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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

Localization of EGFP and YieF-EGFP fusion protein in transient transfectants. Green fluorescence indicated the localization of empty fluorescent vector EGFP (A) and fusion protein YieF-EGFP (D). Nuclei were counterstained with Hoechst 33258 (B,E). C is the overlay of A and B, and F is the overlay of D and E. Scale Bar = 10 μm.
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ijms-16-11892-f004: Localization of EGFP and YieF-EGFP fusion protein in transient transfectants. Green fluorescence indicated the localization of empty fluorescent vector EGFP (A) and fusion protein YieF-EGFP (D). Nuclei were counterstained with Hoechst 33258 (B,E). C is the overlay of A and B, and F is the overlay of D and E. Scale Bar = 10 μm.

Mentions: To visualize the localization of YieF, a fusion expression vector carrying YieF-EGFP was constructed and introduced to HepG2. 24 h after transfection, the expression of EGFP and EGFP-YieF was visualized by laser scanning confocal microscopy. The fluorescence was mainly localized in the nucleus of the cells that expressed EGFP only (Figure 4A–C), whereas the fluorescence in YieF-EGFP-transfected cells was detected in both nucleus and cytoplasm (Figure 4D–F), with slightly higher fluorescence intensity in the cytoplasm. This result demonstrated that fusion protein YieF-EGFP was localized in the cytoplasm where Cr reduction took place, possibly to protect the cell nucleus from Cr damage [28].


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)

Localization of EGFP and YieF-EGFP fusion protein in transient transfectants. Green fluorescence indicated the localization of empty fluorescent vector EGFP (A) and fusion protein YieF-EGFP (D). Nuclei were counterstained with Hoechst 33258 (B,E). C is the overlay of A and B, and F is the overlay of D and E. Scale Bar = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-11892-f004: Localization of EGFP and YieF-EGFP fusion protein in transient transfectants. Green fluorescence indicated the localization of empty fluorescent vector EGFP (A) and fusion protein YieF-EGFP (D). Nuclei were counterstained with Hoechst 33258 (B,E). C is the overlay of A and B, and F is the overlay of D and E. Scale Bar = 10 μm.
Mentions: To visualize the localization of YieF, a fusion expression vector carrying YieF-EGFP was constructed and introduced to HepG2. 24 h after transfection, the expression of EGFP and EGFP-YieF was visualized by laser scanning confocal microscopy. The fluorescence was mainly localized in the nucleus of the cells that expressed EGFP only (Figure 4A–C), whereas the fluorescence in YieF-EGFP-transfected cells was detected in both nucleus and cytoplasm (Figure 4D–F), with slightly higher fluorescence intensity in the cytoplasm. This result demonstrated that fusion protein YieF-EGFP was localized in the cytoplasm where Cr reduction took place, possibly to protect the cell nucleus from Cr damage [28].

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