Khalil Sara, Luciano Jason, Chen Wenjun, Liu Alice Y-C
Rutgers College, Rutgers State University of New Jersey, Piscataway, New Jersey 08854, USA.
J Cell Physiol. 2006 May;207(2):562-9. doi: 10.1002/jcp.20599.
The objective of this study is to better define induction of the heat shock response by arsenite, and to evaluation if induction of heat shock proteins (HSPs) contributes to the carcinogenic activity of arsenite. We show here that arsenite is a ubiquitous inducer of the heat shock response in mammalian cells: that it activated heat shock transcription factor 1 (HSF1) DNA-binding activity, enhanced hsp 70 promoter, and induced hsp70mRNA and synthesis of HSP chaperones. Using a high throughput hsp70 promoter-luciferase reporter assay, we observed a hormetic dose response where low concentrations of arsenite stimulated and high concentrations inhibited. Further, the response was time-dependent such that with longer times of incubation, the dose response shifted to the left. The effect of arsenite in inducing the hsp 70-luciferase reporter absolutely required a functional HSF1 as it was not observed in HSF1 minus cells but re-instated by expression of HSF1. Consistent with the suggestion that arsenic targets vicinal cysteine-SH, we showed that dithiothreitol blocked the effect of arsenite. Assays of cell viability and caspase showed that arsenite caused a dose-dependent increase in cell death by activation of caspase 3/7 and pre-induction of HSPs blunted these effects. Using anchorage independent cell growth as a late stage tumor promotion assay, we showed that low concentrations of arsenite had a growth promoting effect, which was enhanced by moderate heat shock. Our study provides evidence that induction of the heat shock response is a sensitive biomarker of arsenic exposure and that induction of HSPs likely contributes to the tumor promotion effect of arsenic.
本研究的目的是更好地界定亚砷酸盐对热休克反应的诱导作用，并评估热休克蛋白（HSPs）的诱导是否有助于亚砷酸盐的致癌活性。我们在此表明，亚砷酸盐是哺乳动物细胞中热休克反应的普遍诱导剂：它激活热休克转录因子1（HSF1）的DNA结合活性，增强hsp 70启动子，并诱导hsp70mRNA及HSP伴侣蛋白的合成。通过高通量hsp70启动子-荧光素酶报告基因检测，我们观察到一种 hormetic 剂量反应，即低浓度亚砷酸盐刺激而高浓度抑制。此外，该反应具有时间依赖性，随着孵育时间延长，剂量反应向左移动。亚砷酸盐诱导hsp 70-荧光素酶报告基因的效应绝对需要功能性的HSF1，因为在HSF1缺失的细胞中未观察到该效应，但通过HSF1的表达得以恢复。与砷靶向邻近半胱氨酸-SH的观点一致，我们表明二硫苏糖醇可阻断亚砷酸盐的作用。细胞活力和半胱天冬酶检测表明，亚砷酸盐通过激活半胱天冬酶3/7导致细胞死亡呈剂量依赖性增加，而预先诱导HSPs可减弱这些效应。使用非锚定依赖性细胞生长作为晚期肿瘤促进检测方法，我们表明低浓度亚砷酸盐具有生长促进作用，适度热休克可增强该作用。我们的研究提供了证据，表明热休克反应的诱导是砷暴露的敏感生物标志物，并且HSPs的诱导可能有助于砷的肿瘤促进作用。
