T-ALL, clonal TCRB, TCRG and TCRD rearrangements were detected in 83%,78%,39% of patients. There are several features in the pattern of Ig/TCR gene rearrangements in Chinese adult ALL patients. Firstly, IGK rearrangement in adult B-lineage ALL had a lower frequency than in children’s pre-B-ALL (14% vs 41%) in reported literature. Secondly, the frequency of TCRG Vrfl-Jr1.3, Vrfl-Jr1.1 and Vr9-Jr1.3 rearrangement was 65%,6%,29% in adult patients with B-lineage ALL, while 60%,27%,13% was the frequency of those in T-ALL, respectively. In conclusion, our data have shown that several special patterns in Ig/TCR gene rearrangements in Chinese adult ALL patients, may provide a guidance for individual treatment protocol designation and MRD-based risk stratification.
Molecular Monitoring of ERG and BAALC as Minimal Residual Disease Markers in Patients with Acute Leukemia.
Authors: Najima Yuho , Ohashi Kazuteru , Kakihana Kazuhiko , Kikuchi Taku , Nagata Yasunobu , Sakurai Chihiro , Ueki Toshimitsu , Kobayashi Takeshi , Yamashita Takuya , Kawamura Machiko , Akiyama Hideki , Kaku Hidefumi , Sakamaki Hisashi 
Published: October 2019

Abstract
Although expression of the genes ERG (ETS-related gene) and BAALC (brain and acute leukemia, cytoplasmic) predicts outcome in acute leukemia, there is little information about their utility as markers of minimal residual disease (MRD). A real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) was used to examine relative levels of ERG and BAALC gene expression in 11 patients with acute leukemia. Pretreatment and post-chemotherapy bone marrow (BM) samples were analyzed to quantify these transcripts. Peripheral blood (PB) samples were also obtained to evaluate the correlation of ERG and BAALC expression levels in BM and PB samples. Significant levels of ERG gene were expressed in all patients. In contrast with ERG, the levels of BAALC gene expression were significantly lower in some patients. Continuous monitoring of the ERG mRNA was performed and assessed a correlation between MRD levels and subsequent clinical courses. In patients with relative high expression of BAALC transcripts (>7x10exp-3/GAPDH), high levels of ERG gene expressions were consistently observed whether they were in remission status or not. In 2 patients with acute promyelocytic leukemia (APL), MRD was simultaneously monitored by RQ-PCR using primers for PML/RAR alpha. Changes in expression levels of PML/RAR alpha and ERG presented poor correlation. While, in other patients with relative low expression of BAALC transcripts (<7x10exp-3/GAPDH), ERG gene expression levels gradually decreased after the induction therapy and following consolidation treatments. In patients unable to attain remission, ERG expression levels remained high. Thus, the quantitation of the ERG gene expression made it possible to assess MRD in patients with acute leukemia (except for APL) carrying low BAALC expression. To our knowledge, this is the first report concerning the use of ERG mRNA expression for MRD monitoring.