Effect of 5-Fluorouracil on the Regulation of Zebrafish Thymidylate Synthase Gene Expression

2013 ◽  
Vol 641-642 ◽  
pp. 732-735
Author(s):  
Chun Xia Song ◽  
Qian Zhang ◽  
Yu Liang Xiao
Keyword(s):  
Gene Expression ◽  
Thymidylate Synthase ◽  
De Novo ◽  
Mrna Level ◽  
Translation System ◽  
Transcriptional Level ◽  
Important Target ◽  
Reticulocyte Lysate ◽  
First Time ◽  
Translational Level

Thymidylate synthase (TS) is an important target in cancer therapy, which is a folate-dependent enyme, catalyzing the de novo synthesis of dUMP. In this report, the effect of 5-flurorouracil (5-FU) on the regulation of TS gene expression was estimated in zebrafish. The results showed 5-FU could significantly increase the TS expression in zebrafish embryos. However, TS mRNA level were remained unchanged. To determine the effect of 5-FU and 5-FdUMP on translation of TS mRNA, a rabbit reticulocyte lysate translation system was used. Addition of 5-FU, not inhibited the translation of TS mRNA. While addition of 5-FdUMP, completely repressed the translation of TS mRNA. Therefore, induced expression of thymidylate synthase by 5-FU in zebrafish occurred in translational level, not in transcriptional level. The findings demonstrated that zebrafish TS protein was able to bind to its own cogate mRNA and the 5-FU regulated TS in the translational level. This is the first time to confirm that the regulation of TS is affected by TS and its cognant mRNA interaction in the whole animal level.

Transcriptional regulation of the human CAD gene during myeloid differentiation

1987 ◽  
Vol 7 (5) ◽  
pp. 1961-1966
Author(s):  
G N Rao ◽  
E S Buford ◽  
J N Davidson
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Morphological Changes ◽  
Transcriptional Level ◽  
Carbamoyl Phosphate ◽  
Aspartate Transcarbamylase ◽  
Trans Retinoic Acid ◽  
Beta Actin ◽  
Cad Gene ◽  
Cad Protein

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.

Changes in Gene Expression during Drying in a Desiccation-Tolerant Grass Sporobolus stapfianus and a Desiccation-Sensitive Grass Sporobolus pyramidalis

1997 ◽  
Vol 24 (5) ◽  
pp. 617 ◽  
Author(s):  
D.F. Gaff ◽  
D. Bartels ◽  
J.L. Gaff
Keyword(s):  
Gene Expression ◽  
Desiccation Tolerance ◽  
Transcriptional Level ◽  
Novel Proteins ◽  
Sporobolus Stapfianus ◽  
Induced Changes ◽  
First Time ◽  
Dimensional Electrophoresis ◽  
Water Contents

For the first time in the grasses, a desiccation-tolerant species (Sporobolus stapfianus) was examined for evidence of drought-induced changes in gene transcription. Desiccation tolerance (the ability of this species to recover from a water potential of –540 MPa) is induced in the resurrection grass during the drying process itself. Specific mRNA was compared in extracts of air-dry, drying and fully hydrated leaves by comparisons of the encoded proteins translated in vitro and partitioned by 2- dimensional electrophoresis. Forty-one genes, that were not expressed in hydrated leaves, were transcribed during drying, whereas only 25 novel polypeptides (translated in vitro) were detected; this suggests that gene expression was controlled mainly at the transcriptional level, but possibly also at the translational level. Leaves of S. stapfianus become desiccation tolerant as they dry on intact plants with mechanically undisturbed roots, whereas leaves on plants whose roots have been disturbed die during drying. Complements of mRNA from live S. stapfianus leaves changed markedly from full hydration to 70% RWC and to air-dryness; they also differed markedly from drought-sensitive leaves (on plants with disturbed roots) at 70% RWC and dead air-dry S. stapfianus leaves and from leaves of the desiccation sensitive grass S. pyramidalis at the same water contents. Drought-induced injury could not be attributed to low abundance of mRNA in either species. Five criteria which might be involved in desiccation tolerance were applied to specific in vitro proteins of S. stapfianus; 12 novel proteins correlated with desiccation tolerance in a least four of the five criteria.

scholarly journals Murine Cytomegalovirus Stimulates Cellular Thymidylate Synthase Gene Expression in Quiescent Cells and Requires the Enzyme for Replication

