Antioxidant and Antiapoptotic Function of Metallothioneins in HL-60 Cells Challenged with Copper Nitrilotriacetate
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- 作者：Kazuaki Kawai ; Shang-Xi Liu ; Vladimir A. Tyurin ; Yulia Y. Tyurina ; Grigory G. Borisenko ; Jian Fei Jiang ; Claudette M. St. Croix ; James P. Fabisiak ; Bruce R. Pitt ; and Valerian E. Kagan
- 刊名：Chemical Research in Toxicology
- 出版年：2000
- 出版时间：December 2000
- 年：2000
- 卷：13
- 期：12
- 页码：1275 - 1286
- 全文大小：207K
- 年卷期：v.13,no.12(December 2000)
- ISSN：1520-5010
文摘
Antioxidant activity is believed to be an important intracellular function of metallothioneins(MT), yet the specific mechanisms of their antioxidant action are not known. Under conditionswhen cells are challenged with elevated concentrations of free copper as a result of metabolicdisturbances or environmental and occupational exposures, MTs may be ideally suited forantioxidant function as effective copper chelators. In the study presented here, we tested thishypothesis using a recently established model of copper nitrilotriacetate-induced oxidative stressin HL-60 cells. Since copper-induced oxidative stress triggers apoptosis, we further investigatedantiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic partialpurification of MTs from cell homogenates with subsequent immuno-dot-blot assay, we showedthat zinc pretreatment yielded a pronounced induction of MTs in HL-60 cells. We report thatzinc-induced MTs were able to (i) completely bind intracellular copper, (ii) completely quenchredox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative stress inmembrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteristicbiochemical features (cytochrome c release from mitochondria into cytosol, caspase-3 activation,and externalization of phosphatidylserine in plasma membranes). In separate experiments,we used lung fibroblasts derived from MT1, MT2 knockout mice (MT-/-) and MT wild-type(MT+/+) mice. ZnCl2 pretreatment resulted in a more than 10-fold induction of MTs in MT+/+cells, whereas the MT content in MT-/- cells remained low, at levels 
100-fold lower than intheir MT wild-type counterparts. MT-/- cells were very sensitive to Cu-NTA and, mostimportantly, showed no response to ZnCl2 pretreatment. In contrast, MT+/+ cells were relativelymore resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl2 pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.
100-fold lower than intheir MT wild-type counterparts. MT-/- cells were very sensitive to Cu-NTA and, mostimportantly, showed no response to ZnCl2 pretreatment. In contrast, MT+/+ cells were relativelymore resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl2 pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.