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1
LEE, Kuan-Der, Seung Joon BAEK, and Rong-Fong SHEN. "Multiple factors regulating the expression of human thromboxane synthase gene." Biochemical Journal 319, no. 3 (November 1, 1996): 783–91. http://dx.doi.org/10.1042/bj3190783.
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Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, ∼75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (∼55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.
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Siddiqui, Mohammad Aarif, Paradesi Naidu Gollavilli, Vignesh Ramesh, Beatrice Parma, Annemarie Schwab, Maria Eleni Vazakidou, Ramakrishnan Natesan, et al. "Thymidylate synthase drives the phenotypes of epithelial-to-mesenchymal transition in non-small cell lung cancer." British Journal of Cancer 124, no. 1 (October 7, 2020): 281–89. http://dx.doi.org/10.1038/s41416-020-01095-x.
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Abstract Background Epithelial-to-mesenchymal transition (EMT) enhances motility, stemness, chemoresistance and metastasis. Little is known about how various pathways coordinate to elicit EMT’s different functional aspects in non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) has been previously correlated with EMT transcription factor ZEB1 in NSCLC and imparts resistance against anti-folate chemotherapy. In this study, we establish a functional correlation between TS, EMT, chemotherapy and metastasis and propose a network for TS mediated EMT. Methods Published datasets were analysed to evaluate the significance of TS in NSCLC fitness and prognosis. Promoter reporter assay was used to sort NSCLC cell lines in TSHIGH and TSLOW. Metastasis was assayed in a syngeneic mouse model. Results TS levels were prognostic and predicted chemotherapy response. Cell lines with higher TS promoter activity were more mesenchymal-like. RNA-seq identified EMT as one of the most differentially regulated pathways in connection to TS expression. EMT transcription factors HOXC6 and HMGA2 were identified as upstream regulator of TS, and AXL, SPARC and FOSL1 as downstream effectors. TS knock-down reduced the metastatic colonisation in vivo. Conclusion These results establish TS as a theranostic NSCLC marker integrating survival, chemo-resistance and EMT, and identifies a regulatory network that could be targeted in EMT-driven NSCLC.
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Gribaudo, Giorgio, Ludovica Riera, Thomas L. Rudge, Patrizia Caposio, Lee F. Johnson, and Santo Landolfo. "Human cytomegalovirus infection induces cellular thymidylate synthase gene expression in quiescent fibroblasts." Journal of General Virology 83, no. 12 (December 1, 2002): 2983–93. http://dx.doi.org/10.1099/0022-1317-83-12-2983.
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Productive infection of non-proliferating cells by cytomegalovirus (CMV) requires the coordinated stimulation of host biochemical pathways that prepare cells to synthesize DNA. Here we illustrate the ability of human CMV (HCMV) to stimulate cellular thymidylate synthase (TS) gene expression in quiescent human embryonic lung fibroblasts. TS mRNA and protein levels are nearly undetectable in quiescent cells, but are greatly increased following HCMV infection. Inhibition of TS activity was shown to impair HCMV DNA synthesis, demonstrating that TS upregulation is required for efficient HCMV replication in quiescent cells. The increase in TS gene expression was due to an increase in gene transcription, since the expression of a reporter gene driven by the human TS promoter was strongly induced by HCMV infection. Deletion analysis of the human TS promoter identified two positive elements that are important for this increased transcription. We have previously shown that murine CMV (MCMV) stimulates the mouse TS promoter by a mechanism that depends on the presence of an E2F element in the promoter region. However, deletion of the two potential E2F binding sites in the human TS promoter did not prevent the virus-induced increase in TS promoter activity. Our data suggest that HCMV activates human TS gene transcription by mechanisms that are independent of E2F and different from those used by MCMV to stimulate the mouse TS promoter.
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Gribaudo, Giorgio, Ludovica Riera, David Lembo, Marco De Andrea, Marisa Gariglio, Thomas L. Rudge, Lee F. Johnson, and Santo Landolfo. "Murine Cytomegalovirus Stimulates Cellular Thymidylate Synthase Gene Expression in Quiescent Cells and Requires the Enzyme for Replication." Journal of Virology 74, no. 11 (June 1, 2000): 4979–87. http://dx.doi.org/10.1128/jvi.74.11.4979-4987.2000.
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ABSTRACT Herpesviruses accomplish DNA replication either by expressing their own deoxyribonucleotide biosynthetic genes or by stimulating the expression of the corresponding cellular genes. Cytomegalovirus (CMV) has adopted the latter strategy to allow efficient replication in quiescent cells. In the present report, we show that murine CMV (MCMV) infection of quiescent fibroblasts induces both mRNA and protein corresponding to the cellular thymidylate synthase (TS) gene, which encodes the enzyme that catalyzes the de novo synthesis of thymidylic acid. The increase in TS gene expression was due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse TS promoter was induced following MCMV infection. Mutagenesis of the potential E2F-responsive element immediately upstream from the TS essential promoter region abolished the virus-mediated stimulation of the TS promoter, suggesting that the transactivating activity of MCMV infection was E2F dependent. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in TS promoter activity. Finally, MCMV replication and viral DNA synthesis were found to be inhibited by ZD1694, a quinazoline-based folate analog that inhibits TS activity. These results demonstrate that upregulation of cellular TS expression is required for efficient MCMV replication in quiescent cells.
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Kaytor, M. D., and D. M. Livingston. "Saccharomyces cerevisiae RAD52 alleles temperature-sensitive for the repair of DNA double-strand breaks." Genetics 137, no. 4 (August 1, 1994): 933–44. http://dx.doi.org/10.1093/genetics/137.4.933.
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Abstract We have screened for mutations of the Saccharomyces cerevisiae RAD52 gene which confer a temperature-sensitive (ts) phenotype with respect to either the repair of DNA lesions caused by methyl methanesulfonate (MMS) or the recombination of an intrachromosomal recombination reporter. We were readily able to isolate alleles ts for the repair of lesions caused by MMS but were unable to find alleles with a severe ts deficiency in intrachromosomal recombination. We extensively characterized four strains conferring ts growth on MMS agar. These strains also exhibit ts survival when exposed to gamma-radiation or when the HO endonuclease is constitutively expressed. Although none of the four alleles confers a severe ts defect in intrachromosomal recombination, two confer significant defects in tests of mitotic, interchromosomal recombination carried out in diploid strains. The mutant diploids sporulate, but the two strains with defects in interchromosomal recombination have reduced spore viability. Meiotic recombination is not depressed in the two diploids with reduced spore viability. Thus, in the two strains with reduced spore viability, defects in mitotic and meiotic recombination do not correlate. Sequence analysis revealed that in three of the four ts alleles the causative mutations are in the first one-third of the open reading frame while the fourth is in the C-terminal third.
