scholarly journals Inhibition of protein synthesis in baby-hamster kidney cells blocks oxysterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA at a post-transcriptional level

1993 ◽  
Vol 296 (3) ◽  
pp. 859-866 ◽  
Author(s):  
J W Choi ◽  
E N Lundquist ◽  
D M Peffley
Keyword(s):  
Protein Synthesis ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Baby Hamster Kidney ◽  
Hmg Coa Reductase ◽  
Fetal Bovine ◽  
Reductase Mrna ◽  
Coa Reductase

The effects of the protein-synthesis inhibitor cycloheximide on 25-hydroxycholesterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA levels were evaluated in the baby-hamster kidney cell line C100. Cells cultured in medium supplemented with delipidized fetal bovine serum and 25 microM lovastatin for 12-24 h had a 5-fold higher level of HMG-CoA reductase mRNA than cells grown in medium supplemented with non-delipidized fetal bovine serum (FBS). The higher level was due to increased transcription, as determined by run-on assays with isolated nuclei. Addition of 25-hydroxycholesterol to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels within 4 h of treatment to those of cells grown in FBS-supplemented medium. This decrease was due in part to a decrease in gene transcription. Cycloheximide added in conjunction with 25-hydroxycholesterol to lovastatin-treated cells blocked the suppression of mRNA levels, but did not block oxysterol-mediated suppression of transcription. In addition, cycloheximide added to cells grown in FBS-supplemented medium rapidly increased mRNA levels by 10-fold relative to untreated cells, with no comparable increase in transcription. No comparable increase in either the mRNA level or rate of transcription for beta-actin was observed under such conditions. These results indicate that cycloheximide specifically stabilizes HMG-CoA reductase mRNA in the presence of oxysterols and suggests that continuous synthesis of a short lived protein regulator is required for oxysterol-mediated suppression of HMG-CoA reductase mRNA at a post-transcriptional level.

scholarly journals Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1150-1157 ◽  
Author(s):  
AR Migliaccio ◽  
G Migliaccio ◽  
M Brice ◽  
P Constantoulakis ◽  
G Stamatoyannopoulos ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Mrna Level ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Gm Csf ◽  
Gamma Globin ◽  
Fetal Bovine ◽  
Globin Synthesis

Abstract We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBS)-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) gamma-globin and gamma- globin mRNA levels. Reduced gamma-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable gamma-globin by immunofluorescence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of gamma-globin both in FBS- supplemented and in FBS-deprived cultures. These results, obtained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by themselves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s) present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood.

scholarly journals Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1150-1157 ◽  
Author(s):  
AR Migliaccio ◽  
G Migliaccio ◽  
M Brice ◽  
P Constantoulakis ◽  
G Stamatoyannopoulos ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Mrna Level ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Gm Csf ◽  
Gamma Globin ◽  
Fetal Bovine ◽  
Globin Synthesis

We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBS)-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) gamma-globin and gamma- globin mRNA levels. Reduced gamma-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable gamma-globin by immunofluorescence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of gamma-globin both in FBS- supplemented and in FBS-deprived cultures. These results, obtained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by themselves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s) present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood.

75 Improvement of Developmental Competence of Bovine In Vitro-Produced Embryos by Using Charcoal:Dextran-Stripped Fetal Bovine Serum on In Vitro Culture Media

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
A. Mesalam ◽  
R. Kong ◽  
B.-H. Choi ◽  
K.-L. Lee ◽  
B.-Y. Park ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Mrna Levels ◽  
Fetal Bovine

Serum has widely been used as a main supplement to embryo in vitro culture media as it contains embryotrophic factors. Charcoal:dextran treatment of fetal bovine serum (FBS) removes lipophilic chemicals and certain steroid hormones and growth factors. The objective of this study was to investigate the effects of charcoal:dextran-stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium (SOF-BE1 medium supplemented with 10% of serum) on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The experiment was conducted in 6 replicates (350 oocytes per group). The differences in embryo development, integrated optical intensity, and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P < 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% v. 36.85 ± 0.89%, respectively). The total number of cells per Day 8 blastocyst was not significantly different (P > 0.05) between the CDS FBS group (208.40 ± 14.77) and the HI FBS group (195.11 ± 19.15). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly increased mitochondrial activity, as identified by MitoTracker Green, and reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P < 0.05) higher after 24 h in the CDS FBS than in the HI FBS group (85.33 ± 4.84% v. 68.67 ± 1.20%). Quantitative reverse transcription PCR showed that the mRNA levels of lipid metabolism-related genes, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, and the cholesterol metabolism related gene hydroxymethylglutaryl-CoA reductase were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of survival gene sirtuin 1, antioxidant gene superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen–thawed blastocysts were significantly (P < 0.05) higher in the CDS FBS group than in the HI FBS group; however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. In conclusion, these data suggest that supplementation of in vitro culture medium with CDS FBS improves in vitro bovine embryo developmental competence and the quality of blastocysts in terms of their crytolerance and gene expression. This research was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets), BK21plus, and KGSP.

