Thiophosphate and selenite conversely modulate cell death induced by glutathione depletion or cisplatin: effects related to activity and Sec contents of thioredoxin reductase

2012 ◽  
Vol 447 (1) ◽  
pp. 167-174 ◽  
Author(s):  
Xiaoxiao Peng ◽  
Jianqiang Xu ◽  
Elias S. J. Arnér
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Thioredoxin Reductase ◽  
Glutathione Depletion ◽  
3T3 Cells ◽  
Drug Induced ◽  
Direct Cytotoxicity ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

Thiophosphate (SPO3) was recently shown to promote cysteine insertion at Sec (selenocysteine)-encoding UGA codons during selenoprotein synthesis. We reported previously that irreversible targeting by cDDP [cis-diamminedichloroplatinum(II) or cisplatin] of the Sec residue in TrxR1 (thioredoxin reductase 1) contributes to cDDP cytotoxicity. This effect could possibly be attenuated in cells expressing less reactive Sec-to-cysteine-substituted TrxR1 variants, or pronounced in cells with higher levels of Sec-containing TrxR1. To test this, we supplemented cells with either SPO3 or selenium and subsequently determined total as well as specific activities of cellular TrxR1, together with extent of drug-induced cell death. We found that cDDP became less cytotoxic after incubation of A549 or HCT116 cells with lower SPO3 concentrations (100–300 μM), whereas higher SPO3 (>300 μM) had pronounced direct cytotoxicity. NIH 3T3 cells showed low basal TrxR1 activity and high susceptibility to SPO3 cytotoxicity, or to glutathione depletion. Supplementing NIH 3T3 cells with selenite, however, gave increased cellular TrxR1 activity with concomitantly decreased dependence on glutathione, whereas the susceptibility to cDDP increased. The results suggest molecular mechanisms by which the selenium status of cells can affect their glutathione dependence while modulating the cytotoxicity of drugs that target TrxR1.

scholarly journals Cytotoxicity, Antiproliferative Effects, and Apoptosis Induction of Methanolic Extract ofCynometra caulifloraLinn. Whole Fruit on Human Promyelocytic Leukemia HL-60 Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
T-Johari S. A. Tajudin ◽  
Nashriyah Mat ◽  
Abu Bakar Siti-Aishah ◽  
A. Aziz M. Yusran ◽  
Afnani Alwi ◽  
...  
Keyword(s):  
Cell Death ◽  
Methanolic Extract ◽  
Apoptotic Cell ◽  
Mouse Fibroblast ◽  
Promyelocytic Leukemia ◽  
Apoptotic Cell Death ◽  
Apoptotic Cells ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Methanolic extract ofCynometra cauliflorawhole fruit was assayed for cytotoxicity against the human promyelocytic leukemia HL-60 and the normal mouse fibroblast NIH/3T3 cell lines by using the MTT assay. The CD50of the extract for 72 hours was 0.9 μg/mL whereas the value for the cytotoxic drug vincristine was 0.2 μg/mL. The viability of the NIH/3T3 cells was at 80.0% when treated at 15.0 μg/mL. The extract inhibited HL-60 cell proliferation with dose dependence. AO/PI staining of HL-60 cells treated with the extract revealed that majority of cells were in the apoptotic cell death mode. Flow cytometry analysis of HL-60 cells treated at CD50of the extract showed that the early apoptotic cells were 31.0, 26.3 and 19.9% at 24, 48, and 72 hours treatment, respectively. The percentage of late apoptotic cells was increased from 62.0 at 24 hours to 64.1 and 70.2 at 48 and 72 hours, respectively. Meanwhile, percent of necrotic cells were 4.9, 6.6, and 8.5 at 24, 48, and 72 hours, respectively. This study has shown that the methanolic extract ofC. cauliflorawhole fruit was cytotoxic towards HL-60 cells and induced the cells into apoptotic cell death mode, but less cytotoxic towards NIH/3T3 cells.

scholarly journals Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

1993 ◽  
Vol 13 (12) ◽  
pp. 7718-7724 ◽  
Author(s):  
H Cen ◽  
A G Papageorge ◽  
W C Vass ◽  
K E Zhang ◽  
D R Lowy
Keyword(s):  
The Other ◽  
Guanine Nucleotide ◽  
3T3 Cells ◽  
Ras Protein ◽  
Exchange Factor ◽  
Type Iv ◽  
Serum Dependent ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.