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Fenn, Kathleen, Veena M. Singh, Shing Mirn Lee, David Cieremans, Andrew B. Lassman, Dawn L. Hershman, Katherine D. Crew, et al. "Diagnosis of leptomeningeal metastasis (LM) through identification of circulating tumor cells (CTCs) in cerebrospinal fluid (CSF)." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 3567. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.3567.
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3567 Background: Diagnosis of LM from solid tumors can be challenging. The TargetSelector (TS) CTC detection assay has demonstrated highly specific and sensitive CTC capture both for epithelial (CK+) and non-epithelial (CK-) subsets. The assay utilizes a ten-antibody (ab) capture cocktail followed by biotinylated secondary abs that bind to CTCs, enriched in a microfluidic device. TS targeted next-generation sequencing (NGS) assay detects somatic mutations in 12 breast cancer-related genes. The aim was to determine whether TS can improve sensitivity in the diagnosis of LM compared to CSF cytology by lumbar puncture (LP). Methods: CSF was collected prospectively from patients (pts) with a prior solid tumor diagnosis and suspicion of LM. CTCs were isolated from CSF using the TS platform. Cells were stained with cytokeratin (CK), CD45, streptavidin and DAPI. CTCs captured in a microchannel were classified as CK + or -. Peripheral blood samples obtained at time of LP underwent similar CTC analysis. Cell-free total nucleic acids (cfTNA) were extracted from plasma and CSF followed by NGS. Data analysis used the Ion Torrent Suite with annotation and report curation by Ion Reporter and Oncomine Knowledgebase Reporter software respectively. Results: There were 14 pts (13 women and 1 man), median age 56 years (range 32-75) with cancers of the breast (10), lung (1), colon (1), CNS lymphoma (1) or glioma (1). Pts had received a median of 2.5 lines of systemic metastatic therapy (range 0-8). CSF cytology was not sent for 1 pt and TS was not performed for 1 pt. TS and standard cytology had 89% agreement in pts with metastatic breast cancer (MBC, 8/9). Of the 6 pts for whom CTCs were detected in CSF by TS, 3 pts had + cytology (all MBC), 2 pts had - cytology and 1 pt with MBC was not tested by cytology. Of the 3 pts with + CSF by cytology (all MBC), all were detected by TS (Table). Among 5 MBC pts with CTCs present in CSF, ER status was concordant in 2 of 5 (40%). HER2 status was concordant in 3 of 4 (75%) evaluable pts and not determined in 1 pt. Analysis of cfDNA from CSF identified somatic mutations in 3 pts (TP53, PIK3CA, CCND1, respectively). In 1 of 3 pts, the mutation identified in the CSF (PIK3CA) in HR+/HER2- MBC was also identified in the blood. Conclusions: TargetSelector is a viable platform for the detection of breast cancer CTCs in the CSF. NGS performed on CSF samples can identify potentially actionable mutations. [Table: see text]
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Ratinier, Maxime, Steeve Boulant, Christophe Combet, Paul Targett-Adams, John McLauchlan, and Jean-Pierre Lavergne. "Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1." Journal of General Virology 89, no. 7 (July 1, 2008): 1569–78. http://dx.doi.org/10.1099/vir.0.83614-0.
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Since the first report of frameshifting in HCV-1, its sequence has been the paradigm for examining the mechanism that directs alternative translation of the hepatitis C virus (HCV) genome. The region encoding the core protein from this strain contains a cluster of 10 adenines at codons 8–11, which is thought to direct programmed ribosomal frameshifting (PRF), but formal evidence for this process has not been established unequivocally. To identify the mechanisms of frameshifting, this study used a bicistronic dual luciferase reporter system in a coupled transcription/translation in vitro assay. This approach revealed +1 as well as –1 frameshifting, whereas point mutations, selectively introduced between codons 8 and 11, demonstrated that PRF did not readily account for frameshifting in strain HCV-1. Sequence analysis of cDNAs derived from RNA transcribed by T7 RNA polymerase in the dual luciferase reporter system, as well as in both a subgenomic replicon and an infectious clone derived from strain JFH1, identified additions and deletions of adenines between codons 8 and 11 due to transcriptional slippage (TS). Moreover, RNA isolated from cells infected with virus generated by JFH1 containing the A-rich tract also contained heterogeneity in the adenine sequence, strongly suggesting TS by the NS5B viral polymerase. These findings have important implications for insight into frameshifting events in HCV-1 and demonstrate for the first time the involvement of transcriptional slippage in this recoding event.
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Tomikawa, Junko, Shuji Takada, Kohji Okamura, Miho Terao, Hiroko Ogata-Kawata, Hidenori Akutsu, Satoshi Tanaka, Kenichiro Hata, and Kazuhiko Nakabayashi. "Exploring trophoblast-specific Tead4 enhancers through chromatin conformation capture assays followed by functional screening." Nucleic Acids Research 48, no. 1 (November 28, 2019): 278–89. http://dx.doi.org/10.1093/nar/gkz1034.
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Abstract Tead4 is critical for blastocyst development and trophoblast differentiation. We assayed long-range chromosomal interactions on the Tead4 promoter in mouse embryonic stem (ES) cells and trophoblast stem (TS) cells. Using luciferase reporter assays with ES and TS cells for 34 candidate enhancer regions, we identified five genomic fragments that increased Tead4 promoter activity in a TS-specific manner. The five loci consisted of three intra- and two inter-chromosomal loci relative to Tead4 on chromosome 6. We established five mouse lines with one of the five enhancer elements deleted and evaluated the effect of each deletion on Tead4 expression in blastocysts. By quantitative RT-PCR, we measured a 42% decrease in Tead4 expression in the blastocysts with a homozygous deletion with a 1.5 kb genomic interval on chromosome 19 (n = 14) than in wild-type blastocysts. By conducting RNA-seq analysis, we confirmed the trans effect of this enhancer deletion on Tead4 without significant cis effects on its neighbor genes at least within a 1.7 Mb distance. Our results demonstrated that the genomic interval on chromosome 19 is required for the appropriate level of Tead4 expression in blastocysts and suggested that an inter-chromosomal enhancer-promoter interaction may be the underlying mechanism.