Fetal bovine serum promotes the development of in vitro porcine blastocysts by activating the Rho-associated kinase signalling pathway

2019 ◽  
Vol 31 (2) ◽  
pp. 366
Author(s):  
Shimeng Guo ◽  
Shichao Liu ◽  
Gerelchimeg Bou ◽  
Jia Guo ◽  
Liyuan Jiang ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Inner Cell Mass ◽  
Fetal Bovine Serum ◽  
Cell Mass ◽  
Hatching Rate ◽  
Mrna Levels ◽  
Embryonic Cells ◽  
Blastocyst Development ◽  
Fetal Bovine ◽  
Number Of Cells

Fetal bovine serum (FBS) supplementation has beneficial effects on invitro porcine embryonic development, but the underlying mechanisms are unclear. In the present study we found that the addition of FBS to PZM-3 increased the number of cells in porcine blastocysts and hatching rate invitro primarily by promoting proliferation of the inner cell mass and further differentiation. Moreover, based on the following results, we surmise that FBS benefits blastocyst development by activating Rho-associated kinase (ROCK) signalling: (1) the ROCK signalling inhibitor Y-27632 decreased the blastocyst rate and the number of cells in blastocysts, whereas FBS rescued the developmental failure induced by Y-27632; (2) the mRNA levels of two ROCK isoforms, ROCK1 and ROCK2, were significantly increased in blastocysts derived from medium containing FBS; and (3) FBS increased RhoA/Rho-kinase expression in the nucleus of embryonic cells. These results indicate that FBS promotes the invitro development of porcine embryos by activating ROCK signalling in a chemically defined medium.

Gamma-Tocotrienol and Simvastatin Synergistically Induce Cytotoxicity In Leukemia Cell Lines, K-562 and HL-60

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4927-4927 ◽  
Author(s):  
Ko Maung ◽  
Victoria Palau ◽  
Janet Lightner ◽  
Marianne Brannon ◽  
Koyamangalath Krishnan
Keyword(s):  
Cell Lines ◽  
Fetal Bovine Serum ◽  
Cholesterol Biosynthesis ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Hmg Coa Reductase ◽  
Leukemia Cell Lines ◽  
Fetal Bovine ◽  
Coa Reductase ◽  
Gamma Tocotrienol