2000 ◽  
Vol 74 (11) ◽  
pp. 4979-4987 ◽  
Author(s):  
Giorgio Gribaudo ◽  
Ludovica Riera ◽  
David Lembo ◽  
Marco De Andrea ◽  
Marisa Gariglio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Thymidylate Synthase ◽  
De Novo ◽  
Present Report ◽  
Murine Cytomegalovirus ◽  
Mcmv Infection ◽  
Quiescent Cells ◽  
Cellular Genes ◽  
Efficient Replication ◽  
Responsive Element

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.

scholarly journals The Expression of the PDZ Protein MALS-1/Velis Is Regulated by Calcium and Calcineurin in Cerebellar Granule Cells

2002 ◽  
Vol 277 (51) ◽  
pp. 49585-49590 ◽  
Author(s):  
Bastiano Sanna ◽  
Dana Kramer ◽  
Armando A. Genazzani
Keyword(s):  
Gene Expression ◽  
Neuronal Development ◽  
Granule Cells ◽  
De Novo ◽  
Cerebellar Granule Cells ◽  
Transcriptional Level ◽  
Synaptic Density ◽  
Cerebellar Granule ◽  
Activity Dependent ◽  
Post Synaptic Density

Activity-dependent gene expression is thought to be important in shaping neuronal development and in modifying the protein content of neurons. Ca2+entry into neurons appears to be one of the key effectors of activity-dependent gene expression. Among the possible downstream targets of calcium, the protein phosphatase calcineurin represents a prime candidate. We hereby report that in cultured cerebellar granule cells the activation of the Ca2+/calcineurin pathway via either voltage- or ligand- operated Ca2+channels regulates MALS-1 and MALS-2 expression at the transcriptional level. These proteins are integral parts of the post-synaptic density and are also involved in receptor trafficking. MALS regulation is not at the level of mRNA stability and does not requirede novoprotein synthesis, thereby suggesting a direct pathway. These data suggest that Ca2+entry by means of calcineurin is capable of controlling the structure of the post-synaptic density by controlling the expression of key components at the transcriptional level.

Nitrogen(II) Oxide (Nitric Oxide, NO): Its Origin, Fate and Physiological Significance. A Review

1997 ◽  
Vol 62 (9) ◽  
pp. 1355-1383 ◽  
Author(s):  
Petr Vetrovsky ◽  
Gustav Entlicher
Keyword(s):  
Nitric Oxide ◽  
Superoxide Anion ◽  
De Novo ◽  
Soluble Guanylate Cyclase ◽  
No Synthase ◽  
Transcriptional Level ◽  
Immune Systems ◽  
Important Target ◽  
Target Molecules

The nitrogen oxide (NO), a free radical molecule, plays a key role in the regulation of mammalian physiology and pathophysiology, e.g., in cardiovascular, nervous or immune systems. This molecule is produced from guanidino moiety of amino acid L-arginine with NG-hydroxy-L-arginine as intermediate and L-citrulline as a co-product of this reaction. This conversion is catalyzed by an ezyme called NO-synthase. The NO-synthase belongs to the cytochrome P450 superfamily and four its isoenzymes are known so far. Two (denoted NOS-I and III) are constitutive, generate lower amounts (pmol) of NO and are regulated by Ca++/calmodulin system. The others (NOS-II and IV) are inducible, produce a larger quantity of NO (nmol) and are regulated at a transcriptional level. The constitutive form is present, for example, in endothelial and neuronal cells whereas the inducible form is de novo synthesized as a consequence of certain stimuli (including cytokines) in macrophages, vascular smooth muscle cells and other cells. There are several target molecules for NO depending on cells. The most frequent target is supposed to be the soluble guanylate cyclase. However, superoxide anion is a very important target for NO, too. The reaction between these two molecules leads to the production of peroxynitrite, the fate of which depends on environmental conditions. Therefore, the importance of this reaction is still debated. This review deals with the nature of NO, the mechanisms of its production, the role of intermediate NG-hydroxy-L-arginine and summarizes the biology of superoxide anion with respect to its reaction with NO. A review with 218 references.

scholarly journals Heat shock response of Trichinella spiralis and T. pseudospiralis

Parasitology ◽  
1996 ◽  
Vol 112 (1) ◽  
pp. 89-95 ◽  
Author(s):  
R. C. Ko ◽  
L. Fan
Keyword(s):  
Heat Shock ◽  
Trichinella Spiralis ◽  
Transcriptional Level ◽  
Sds Page ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Shock Proteins ◽  
First Time ◽  
Laser Densitometry