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Sablina, A. A., G. V. Ilyinskaya, S. N. Rubtsova, L. S. Agapova, P. M. Chumakov, and B. P. Kopnin. "Activation of p53-mediated cell cycle checkpoint in response to micronuclei formation." Journal of Cell Science 111, no. 7 (April 1, 1998): 977–84. http://dx.doi.org/10.1242/jcs.111.7.977.
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Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chromosomes. In order to better define the role of p53 function in maintaining genome integrity we investigated the involvement of p53 in the control of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensitive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) we showed that inhibition of p53 function increases the frequency of cells with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei the proportion of p53-positive cells is considerably higher among micronucleated variants as compared with their mononuclear counterparts. Analysis of 12(1)ConA cells expressing the beta-galactosidase reporter gene under the control of a p53-responsive promoter showed activation of p53-regulated transcription in the cells with micronuclei. Importantly, the percentage of cells manifesting specific p53 activity in colcemid-treated cultures increased with an augmentation of the number of micronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was determined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorporation by the cells with micronuclei and their mononuclear counterparts. Inhibition of p53 function in the cells with tetracycline-regulated p53 gene expression, as well as in the cells expressing ts-p53 or p53-GSE, abolished cell cycle arrest in micronucleated cells. These results along with the data showing no increase in the frequency of chromosome breaks in REF52 cells after colcemid treatment suggest the existence of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome segregation during mitosis.
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Prima, Victor, Lia Gore, Aimee Caires, Theresa Boomer, Miyako Yoshinari, Imaizume Masue, Varella-Garcia Marileila, and Stephen P. Hunger. "Chimeric MEF2D and Dazap1 Fusion Proteins Are Created by a Variant t(1;19)(q23;p13.3) in Acute Lymphoblastic Leukemia (ALL)." Blood 104, no. 11 (November 16, 2004): 548. http://dx.doi.org/10.1182/blood.v104.11.548.548.
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Abstract The t(1;19)(q23;p13) is one of the most common chromosome translocations in ALL. In 90–95% of ALL cases with a t(1;19), the 19p13.3 gene E2A is fused to PBX1 located at 1q23, producing E2A-PBX1 chimeric proteins that possess transforming properties. The molecular abnormalities present in the 5–10% of ALL cases with a t(1;19) but no E2A-PBX1 fusion are unknown. TS-2 is an ALL cell line with a t(1;19)(q23;p13.3) but no E2A-PBX1 fusion. We used fluoresence in situ hybridization to localize the chromosome 19 breakpoint in TS-2 to a region approximately 400 kilobases telomeric to E2A and found that the t(1;19) in TS-2 fuses the 19p13 gene DAZAP1 (deleted in azoospermia associated protein 1) to the 1q23 gene MEF2D (myocte enhancer factor 2D). We cloned and sequenced the fusion genes and found they encode for reciprocal in-frame DAZAP1/MEF2D and MEF2D/DAZAP1 fusion transcripts, both of which are expressed in TS-2. MEF2D is a member of the MEF2 family of DNA binding proteins, which were originally characterized as muscle-specific transcription factors that regulated transcription of genes involved in myogenic differentiation. MEF2 proteins are now recognized to have more diverse functions: they are transcriptional effectors of mitogenic signaling pathways and inflammation, play critical roles in calcium-regulated signaling pathways that mediate survival of neurons and T-lympocytes, and participate in neuronal plasticity. DAZAP1 is a protein with novel RNA binding properties that is expressed most abundantly in testis and to a lesser extent in thymus. MEF2D-DAZAP1 includes the MEF2D MADS (MCM1, agamous, deficiens, and serum response factor) box and adjacent MEF2D domain that mediate sequence-specific DNA binding and protein-protein interactions, as well as one of two MEF2D transcriptional activation domains (TAD) fused to the C-terminus of DAZAP1. The DAZAP1-MEF2D chimera includes an intact first and truncated second RNA recognition motif from DAZAP1 joined to the C-terminus of MEF2D that includes its second TAD. We performed electrophoretic mobility shift assays using cognate and mutant MEF2D DNA recognition sites and found that MEF2D/DAZAP1 binds avidly and specifically to DNA in a manner indistinguishable from that of native MEF2D. We found that MEF2D/DAZAP1 activated transcription of a luciferase reporter gene under control of MEF2D recognition elements with substantially more potency than did wild type MEF2D. We also show that DAZAP1/MEF2D proteins bind RNA in a sequence specific manner analogous to that of wild type DAZAP1. MEF2D has been identified as a candidate oncogene involved in development of leukemia/lymphoma via murine retroviral insertional mutagenesis studies. Our data implicate MEF2D in human cancer and suggest that MEF2D/DAZAP1 and/or DAZAP1/MEF2D contributes to leukemogenesis by altering signaling pathways normally regulated by wild type MEF2D and DAZAP1.
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Zhou, Z., and S. J. Elledge. "Isolation of crt mutants constitutive for transcription of the DNA damage inducible gene RNR3 in Saccharomyces cerevisiae." Genetics 131, no. 4 (August 1, 1992): 851–66. http://dx.doi.org/10.1093/genetics/131.4.851.