Abstract Introduction Statins and tocotrienols modulate the cholesterol biosynthesis pathway by inhibiting the 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase. Tocotrienols modulate HMG-CoA reductase by post-transcriptional downregulation. In addition, tocotrienols contain a farnesol moiety in its side-chain that triggers degradation of HMG-CoA reductase. These effects lead to suppression of cell proliferation, cell cycle arrest, and apoptosis. Several studies have shown that statins have suppressive effects in in vitro experiments on acute myelocytic leukemia cell lines. Since both statins and gamma-tocotrienol are associated with decreased cholesterol biosynthesis, we hypothesized that if the cytotoxicity of these drugs on cancer cells is related to impaired biosynthesis of cholesterol, combination of them could synergize in cytotoxicity on leukemic cells. Materials and Methods K-562 and HL-60 leukemia cells were grown in Iscove's Modified Dulbecco's medium with penicillin/streptomycin, 10% fetal bovine serum and 20% fetal bovine serum added respectively. K-562 and HL-60 leukemia cells were seeded in 96 well plates, grown overnight, and treated for 24, 48 and 72 hours with simvastatin in concentrations of 1,2,4, and5 µM; gamma-tocotrienol in concentrations of 20, 40, and 80 µM; and a combination of the two drugs in the same concentrations. For 24 hour dose, cells were seeded at a density of 5000/well and for 48 and 72 hour doses, at  3500/well. Following the treatment, MTS/PMS reagent (Promega, Madison, WI), [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] mixed with and an electron coupling reagent (phenazine methosulfate), was added at 40 µg/well and incubated at 37°C for 2 hours. We measured the solubilized formazan crystals at 450 nm as an indicator of cytotoxicity. The presence of ATP  as an indicator of cell viability was measured with the CellTiter Glo®  assay (Promega). Cells were again seeded, grown overnight, and dosed as indicated above.  Following treatment, the assay was conducted as specified by the manufacturer. Results Both simvastatin and gamma-tocotrienol induce cytotoxicity in K-562 and HL-60 cell lines by the MTS and Cell Titer Glo Assays.  The IC50 at 72 hour incubation are as follows 1) HL-60: simvastatin - 16.543 uM, gamma tocotrienol - 36.297 uM;  2) K-562: simvastatin - 5.235 uM, gamma tocotrienol - 34.947 uM.  We used CompuSyn to calculate the IC50.  When combined, simvastatin and gamma-tocotrienol exhibit synergy at lower concentrations when examined by isobologram analysis. Conclusion Gamma-tocotrienol, an isoform of vitamin E, and simvastatin, a cholesterol lowering drug exhibit synergy in induction of cytotoxicity in K-562 and HL-60 leukemia cell lines. Rescue experiments and mechanistic pathway analysis are being explored to confirm these observations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 3′-untranslated sequences mediate post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA by 25-hydroxycholesterol

1995 ◽  
Vol 307 (1) ◽  
pp. 233-238 ◽  
Author(s):  
J W Choi ◽  
D M Peffley
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Regulation ◽  
Untranslated Region ◽  
Simian Virus 40 ◽  
Beta Globin ◽  
Hmg Coa Reductase ◽  
Reductase Mrna ◽  
Coa Reductase ◽  
Post Transcriptional Regulation ◽  
Rna Levels

In an earlier study [Choi, Lundquist and Peffley (1993) Biochem. J. 296, 859-866], we determined that 25-hydroxycholesterol regulates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA through a post-transcriptional mechanism that requires protein synthesis. To investigate whether 3′-untranslated sequences play a role in 25-hydroxycholesterol-mediated post-transcriptional control, we ligated approx. 1400 bp of the 3′-untranslated region of HMG-CoA reductase cDNA to the coding region of human beta-globin DNA. beta-Globin-3′-untranslated reductase fusion constructs were then transiently expressed in Chinese hamster ovary fibroblasts under conditions known to regulate reductase mRNA. There were no differences in beta-globin RNA levels in transfected cells incubated with or without lovastatin, a competitive inhibitor of reductase. However, in the presence of lovastatin and an oxysterol, 25-hydroxycholesterol, beta-globin RNA levels were decreased approx. 2-fold. Inhibition of protein synthesis with cycloheximide blocked the effects of 25-hydroxycholesterol on beta-globin RNA. Moreover, replacing the 3′-untranslated sequences with 1367 bp of the simian virus 40 enhancer region eliminated the regulatory effect of 25-hydroxycholesterol. Because the fusion construct has no sterol regulatory elements necessary for transcription, our results indicate that the change in beta-globin RNA occurred at a post-transcriptional level. In addition, we have shown that the 3′-untranslated region of HMG-CoA reductase cDNA imparted oxysterol-mediated post-transcriptional regulation to beta-globin RNA, an effect that required protein synthesis.

scholarly journals Differential transcriptional and posttranscriptional regulation of gene expression of the colony-stimulating factors by interleukin-1 and fetal bovine serum in murine fibroblasts

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 658-665 ◽  
Author(s):  
JH Falkenburg ◽  
MA Harrington ◽  
RA de Paus ◽  
WK Walsh ◽  
R Daub ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Interleukin 1 ◽  
Transcription Rate ◽  
Colony Stimulating Factors ◽  
Gm Csf ◽  
Murine Fibroblasts ◽  
Fetal Bovine