Abstract 268: Transforming Growth Factor-β1 Increases Resistance of Fibroblasts to Apoptotic Cell Death

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Ulugbek Negmadjanov ◽  
Zarko Godic ◽  
Mahek Mirza ◽  
Larisa Emelyanova ◽  
Farhan Rizvi ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cell Death ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Tgf Β1 ◽  
Nih 3T3 Cells

Introduction: Cardiac injury results in the death of cardiac myocytes and subsequent scar formation through extracellular matrix (ECM) deposition by fibroblasts (FB) and myofibroblasts (myoFB). Excessive fibrosis results in pathological scarring that predisposes to arrhythmogenesis and heart failure, particularly in the elderly. Strategies to limit adverse ECM remodeling are urgently needed to curtail the growing epidemic of atrial fibrillation and heart failure in the aging population. Persistence of myoFB and resistance to apoptotic cell death has been proposed to underlie the mechanism of excessive fibrosis, yet is not fully characterized. Methods: Cultured NIH/3T3 cells (control and TGF-β1 treated) have been challenged with activators of extrinsic (FAS-Ligand, 1 μg/mL) or intrinsic (Thapsigargin 10 μM and Staurosporine 5 μM) apoptotic pathways and Caspase-3 activity was measured in cellular lysate. Results: FAS-L exposure induced ~40-fold suppression of Caspase-3 activity in TGF-β1 treated cells as compared with control (17±12 vs 686±5 nmol AMC/min/106 cells, respectively). Similarly, Staurosporine activated Caspase-3 in TGF-β1 treated cells ~3-fold (171±38 vs 536±29 nmol AMC/min/106 cells), and Thapsigargin ~10-fold (73±33 vs 742±8 nmol AMC/min/106 cells). Conclusion: TGF-β1 treatment increased the sensitivity of NIH/3T3 cells toward extrinsic and intrinsic apoptotic stimuli. Although, TGF-β1 treatment increased overall resistance of NIH/3T3 cells to apoptosis, the responsiveness of cells to extrinsic vs intrinsic pathways was differentially affected. This data support the hypothesis that persistence of myoFB results in pathological scarring.

Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos

1988 ◽  
Vol 8 (10) ◽  
pp. 4212-4216
Author(s):  
K Maly ◽  
W Doppler ◽  
H Oberhuber ◽  
H Meusburger ◽  
J Hofmann ◽  
...  
Keyword(s):  
Growth Factors ◽  
Intracellular Ph ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
3T3 Cells ◽  
Ras Gene ◽  
Phosphatidylinositol Turnover ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

scholarly journals Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

1988 ◽  
Vol 8 (10) ◽  
pp. 4212-4216 ◽  
Author(s):  
K Maly ◽  
W Doppler ◽  
H Oberhuber ◽  
H Meusburger ◽  
J Hofmann ◽  
...  
Keyword(s):  
Growth Factors ◽  
Intracellular Ph ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
3T3 Cells ◽  
Ras Gene ◽  
Phosphatidylinositol Turnover ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

scholarly journals Intracellular Restriction of a Productive Noncytopathic Coronavirus Infection

2007 ◽  
Vol 82 (1) ◽  
pp. 451-460 ◽  
Author(s):  
Olga Slobodskaya ◽  
Alexander Laarman ◽  
Willy J. M. Spaan
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Host Cells ◽  
3T3 Cells ◽  
Rna Amplification ◽  
Coronavirus Infection ◽  
Nih 3T3 ◽  
The Impact ◽  
Nih 3T3 Cells ◽  
In Cells

ABSTRACT Virus infection in vitro can either result in a cytopathic effect (CPE) or proceed without visible changes in infected cells (noncytopathic infection). We are interested in understanding the mechanisms controlling the impact of coronavirus infection on host cells. To this end, we compared a productive, noncytopathic infection of murine hepatitis virus (MHV) strain A59 in the fibroblastlike cell line NIH 3T3 with cytopathic MHV infections. Infected NIH 3T3 cells could be cultured for up to 4 weeks without apparent CPE and yet produce virus at 107 to 108 PFU/ml. Using flow cytometry, we demonstrated that NIH 3T3 cells expressed as much MHV receptor CEACAM1 as other cell lines which die from MHV infection. In contrast, using quantitative reverse transcription-PCR and metabolic labeling of RNA, we found that the rate of viral RNA amplification in NIH 3T3 cells was lower than the rate in cells in which MHV induces a CPE. The rate of cellular RNA synthesis in contact-inhibited confluent NIH 3T3 cells was also lower than in cells permissive to cytopathic MHV infection. However, the induction of cellular RNA synthesis in growing NIH 3T3 cells did not result in an increase of either viral RNA amplification or CPE. Our results suggest that a specific, receptor CEACAM1-independent mechanism restricting coronaviral RNA synthesis and CPE is present in NIH 3T3 and, possibly, other cells with preserved contact inhibition.

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor

1993 ◽  
Vol 13 (12) ◽  
pp. 7718-7724
Author(s):  
H Cen ◽  
A G Papageorge ◽  
W C Vass ◽  
K E Zhang ◽  
D R Lowy
Keyword(s):  
The Other ◽  
Guanine Nucleotide ◽  
3T3 Cells ◽  
Ras Protein ◽  
Exchange Factor ◽  
Type Iv ◽  
Serum Dependent ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.

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