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Abstract Ribonucleotide reductase is an essential enzyme that catalyzes the rate limiting step for production of the deoxyribonucleotides required for DNA synthesis. It is encoded by three genes, RNR1, RNR2 and RNR3, each of which is inducible by agents that damage DNA or block DNA replication. To probe the signaling pathway mediating this DNA damage response, we have designed a general selection system for isolating spontaneous trans-acting mutations that alter RNR3 expression using a chromosomal RNR3-URA3 transcriptional fusion and an RNR3-lacZ reporter plasmid. Using this system, we have isolated 202 independent trans-acting crt (constitutive RNR3 transcription) mutants that express high levels of RNR3 in the absence of DNA damaging agents. Of these, 200 are recessive and fall into 9 complementation groups. In some crt groups, the expression of RNR1 and RNR2 are also elevated, suggesting that all three RNR genes share a common regulatory pathway. Mutations in most CRT genes confer additional phenotypes, among these are clumpiness, hydroxyurea sensitivity, temperature sensitivity and slow growth. Five of the CRT genes have been identified as previously cloned genes; CRT4 is TUP1, CRT5 is POL1/CDC17, CRT6 is RNR2, CRT7 is RNR1, and CRT8 is SSN6. crt6-68 and crt7-240 are the first ts alleles of RNR2 and RNR1, respectively, and arrest with a large budded, cdc terminal phenotype at the nonpermissive temperature. The isolation of crt5-262, an additional cdc allele of POL1/CDC17, suggests for the first time that directly blocking DNA replication can provide a signal to induce the DNA damage response. crt2 mutants show a defect in basal level expression of RNR1-lacZ reporter constructs. These are the first mutants isolated in yeast that alter the regulation of DNA damage inducible genes and the identification of their functions sheds light on the DNA damage sensory network.
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Chang, Hye Jung, Moon Young Choi, Min-Hee Cho, Kyoung Eun Lee, and Soon-Nam Lee. "Molecular mechanism of chemoresistance and restoration in human gastric cancer cells." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e15544-e15544. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e15544.
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e15544 Background: Gastric cancer is characterized by a high rate of relapse and failure of chemotherapy because of the emergence of drug resistant cells. Hence, resistance to chemotherapy is a major obstacle for the management of gastric cancer. It might be related with the development of cancer stem cells (CSCs). The aim of this study is to investigate the characteristics of the 5-fluorouracil (FU) resistant gastric cancer and to study how to restore the chemosensitivity. Methods: We used the AGS gastric cancer cell line (AGS) and transformed it into a 5-FU resistant cell line (AGS-R). AGS-R was established by continuous exposure of the cells to progressively increasing concentrations of 5-FU for about 1 year and modulating mRNA expression levels of four genes associated with thymidylate synthase (TS). The research methods used were MTT assay, flow cytometry analysis, luciferase reporter assay, western blotting, and siRNA transfection. Results: 5-FU-resistant gastric cancer cell, AGS-R was established by continuous exposure to 5-FU with gradual increase of its concentration for about 1 year. Comparing with AGS, thymidylate synthase (TS) expression in AGS-R was highly increase at transcriptional and translational level. And decreased transcriptional activity of β-catenin in AGS-R was confirmed by Western blotting and luciferase assay. Even though treatment with ICG-001, an inhibitor of β-catenin, showed growth inhibition of AGS in a dose- and time-dependent manner, but it could not in AGS-R. The expression of CD44, well-known CSCs marker, was significantly higher on AGS-R compared to AGS. And the Notch pathway was remarkably up-regulated. Flow cytometry analysis revealed that CD44 expression was reduced on AGS-R transfected with specific siRNA for notch intracellular domain (NICD). Conclusions: This study suggests that chemoresistance of gastric cancer against continuous exposure to 5-FU is associated with decrease of β-catenin activity and development of stemness via the activation of Notch pathway. Therefore, the inhibition of Notch pathway might be a potential therapeutic target in 5-FU-resistant gastric cancer.
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Gadal, Olivier, Daniela Strauss, Elisabeth Petfalski, Pierre-Emmanuel Gleizes, Nicole Gas, David Tollervey, and Ed Hurt. "Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing." Journal of Cell Biology 157, no. 6 (June 10, 2002): 941–52. http://dx.doi.org/10.1083/jcb.200111039.
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Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.
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Akada, Rinji, Lorena Kallal, Douglas I. Johnson, and Janet Kurjan. "Genetic Relationships Between the G Protein βγ Complex, Ste5p, Ste20p and Cdc42p: Investigation of Effector Roles in the Yeast Pheromone Response Pathway." Genetics 143, no. 1 (May 1, 1996): 103–17. http://dx.doi.org/10.1093/genetics/143.1.103.
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Abstract The Saccharomyces cerevisiae G protein βγ dimer, Ste4p/Ste18p, acts downstream of the a subunit, Gpalp, to activate the pheromone response pathway and therefore must interact with a downstream effector. Synthetic sterile mutants that exacerbate the phenotype of ste4-ts mutations were isolated to identify proteins that functionally interact with Ste4p. The identification of a stel8 mutant indicated that this screen could identify proteins that interact directly with Ste4p. The other mutations were in STE5 and the STE20 kinase gene, which act near Ste4p in the pathway, and a new gene called STE21. ste20 null mutants showed residual mating, suggesting that another kinase may provide some function. Overexpression of Ste5p under galactose control activated the pheromone response pathway. This activation was dependent on Ste4p and Ste18p and partially dependent on Ste20p. These results cannot be explained by the linear pathway of Ste4p → Ste20p → Ste5p. Overexpression of Cdc42p resulted in a slight increase in pheromone induction of a reporter gene, and overexpression of activated forms of Cdc42p resulted in a further twofold increase. Mutations in pheromone response pathway components did not suppress the lethality associated with the activated CDC42 mutations, suggesting that this effect is independent of the pheromone response pathway.
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Gil-Varea, Elia, Maria Fedetz, Herena Eixarch, Nino Spataro, Luisa María Villar, Elena Urcelay, Albert Saiz, et al. "A New Risk Variant for Multiple Sclerosis at 11q23.3 Locus Is Associated with Expansion of CXCR5+ Circulating Regulatory T Cells." Journal of Clinical Medicine 9, no. 3 (February 26, 2020): 625. http://dx.doi.org/10.3390/jcm9030625.
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Genome-wide association studies and meta-analysis have contributed to the identification of more than 200 loci associated with multiple sclerosis (MS). However, a proportion of MS heritability remains unknown. We aimed to uncover new genetic variants associated with MS and determine their functional effects. For this, we resequenced the exons and regulatory sequences of 14 MS risk genes in a cohort of MS patients and healthy individuals (n = 1070) and attempted to validate a selection of signals through genotyping in an independent cohort (n = 5138). We identified three new MS-associated variants at C-X-C motif chemokine receptor 5 (CXCR5), Ts translation elongation factor, mitochondrial (TSFM) and cytochrome P450 family 24 subfamily A member 1 (CYP24A1). Rs10892307 resulted in a new signal at the CXCR5 region that explains one of the associations with MS within the locus. This polymorphism and three others in high linkage disequilibrium mapped within regulatory regions. Of them, rs11602393 showed allele-dependent enhancer activity in the forward orientation as determined by luciferase reporter assays. Immunophenotyping using peripheral blood mononuclear cells from MS patients associated the minor allele of rs10892307 with increased percentage of regulatory T cells expressing CXCR5. This work reports a new signal for the CXCR5 MS risk locus and points to rs11602393 as the causal variant. The expansion of CXCR5+ circulating regulatory T cells induced by this variant could cause its MS association.