Colony-stimulating factors (CSF) are important factors in the proliferation and differentiation of hematopoietic progenitor cells (HPC), and in the survival and activation of mature blood cells. Interleukin-1 (IL-1) combined with fetal bovine serum (FBS) strongly induces the expression of macrophage-CSF (M-CSF), granulocyte-CSF (G- CSF), and granulocyte-macrophage-CSF (GM-CSF) in fibroblasts. Here, we report on the regulation of CSF gene expression in murine fibroblasts following IL-1 and FBS stimulation. We demonstrate that 10T1/2 murine fibroblasts induced by FBS or IL-1 accumulate M-CSF messenger RNA (mRNA). G-CSF mRNA expression was induced by IL-1, and not by FBS. For GM-CSF expression, induction with both FBS and IL-1 was required. Blocking studies with actinomycin-D showed that active transcription is essential for accumulation of all three CSF mRNAs. After blocking protein synthesis with cycloheximide, IL-1- or FBS-induced M-CSF expression and IL-1 plus FBS-induced GM-CSF expression still occurred and was increased. IL-1-induced G-CSF expression was completely prevented in these cells by pretreatment with cycloheximide, illustrating that, for this effect, intermediate protein synthesis was required. The half-lives of M-CSF transcripts were not substantially altered by addition of IL-1, FBS, or FBS plus IL-1. Using nuclear run- on assays, we demonstrated that the transcription rate of M-CSF was increased up to 20-fold by the addition of FBS, IL-1, or FBS plus IL-1. After blocking protein synthesis with cycloheximide, IL-1-or FBS- induced increase in M-CSF transcription rate was also observed. GM-CSF transcription increased up to fourfold after induction with FBS or IL- 1. G-CSF transcription rate was not altered by FBS or IL-1. Our results indicate that M-CSF expression induced by FBS or IL-1 in these fibroblasts is primarily regulated at the transcriptional level. GM-CSF expression appears to be regulated both transcriptionally and posttranscriptionally, and G-CSF expression is regulated mainly at the posttranscriptional level.

scholarly journals Differential transcriptional and posttranscriptional regulation of gene expression of the colony-stimulating factors by interleukin-1 and fetal bovine serum in murine fibroblasts

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 658-665 ◽  
Author(s):  
JH Falkenburg ◽  
MA Harrington ◽  
RA de Paus ◽  
WK Walsh ◽  
R Daub ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Interleukin 1 ◽  
Transcription Rate ◽  
Colony Stimulating Factors ◽  
Gm Csf ◽  
Murine Fibroblasts ◽  
Fetal Bovine

Abstract Colony-stimulating factors (CSF) are important factors in the proliferation and differentiation of hematopoietic progenitor cells (HPC), and in the survival and activation of mature blood cells. Interleukin-1 (IL-1) combined with fetal bovine serum (FBS) strongly induces the expression of macrophage-CSF (M-CSF), granulocyte-CSF (G- CSF), and granulocyte-macrophage-CSF (GM-CSF) in fibroblasts. Here, we report on the regulation of CSF gene expression in murine fibroblasts following IL-1 and FBS stimulation. We demonstrate that 10T1/2 murine fibroblasts induced by FBS or IL-1 accumulate M-CSF messenger RNA (mRNA). G-CSF mRNA expression was induced by IL-1, and not by FBS. For GM-CSF expression, induction with both FBS and IL-1 was required. Blocking studies with actinomycin-D showed that active transcription is essential for accumulation of all three CSF mRNAs. After blocking protein synthesis with cycloheximide, IL-1- or FBS-induced M-CSF expression and IL-1 plus FBS-induced GM-CSF expression still occurred and was increased. IL-1-induced G-CSF expression was completely prevented in these cells by pretreatment with cycloheximide, illustrating that, for this effect, intermediate protein synthesis was required. The half-lives of M-CSF transcripts were not substantially altered by addition of IL-1, FBS, or FBS plus IL-1. Using nuclear run- on assays, we demonstrated that the transcription rate of M-CSF was increased up to 20-fold by the addition of FBS, IL-1, or FBS plus IL-1. After blocking protein synthesis with cycloheximide, IL-1-or FBS- induced increase in M-CSF transcription rate was also observed. GM-CSF transcription increased up to fourfold after induction with FBS or IL- 1. G-CSF transcription rate was not altered by FBS or IL-1. Our results indicate that M-CSF expression induced by FBS or IL-1 in these fibroblasts is primarily regulated at the transcriptional level. GM-CSF expression appears to be regulated both transcriptionally and posttranscriptionally, and G-CSF expression is regulated mainly at the posttranscriptional level.

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