SUMMARYHeat shock proteins (HSPs) were documented for the first time in both somatic extracts and excretory/secretory (ES) products of the infective-stage larvae of Trichinella spiralis and T. pseudospiralis. Larvae recovered from muscles of infected mice were heat shocked at 37, 40, 43 and 45 °C in RPMI 1640 medium containing L-[35S]methionine. Somatic extracts and ES products of heat-shocked worms were then analysed by SDS-PAGE, autoradiography and laser densitometry. Prominent bands of HSPs were observed at 43 °C which is the optimal heat shock temperature. The major HSPs in somatic extracts of T. spiralis were 20, 47, 50, 70, 80 and 86 kDa. When the temperature was increased from 37 to 43 °C, the greatest increase in absorbance was observed in HSPs 70 and 86. In vitro translation of mRNA in a nuclease-treated rabbit reticulocyte lysate system showed an increase in the synthesis of the 80 kDa protein. This suggests that the production of HSP 80 is regulated at the transcriptional level. The major HSPs in the ES products were 11, 45, 53 and 64 kDa. In T. pseudospiralis, the major HSPs in the somatic extracts were 20, 26, 31, 50, 53, 70, 80 and 86 kDa, and in the ES products, 11, 35, 37, 41 and 64 kDa.

scholarly journals IMiD® Compounds Affect the Hematopoiesis Via CRBN Dependent Degradation of IKZF1 Protein in CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 418-418
Author(s):  
Shirong Li ◽  
Jing Fu ◽  
Markus Mapara ◽  
Suzanne Lentzsch
Keyword(s):  
Transcription Factors ◽  
Ubiquitin Ligase ◽  
Mrna Level ◽  
Proteasome Inhibitors ◽  
Lineage Commitment ◽  
Transcriptional Level ◽  
Promoter Regions ◽  
Cd34 Cells ◽  
Myeloid Development ◽  
First Time

Abstract Introduction: Lenalidomide (LEN) and pomalidomide (POM) are derivatives of thalidomide (IMiD® compounds) and currently used to treat multiple myeloma (MM) and B-cell malignancies. We have shown before that IMiD® compounds shift lineage commitment of CD34+ cells towards myeloid development by affecting critical transcription factors such as GATA1 and PU.1 with concomitant inhibition of cell maturation resulting in anemia and neutropenia. Nonetheless the underlying pathomechanism is still unknown. Recently, IMiD® compounds were shown to bind to cereblon (CRBN) in MM cells, which is the substrate recognition component of cullin-dependent ubiquitin ligase and LEN leads to ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IZKF3 by the CRBN-CRL4 ubiquitin ligase. We investigated here the role of CRBN, IKZF1 and IKZF3 in IMiD®-induced effects on lineage commitment and maturation of CD34+ cells. Methods and Results: By western blot analysis we found that CRBN and IKZF1, but not IKZF3 are expressed in CD34+ cells. Treatment of CD34+ cells with LEN and POM for only 1h almost completely decreased the expression of IKZF1 without affecting CRBN protein expression. By using a thalidomide analog bead assay, we found that IMiD® compounds directly bind CRBN in CD34+ cells. In contrast to our protein studies, IKZF1-mRNA level was not altered in real-time PCR, suggesting that IMiD® compounds regulate IKZF1 at post-transcriptional level. Treatment with proteasome inhibitors MG132, PS341 or MLN4924 which function as cullin-dependent ubiquitin ligase inhibitors blocked LEN and POM induced IKZF1 degradation, confirming that IMiD® compounds induce ubiquitination and subsequent protein degradation of endogenous IKZF1. Next we generated lentiviral constructs to knockdown the expression of CRBN in CD34+ cells. Knockdown of CRBN in CD34+ cells induced resistance to POM-induced IKZF1 downregulation and subsequently reversed the POM-induced lineage shift in colony-formation assays, suggesting that POM-induced degradation of IKZF1 in HSC requires CRBN.Knowing that PU.1 and GATA-1 are critically involved in the IMID-induced lineage shift in CD34+ cells we demonstrated in chromatin immunoprecipitation assays that IKZF1 binds to promoter regions of PU.1 and GATA-1, suggesting that PU.1 and GATA-1 are direct downstream targets of IKZF1 in CD34+ cells . Conclusion: Our findings show that CRBN and IKZF1 mediate the effects of IMiD® compounds on hematopoietic progenitors. IMiD® compounds promote CRBN dependent degradation of IKZF1 protein in CD34+ cells that subsequently decreases transcription factors such as GATA1 and PU.1 which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Our findings that IMiD® compounds mediate their effects via CRBN and IKZF1 provide for the first time the pathomechanism how LEN and POM affect hematopoiesis and induce neutropenia, thrombocytopenia as well as anemia. Disclosures Lentzsch: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Research Funding.