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Ao, Wanyuan, and Dave Pilgrim. "Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a." Journal of Cell Biology 148, no. 2 (January 24, 2000): 375–84. http://dx.doi.org/10.1083/jcb.148.2.375.
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In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B–dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.
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Falcone, G., S. Alemà, and F. Tatò. "Transcription of muscle-specific genes is repressed by reactivation of pp60v-src in postmitotic quail myotubes." Molecular and Cellular Biology 11, no. 6 (June 1991): 3331–38. http://dx.doi.org/10.1128/mcb.11.6.3331.
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Quail myogenic cells infected with temperature sensitive (ts) mutants of Rous sarcoma virus (RSV) exhibit a temperature-dependent transformation and block of differentiation. When the cells are allowed to differentiate at the restrictive temperature (41 degrees C) and then shifted back to the permissive temperature (35 degrees C), a sharp reduction in the accumulation of muscle-specific mRNAs is observed, following reactivation of the transforming protein pp60v-src. A kinetic analysis of this down-regulation reveals that the reduction in the accumulation of muscle-specific transcripts occurs fairly rapidly within 6 to 20 h after the shift back, depending on the mRNA analyzed. Studies on transcription of endogenous muscle-specific genes and a transfected chloramphenicol acetyltransferase reporter gene under the control of muscle-specific promoters, at the different temperatures, suggest that the oncogene exerts its control mainly at the transcriptional level. On the contrary, transcription of the CMD1 gene, the avian homolog of the mouse muscle regulatory MyoD gene, is not significantly affected by the oncogene both in proliferating myoblasts and in myotubes shifted back to 35 degrees C. These findings are consistent with the conclusion that v-src blocks myogenesis by controlling transcription of muscle-specific genes independently of cell proliferation. Furthermore, they suggest the existence of an alternative pathway, not requiring the silencing of CMD1 transcription, through which the oncogene exerts its effect.
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Falcone, G., S. Alemà, and F. Tatò. "Transcription of muscle-specific genes is repressed by reactivation of pp60v-src in postmitotic quail myotubes." Molecular and Cellular Biology 11, no. 6 (June 1991): 3331–38. http://dx.doi.org/10.1128/mcb.11.6.3331-3338.1991.
Full textAbstract:
Quail myogenic cells infected with temperature sensitive (ts) mutants of Rous sarcoma virus (RSV) exhibit a temperature-dependent transformation and block of differentiation. When the cells are allowed to differentiate at the restrictive temperature (41 degrees C) and then shifted back to the permissive temperature (35 degrees C), a sharp reduction in the accumulation of muscle-specific mRNAs is observed, following reactivation of the transforming protein pp60v-src. A kinetic analysis of this down-regulation reveals that the reduction in the accumulation of muscle-specific transcripts occurs fairly rapidly within 6 to 20 h after the shift back, depending on the mRNA analyzed. Studies on transcription of endogenous muscle-specific genes and a transfected chloramphenicol acetyltransferase reporter gene under the control of muscle-specific promoters, at the different temperatures, suggest that the oncogene exerts its control mainly at the transcriptional level. On the contrary, transcription of the CMD1 gene, the avian homolog of the mouse muscle regulatory MyoD gene, is not significantly affected by the oncogene both in proliferating myoblasts and in myotubes shifted back to 35 degrees C. These findings are consistent with the conclusion that v-src blocks myogenesis by controlling transcription of muscle-specific genes independently of cell proliferation. Furthermore, they suggest the existence of an alternative pathway, not requiring the silencing of CMD1 transcription, through which the oncogene exerts its effect.
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Da Costa, Bruno, Alix Sausset, Sandie Munier, Alexandre Ghounaris, Nadia Naffakh, Ronan Le Goffic, and Bernard Delmas. "Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation." Journal of Virology 89, no. 12 (April 8, 2015): 6376–90. http://dx.doi.org/10.1128/jvi.00589-15.
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ABSTRACTThe influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. Thetsphenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed thattsand lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and β-importin IPO5 tagged with fragments of theGaussia princepsluciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Twotsmutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a “hot spot” to engineertsmutants that could be used to design novel attenuated vaccines.IMPORTANCEBy targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number oftsmutations were engineered in such a short domain, demonstrating that rational design oftsmutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of thesetsmutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs.
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Zee, Rebecca S., Evaristus C. Mbanefo, Loc H. Le, Luke F. Pennington, Justin I. Odegaard, Theodore S. Jardetzky, Abdulaziz Alouffi, Jude Akinwale, Franco H. Falcone, and Michael H. Hsieh. "IPSE, a parasite-derived host immunomodulatory protein, is a potential therapeutic for hemorrhagic cystitis." American Journal of Physiology-Renal Physiology 316, no. 6 (June 1, 2019): F1133—F1140. http://dx.doi.org/10.1152/ajprenal.00468.2018.
