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Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

Liu X, Wu G, Zhang Y, Wu D, Li X, Liu P - Int J Mol Sci (2015)

Bottom Line: The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction.This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF.Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China. kathyliu0505@gmail.com.

ABSTRACT
Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.

No MeSH data available.


Related in: MedlinePlus

(A) The expression levels of gene yieF in different conditions determined by qRT-PCR. Relative level of yieF mRNA was measured in HepG2 (▤), Cr(VI)-treated HepG2 (■), HepG2-YieF (▢) and Cr(VI)-treated HepG2-YieF (▨) cells. Data were normalized to β-actin expression as a housekeeping gene. The values are the mean of four replicates. There are significant differences (p < 0.05) between the two bars marked a and b; (B) Detection of GFP-YieF fusion protein by western blotting. Molecular weight of GFP-YieF is about 50 kDa; (C) The integration of yieF in human chromosome 1. Grey boxes on the 5ʹ and 3ʹ sides of yieF indicated genes located on Chr 1. Grey line immediately upstream of yieF marked 1 indicate predicted TF binding sites including TBP, Sp1 and CBP100 binding sites. The diagram was not drawn to scale. ADAR, adenosine deaminase, RNA-specific; KCNN3, potassium channel, calcium activated intermediate/small conductance subfamily N α, member 3.
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ijms-16-11892-f001: (A) The expression levels of gene yieF in different conditions determined by qRT-PCR. Relative level of yieF mRNA was measured in HepG2 (▤), Cr(VI)-treated HepG2 (■), HepG2-YieF (▢) and Cr(VI)-treated HepG2-YieF (▨) cells. Data were normalized to β-actin expression as a housekeeping gene. The values are the mean of four replicates. There are significant differences (p < 0.05) between the two bars marked a and b; (B) Detection of GFP-YieF fusion protein by western blotting. Molecular weight of GFP-YieF is about 50 kDa; (C) The integration of yieF in human chromosome 1. Grey boxes on the 5ʹ and 3ʹ sides of yieF indicated genes located on Chr 1. Grey line immediately upstream of yieF marked 1 indicate predicted TF binding sites including TBP, Sp1 and CBP100 binding sites. The diagram was not drawn to scale. ADAR, adenosine deaminase, RNA-specific; KCNN3, potassium channel, calcium activated intermediate/small conductance subfamily N α, member 3.

Mentions: Stable HepG2-YieF cells and controls were cultured with or without 5 μM Cr(VI) to determine the relative expression level of yieF. The expression of yieF was found upregulated by 3.5-fold with Cr(VI) treatment while no induction was observed in control cells (Figure 1A). This result indicated that gene yieF was expressed in HepG2 and the expression could be induced by the addition of chromate although the expression vector carrying yieF was non-inducible. The presence of YieF protein was confirmed by flow cytometry and western blotting using antibodies against Enhanced Green Fluorescent Protein (EGFP) in EGFP-YieF-HepG2 cells transiently expressing EGFP-YieF fusion protein (Figure 1B). The fluorescence intensity was 22.5%, reflecting relatively low efficiency of transfection (Figure S1).


Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

Liu X, Wu G, Zhang Y, Wu D, Li X, Liu P - Int J Mol Sci (2015)

(A) The expression levels of gene yieF in different conditions determined by qRT-PCR. Relative level of yieF mRNA was measured in HepG2 (▤), Cr(VI)-treated HepG2 (■), HepG2-YieF (▢) and Cr(VI)-treated HepG2-YieF (▨) cells. Data were normalized to β-actin expression as a housekeeping gene. The values are the mean of four replicates. There are significant differences (p < 0.05) between the two bars marked a and b; (B) Detection of GFP-YieF fusion protein by western blotting. Molecular weight of GFP-YieF is about 50 kDa; (C) The integration of yieF in human chromosome 1. Grey boxes on the 5ʹ and 3ʹ sides of yieF indicated genes located on Chr 1. Grey line immediately upstream of yieF marked 1 indicate predicted TF binding sites including TBP, Sp1 and CBP100 binding sites. The diagram was not drawn to scale. ADAR, adenosine deaminase, RNA-specific; KCNN3, potassium channel, calcium activated intermediate/small conductance subfamily N α, member 3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490421&req=5

ijms-16-11892-f001: (A) The expression levels of gene yieF in different conditions determined by qRT-PCR. Relative level of yieF mRNA was measured in HepG2 (▤), Cr(VI)-treated HepG2 (■), HepG2-YieF (▢) and Cr(VI)-treated HepG2-YieF (▨) cells. Data were normalized to β-actin expression as a housekeeping gene. The values are the mean of four replicates. There are significant differences (p < 0.05) between the two bars marked a and b; (B) Detection of GFP-YieF fusion protein by western blotting. Molecular weight of GFP-YieF is about 50 kDa; (C) The integration of yieF in human chromosome 1. Grey boxes on the 5ʹ and 3ʹ sides of yieF indicated genes located on Chr 1. Grey line immediately upstream of yieF marked 1 indicate predicted TF binding sites including TBP, Sp1 and CBP100 binding sites. The diagram was not drawn to scale. ADAR, adenosine deaminase, RNA-specific; KCNN3, potassium channel, calcium activated intermediate/small conductance subfamily N α, member 3.
Mentions: Stable HepG2-YieF cells and controls were cultured with or without 5 μM Cr(VI) to determine the relative expression level of yieF. The expression of yieF was found upregulated by 3.5-fold with Cr(VI) treatment while no induction was observed in control cells (Figure 1A). This result indicated that gene yieF was expressed in HepG2 and the expression could be induced by the addition of chromate although the expression vector carrying yieF was non-inducible. The presence of YieF protein was confirmed by flow cytometry and western blotting using antibodies against Enhanced Green Fluorescent Protein (EGFP) in EGFP-YieF-HepG2 cells transiently expressing EGFP-YieF fusion protein (Figure 1B). The fluorescence intensity was 22.5%, reflecting relatively low efficiency of transfection (Figure S1).

Bottom Line: The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction.This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF.Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China. kathyliu0505@gmail.com.

ABSTRACT
Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.

No MeSH data available.


Related in: MedlinePlus