scholarly journals Hormonal control of the transcription factor Pax8 and its role in the regulation of thyroglobulin gene expression in thyroid cells

2002 ◽  
Vol 172 (1) ◽  
pp. 163-176 ◽  
Author(s):  
A Mascia ◽  
L Nitsch ◽  
R Di Lauro ◽  
M Zannini
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
De Novo ◽  
Hormonal Control ◽  
Thyroid Cell ◽  
Transcriptional Level ◽  
Thyroid Cells ◽  
Tsh Regulation ◽  
Pax8 Expression ◽  
Rat Thyroid

The transcription factor Pax8 plays an important role in the expression of the differentiated phenotype of thyroid follicular cells. It has recently been shown that Pax8 is necessary for thyroglobulin (Tg) gene expression in the fully differentiated rat thyroid cell line PC. We have used the PC model system to investigate the role of Pax8 as a mediator of TSH regulation of Tg gene expression. We have demonstrated that Pax8 expression, as well as Tg expression, is severely reduced in cells grown in the absence of hormones and serum. The re-addition of TSH or forskolin to the culture medium is able to restore to wild-type levels the expression of both Pax8 and Tg. We have determined that the action of TSH/forskolin on Pax8 is at the transcriptional level. However, the re-expression of Pax8 can be observed several hours before that of Tg, suggesting that either another factor is needed or that Pax8 itself must be post-translationally modified by a newly synthesized protein to become active. To distinguish between these two possibilities we have stably transfected into PC cells an exogenous Pax8 that is expressed independently of TSH. Our results indicate that in these cells the Tg promoter is still dependent on TSH despite the constitutive presence of Pax8. Furthermore, we also show that in this condition Tg gene transcription requires de novo protein synthesis. In conclusion, TSH regulates the expression of Pax8 at a transcriptional level and also regulates the activity of Pax8 by controlling the expression of one or more as yet unknown factors.

scholarly journals Transcriptional regulation of the human CAD gene during myeloid differentiation.

1987 ◽  
Vol 7 (5) ◽  
pp. 1961-1966 ◽  
Author(s):  
G N Rao ◽  
E S Buford ◽  
J N Davidson
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Morphological Changes ◽  
Transcriptional Level ◽  
Carbamoyl Phosphate ◽  
Aspartate Transcarbamylase ◽  
Trans Retinoic Acid ◽  
Beta Actin ◽  
Cad Gene ◽  
Cad Protein

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.

Directed De Novo DNA Methylation of a Genomic Locus Leads to Heritable Transcriptional Repression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 343-343
Author(s):  
Kevin G. Ford ◽  
Paul J. Hurd ◽  
Andrew J. Bannister ◽  
Tony Kouzarides ◽  
Alexander E. Smith
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Transcriptional Repression ◽  
Cytosine Methylation ◽  
De Novo ◽  
Genomic Locus ◽  
Repressive Chromatin ◽  
First Time ◽  
Dna Cytosine Methylation ◽  
De Novo Methylation

Abstract The ability to impose exogenous targeted epigenetic changes in the genome represents an attractive goal in gene therapy for the heritable repression of target genes, while potentially enabling the generation and subsequent study of the downstream effects of de novo epigenetic events, which are known to occur in disease. Here we demonstrate the ability of zinc-finger peptides to deliver DNA cytosine methylation in vivo to a genomic target promoter, when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular de novo methylation events. We show for the first time targeted gene silencing in response to directed DNA cytosine methylation via initiation of a repressive chromatin signature at a targeted genomic locus, characterised by elevation of histone H3K9Me2 and reduction of H3K4Me3 levels at that region. This transcriptional repression is maintained in the absence of sustained targeted methyltransferase action, confirming epigenetic maintenance by the cells own machinery. The inherited DNA methylation pattern is restricted to specific target sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. Therefore, we demonstrate for the first time, targeted DNA methyltransferases as potential tools for the exogenous and heritable control of gene expression at the chromosomal level, while providing the clearest and most direct confirmation to date of the functional and mechanistic consequences of de novo DNA methylation in the cell. This work represents an important step towards the longer term goal of controlling gene expression through the inheritance of a repressive DNA state, as well as providing a valuable tool for studying spatial and temporal issues associated with ‘genuine’ de novo methylation, on transcription and chromatin structure.

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