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Chemotherapy-induced hemorrhagic cystitis is characterized by bladder pain and voiding dysfunction caused by hemorrhage and inflammation. Novel therapeutic options to treat hemorrhagic cystitis are needed. We previously reported that systemic administration of the Schistosomiasis hematobium-derived protein H-IPSEH06 (IL-4-inducing principle from Schistosoma mansoni eggs) is superior to three doses of MESNA in alleviating hemorrhagic cystitis (Mbanefo EC, Le L, Pennington LF, Odegaard JI, Jardetzky TS, Alouffi A, Falcone FH, Hsieh MH. FASEB J 32: 4408–4419, 2018). Based on prior reports by others on S. mansoni IPSE (M-IPSE) and additional work by our group, we reasoned that H-IPSE mediates its effects on hemorrhagic cystitis by binding IgE on basophils and inducing IL-4 expression, promoting urothelial proliferation, and translocating to the nucleus to modulate expression of genes implicated in relieving bladder dysfunction. We speculated that local bladder injection of the S. hematobium IPSE ortholog IPSEH03, hereafter called H-IPSEH03, might be more efficacious in preventing hemorrhagic cystitis compared with systemic administration of IPSEH06. We report that H-IPSEH03, like M-IPSE and H-IPSEH06, activates IgE-bearing basophils in a nuclear factor of activated T-cells reporter assay, indicating activation of the cytokine pathway. Furthermore, H-IPSEH03 attenuates ifosfamide-induced increases in bladder wet weight in an IL-4-dependent fashion. H-IPSEH03 relieves hemorrhagic cystitis-associated allodynia and modulates voiding patterns in mice. Finally, H-IPSEH03 drives increased urothelial cell proliferation, suggesting that IPSE induces bladder repair mechanisms. Taken together, H-IPSEH03 may be a potential novel therapeutic to treat hemorrhagic cystitis by basophil activation, attenuation of allodynia, and promotion of urothelial cell proliferation.
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Porcu, P., A. Ferber, Z. Pietrzkowski, C. T. Roberts, M. Adamo, D. LeRoith, and R. Baserga. "The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor." Molecular and Cellular Biology 12, no. 11 (November 1992): 5069–77. http://dx.doi.org/10.1128/mcb.12.11.5069.
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We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).
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Porcu, P., A. Ferber, Z. Pietrzkowski, C. T. Roberts, M. Adamo, D. LeRoith, and R. Baserga. "The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor." Molecular and Cellular Biology 12, no. 11 (November 1992): 5069–77. http://dx.doi.org/10.1128/mcb.12.11.5069-5077.1992.
Full textAbstract:
We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).
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Meyer, Léa, Alix Sausset, Laura Sedano, Bruno Da Costa, Ronan Le Goffic, and Bernard Delmas. "Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses." Journal of Virology 90, no. 7 (January 20, 2016): 3684–93. http://dx.doi.org/10.1128/jvi.03101-15.
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ABSTRACTThe influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA–PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for furtherin vitroandin vivocharacterization. Four viable mutants with single-codon deletions were generated; all of them exhibited atsphenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serialin vitropassages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stabletsmutants that could be used to design novel attenuated vaccines.IMPORTANCEIn order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA–PB1 dimer to the nucleus, where viral replication occurs. Thesetsdeletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.
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Zhong, Hua, Chun Wang, Chen Fangyuan, Lan Xu, Jihua Zhong, and HU Jiong. "MiR-146a Expression Level As a Molecular Marker in Acute Promyleocytic Leukemia." Blood 120, no. 21 (November 16, 2012): 4821. http://dx.doi.org/10.1182/blood.v120.21.4821.4821.
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Abstract Abstract 4821 MiR-146a which is located on chromosomes 5 may be involved in normal hemtopoisis and the pathogenesis of several hematopoietic diseases through inhibiting the expression of its targets. Acute promyelocytic leukemia (APL) cell line NB4 was taken as research model to find out whether miR-146a expression was associated to cell differentiation and proliferation. The miR-146a expression level of NB4 cells decreased after ATRA treatment, meanwhile the expression level of its predicted target gene Smad4 increased simultaneously. MiR-146a expression plasmid, no related negative control sequence plasmid and recombined luciferase reporter gene vector containing the miR-146a complementary combined site on Smad4 plasmid were constructed by DNA recombination technical. Both recombined plasmids were transiently co-transfected into 293T cells with control plasmid pRL-TK. The interaction between miR-146a and its predicted target gene Smad4 was confirmed by the relative activities of dual luciferase detection. This interaction relationship was further confirmed by liposome entrapment transfection of miR-146a and its antisense molecular in NB4 cells. The expression level of miR-146a in NB4 cells did no influence on Smad4 gene expression but Smad4 protein expression levels (fig 1). The changes of NB4 cells on biological behaviors were observed by cell morphology, proliferation curve and flow cytometry methods under different miR-146a expression status. The results showed that cell proliferation ratio decreased and cell apoptosis ratio increased after miR-146a expression suppression in NB4 cells. The investigation of miR-146a expression levels in mononuclear cells of APL patients' bone marrow and clinical data were carried out. It was showed that the expression levels of miR-146a were higher in APL patients than normal controls. The expression level of miR-146a was positive correlated with peripheral blood white cell count (fig 2). MiR-146a expression level was negative correlated with Th/Ts ratio among these patients (fig 3). MiR-146a/Smad4 signal pathway is expressed in acute promyelocytic leukemia. The proliferation and apoptosis of the cells are modified by the Smad4 protein expression regulated by miR-146a expression. MiR-146a expression is somewhat associated with acknowledged clinical prognosis factors. It has potential possibility to take miR-146a as prognosis factor. Disclosures: No relevant conflicts of interest to declare.
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Asare, Emmanuel, JoAnn Mugavero, Ping Jiang, Eckard Wimmer, and Aniko V. Paul. "A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPaseCauses Conditional Defects in Encapsidation and Uncoating." Journal of Virology 90, no. 14 (April 13, 2016): 6174–86. http://dx.doi.org/10.1128/jvi.02877-15.
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ABSTRACTThe specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252of poliovirus 2CATPaseinteracts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPasehas important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an α helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus.In vivoprotein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2CATPase(K259A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection.IMPORTANCEEnterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackieviruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2CATPase. In this study, we have searched for residues in poliovirus 2CATPase, near a presumed capsid-interacting site, important for encapsidation. An unusual cold-sensitive mutant of 2CATPasepossessed a defect in encapsidation at 37°C and subsequently in uncoating during the next cycle of infection at 33°C. These studies not only reveal a new site in 2CATPasethat is involved in encapsidation but also identify a link between encapsidation and uncoating.
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Krabbendam, Hans. "Elke van Cassel, A Cold War Magazine of Causes: A critical history of the Reporter, 1949-1968." Tijdschrift voor Tijdschriftstudies, no. 23 (June 1, 2008): 47. http://dx.doi.org/10.18352/ts.232.
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Pardee, Timothy, Evan Gomes, Jamie Jennings-Gee, David L. Caudell, and William Gmeiner. "The Novel Fluoropyrimidine FdUMP[10] Is Highly Active Against Acute Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 3302. http://dx.doi.org/10.1182/blood.v116.21.3302.3302.
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Abstract Abstract 3302 Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that leads to marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of research, therapy remains essentially unchanged and outcomes are poor. In patients over the age of 60 less then 10% of patients survive 5 years from diagnosis. There is a desperate need for the identification of new active agents with favorable toxicity profiles. The novel polymeric fluoropyrimidine (FP) FdUMP[10] is an oligodeoxynucleotide pro-drug of the thymidylate synthase (TS)-inhibitory FP metabolite 5-fluoro-2'-deoxyuridine-5`-O-monophosphate (FdUMP). The observation that this compound was highly active against several leukemia lines in the NCI 60 cell line screen prompted us to evaluate its activity in several preclinical models of AML. In vitro, FdUMP[10] exhibited remarkable activity against 3 human acute leukemia cell lines, HL60, Jurkat and THP-1, with IC50 values of 3.378 nM (95% CI 2.984 to 3.825), 5.438 nM (4.609 to 6.417) and 4.093 nM (3.413 to 4.907) respectively. We next tested its efficacy against a more genetically defined murine model of AML driven by expression of MLL-ENL. FdUMP[10] exhibited even greater activity against all murine lines tested. The IC50 values of FdUMP[10] against two MLL-ENL driven murine AML cell lines were 214 pM (95%CI 178.9 to 255.9) and 292.3 pM (251.8 to 339.4). The IC50 values observed for FdUMP[10] for all the murine lines tested were lower than both Ara-C (30-40 nM) and doxorubicin (2-4 nM). We then determined the cytotoxic mechanism for FdUMP[10] in vitro. Upon treatment with FdUMP[10] both the human and murine cell lines undergo extensive apoptosis as indicated by Annexin V and propidium iodide staining. Treated cells developed γH2AX foci, rapid and complete TS inhibition and display trapped Topoisomerase I (Topo I) cleavage complexes. FdUMP[10]-mediated induction of apoptosis was p53 independent as murine AML cells that had p53 knocked down by RNAi demonstrated resistance to both Ara-C and doxorubicin, but not to FdUMP[10]. We next tested the efficacy of FdUMP[10] in vivo. The MLL-ENL driven murine AML model results in blasts that can be transplanted into sublethally irradiated, immunocompetent, syngeneic recipients. The recipients develop a fatal and therapy-resistant AML. Lines were generated that expressed a luciferase reporter. Animals were imaged 6–7 days after injection of the leukemias to ensure engraftment and then began treatment with either the combination of Ara-C plus doxorubicin, single-agent FdUMP[10], or observation. Studies were performed using 2 doses of FdUMP[10] at 150 or 300 mg/kg injected on days 1 and 3 and compared to animals treated with 100 mg/kg Ara-C and 3mg/kg doxorubicin injected on days 1 through 5. Both treatments resulted in a statistically significant survival advantage over observation. A preliminary toxicology study compared FdUMP[10], 150 mg/kg daily, to 5-fluorouracil (5 FU), 150 mg/kg daily, or the combination of Ara-C at 100 mg/kg plus doxorubicin at 3 mg/kg daily. All groups were treated for 3, 4 or 5 days. On day 6 animals were sacrificed and organs harvested, sectioned, and stained. Slides were then reviewed by a veterinary pathologist. Tissues most affected were the small intestine, colon, and the bone marrow. The 5FU-treated animals had severe villous blunting and fusion with crypt necrosis in both large and small intestine. In contrast, FdUMP[10]-treated animals had only mild crypt epithelial apoptosis with mitoses. The 5 FU and Ara-C plus doxorubicin groups had a severe pan-cytopenia in the marrow compared to FdUMP[10] treated animals that showed only minimal to mild apoptosis. These data support the assertion that FdUMP[10] has lower toxicity then either Ara-C plus doxorubicin or identically dosed 5 FU. In summary FdUMP[10] exhibited remarkable activity against AML cells in vitro and in vivo. Additionally, FdUMP[10] had decreased toxicity compared to treatment with either single agent 5 FU or combination treatment with Ara-C plus doxorubicin. Disclosures: Gmeiner: Salzburg Therapeutics: Equity Ownership.
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Ocias, Lukas Frans, Dennis Lund Hansen, Thomas Kielsgaard Kristensen, Karin de Stricker, Daniel El Fassi, Jesper Stentoft, Jørn Starklint, et al. "No Development of Neutralizing Antibodies Against Recombinant Interferon-Alpha in Ph-Negative Myeloproliferative Neoplasms - a Prospective Study." Blood 126, no. 23 (December 3, 2015): 5177. http://dx.doi.org/10.1182/blood.v126.23.5177.5177.
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Abstract Background Treatment of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPNs) with recombinant pegylated interferon alpha2a/b (rIFN-alpha) has proven effective. It is well known that prolonged therapy with recombinant type 1 interferons (IFN-alpha and IFN-beta) may induce neutralizing antibodies (nAbs) against the drug leading to treatment failure. Most data on type 1 IFN immunogenicity are available from studies of patients with multiple sclerosis treated with rIFN-beta, and patients with hepatitis C treated with rIFN-alpha. A few reports have demonstrated nAbs in MPN patients not responding adequately to rIFN-alpha treatment, but the phenomenon has not been thoroughly investigated in MPNs. Patients and Methods Newly diagnosed patients with MPNs enrolled in the Danish multicenter trial - DALIAH (Low-dose rIFN-alpha versus Hydroxyurea in The Treatment of Ph-Negative MPNs) were included. Patients were randomized to treatment with either rIFN-alpha 2a or 2b at a starting dose of 45 and 35 mikrograms once weekly, respectively. The occurrence of neutralizing Abs (nAbs) against rIFN-alpha was investigated at baseline, month 12 and month 24 by reporter gene assays (iLiteTM alphabeta and iLiteTM antialpha, Biomonitor A/S, Copenhagen, Denmark). JAK2 V617F quantitative mutation analyses were performed as previously described (Larsen TS, BJH 2007). Statistical analyses were performed using STATA version 9.0. Results Ninety-two patients on sustained treatment with rIFN-alpha2a (n=48) and rIFN-alpha2b (n=44) for 12 months were analyzed for this study. Forty-five patients had ET, 39 patients had PV and 8 patients had proliferative PMF. Thirty-six out of 39 (92%) PV patients, 22 out of 45 (49%) ET patients and 4 out of 8 (50%) PMF patients were JAK2V617F mutated. Hematological responses at 12 months: ET: 67% CR, 29 % PR; PV: 64% CR, 31% PR (ELN 2009 criteria); PMF: 50% had at least a minor response (EUMNET). The median serum concentration of bioactive IFN-alpha at 12 months was 12,4 (range <2,4-86,4) and 2,6 (range <2,4-12,8) IU/mL serum, for patients treated with rIFN-alpha 2a and -2b respectively. No significant association between hematological or molecular response and serum IFN-alpha activity was found. Serum from 92 patients was analyzed at 12 months and 33 patients were analyzed at both 12 and 24 months and no occurrence of nAbs was seen during treatment. Twenty-four patients had pre-treatment levels of IFN nAbs measured. Notably, one patient was tested positive for the presence of nAbs before rIFN-alpha exposure. This autoAb-positive patient remained positive throughout the study and has shown low IFN serum activity (< 2,4 IU/mL) and only partial hematological and molecular response after 24 months of treatment. Conclusions Development of nAbs in MPN patients completing treatment for 12 months with rIFN-alpha seems exceedingly rare as no patients, neither complete responders nor patients not meeting criteria for complete hematological remission developed nAbs after 12 (24) months of therapy. Its apparent rarity does not justify a routine investigation of nAbs in patients not responding to rIFN-alpha treatment. There was no significant correlation between serum concentration of rIFN-alpha 2a and -2b and clinical or molecular responses. The intriguing finding that one of 24 patients had pre-existing cross reacting nAbs against rIFN-alpha 2a and 2b before commencing rIFN-alpha treatment is interesting and was associated with an insufficient response. Disclosures Off Label Use: Recombinant interferon-alpha 2a and -2b in the treatment of chronic Philadelphia-negative myeloproliferative neoplasms.. El Fassi:Novartis Denmark: Honoraria, Other: Have conducted an educational session for Novartis Denmark, regarding MPNs and ruxolitinib, for this a honorarium was received.. Bjerrum:Bristoll Myers Squibb, Novartis and Pfizer: Other: educational activities. Hasselbalch:Novartis: Research Funding. Bendtzen:Pfizer: Honoraria; Eurodiagnostica AB: Equity Ownership; Novo-Nordisk: Equity Ownership.
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Li, Chuanling, Jian-Xiu Shang, Chenlei Qiu, Baowen Zhang, Jinxue Wang, Shuo Wang, and Yu Sun. "Plastid-Localized EMB2726 Is Involved in Chloroplast Biogenesis and Early Embryo Development in Arabidopsis." Frontiers in Plant Science 12 (July 23, 2021). http://dx.doi.org/10.3389/fpls.2021.675838.
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Embryogenesis is a critical developmental process that establishes the body organization of higher plants. During this process, the biogenesis of chloroplasts from proplastids is essential. A failure in chloroplast development during embryogenesis can cause morphologically abnormal embryos or embryonic lethality. In this study, we isolated a T-DNA insertion mutant of the Arabidopsis gene EMBRYO DEFECTIVE 2726 (EMB2726). Heterozygous emb2726 seedlings produced about 25% albino seeds with embryos that displayed defects at the 32-cell stage and that arrested development at the late globular stage. EMB2726 protein was localized in chloroplasts and was expressed at all stages of development, such as embryogenesis. Moreover, the two translation elongation factor Ts domains within the protein were critical for its function. Transmission electron microscopy revealed that the cells in emb2726 embryos contained undifferentiated proplastids and that the expression of plastid genome-encoded photosynthesis-related genes was dramatically reduced. Expression studies of DR5:GFP, pDRN:DRN-GFP, and pPIN1:PIN1-GFP reporter lines indicated normal auxin biosynthesis but altered polar auxin transport. The expression of pSHR:SHR-GFP and pSCR:SCR-GFP confirmed that procambium and ground tissue precursors were lacking in emb2726 embryos. The results suggest that EMB2726 plays a critical role during Arabidopsis embryogenesis by affecting chloroplast development, possibly by affecting the translation process in plastids.
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Tan, Bibo, Yong Li, Qun Zhao, Liqiao Fan, and Dong Wang. "ZNF139 increases multidrug resistance in gastric cancer cells by inhibiting miR-185." Bioscience Reports 38, no. 5 (September 5, 2018). http://dx.doi.org/10.1042/bsr20181023.
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It has been reported that the expression of zinc finger protein 139 (ZNF139) and microRNA-185 (miR-185) were associated with proliferation, drug resistance of gastric cancer (GC) cells. However, the detailed mechanisms have not been fully investigated. The expression of ZNF139 in both GC tissues and cell lines was tested, then SGC7901/ADR or SGC7901 cells were transfected with ZNF139-siRNA, miR-185 analog, or pcDNA-ZNF139. Cell activity was determined by MTT assay. Real-time PCR and Western blot were utilized to detect ZNF139, miR-185, and multidrug resistance (MDR) related genes including MDR1/P-gp, GST-π, MRP-1, Bcl-2, TS and Bax. ChIP and dual luciferase activity assay were used to investigate regulation between ZNF139 and miR-185. Increased ZNF139 and decreased miR-185 expression were detected in GC tissues and cell lines. Transfection with ZNF139-siRNA into SGC7901/ADR cells markedly increased expression of miR-185, and treating with chemotherapeutic drugs ADR, 5-FU, L-OHP, the survival rate of SGC7901/ADR cells obviously decreased after ZNF139-siRNA transfection. On the other hand, transfection with pcDNA-ZNF139 in GC cell line SGC7901 resulted in an increased expression level of ZNF139 and a decline in the expression level of miR-185, meanwhile drug resistance of GC cells was clearly enhanced to ADR, 5-FU, L-OHP. Dual luciferase activity assay demonstrated that ZNF139 inhibited transcriptional activities of miR-185’s promoter in cells transfected with the reporter plasmid encompassing the upstream promoter region of miR-185 along with pcDNA-ZNF139. Our data reveal that ZNF139 might promote MDR gene MDR1/P-gp, MRP-1 and Bcl-2 by prohibiting miR-185.