Development of HSPA1A promoter-driven luciferase reporter gene assays in human cells for assessing the oxidative damage induced by silver nanoparticles

Xin, Lili; Wang, Jianshu; Zhang, Leshuai W.; Che, Bizhong; Dong, Guoying; Fan, Guoqiang; Cheng, Kaiming

doi:10.1016/j.taap.2016.05.010

Cited by 14 publications

(11 citation statements)

References 32 publications

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“…Target genes like HSPA1A are strongly induced by this transcription factor, however, their promoters typically integrate signals from several different signalling pathways [17,18]. HSPA1A reporter cell lines have already been used to detect cellular stress [37–39] and the response to heavy metals [40], however, in this study we intended to concentrate on the effect of heavy metals solely on the HSR. An ideal tool for this purpose is the artificial HSE promoter, consisting exclusively of consensus HSEs [28].…”

Section: Discussionmentioning

confidence: 99%

HSF1 mediated stress response of heavy metals

et al. 2018

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The heat shock response (HSR) pathway is a highly conserved cellular stress response and mediated by its master regulator HSF1. Activation of the pathway results in the expression of chaperone proteins (heat shock proteins; HSP) to maintain protein homeostasis. One of the genes strongest upregulated upon stress is HSPA1A (HSP72). Heavy metals are highly toxic to living organisms and known as environmental contaminants, due to industrialisation. Furthermore, many of them are well-described inducers of the HSR pathway. Here we compare the effect of different heavy metals, concerning their potential to activate HSF1 with a sensitive artificial heat shock reporter cell line, consisting of heat shock elements (HSE). In general the responses of the artificial promoter to heavy metal stress were in good agreement with those of well-established HSF1 target genes, like HSPA1A. Nevertheless, differences were observable when effects of heat and heavy metal stress were compared. Whereas heat stress preferentially activated the HSE promoter, heavy metals more strongly induced the HSPA1A promoter. We therefore analysed the HSPA1A promoter in more detail, by isolating and mutating the HSEs. The results indicate that the importance of the individual binding sites for HSF1 is determined by their sequence similarity to the consensus sequence and their position relative to the transcription start site, but they were not differentially affected by heat or heavy metal stress. In contrast, we found that other parts of the HSPA1A promoter have different impact on the response under different stress conditions. In this work we provide deeper insights into the regulation of HSP72 expression as a well as a method to quantitatively and sensitively evaluate different stressor on their potential to activate HSF1.

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Section: Discussionmentioning

confidence: 99%

HSF1 mediated stress response of heavy metals

et al. 2018

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“…The cellular uptake and localization of AgNPs were detected by TEM and sector field inductively coupled plasma mass spectrometry (SF‐ICP‐MS, Thermo Fisher, Waltham, MA) . For TEM analysis, the HepG2‐ and A549‐luciferase cells were treated with 0 or 25 µg/mL AgNPs for 48 hours.…”

Section: Methodsmentioning

confidence: 99%

“…After the treatments, 100 µL of lysis buffer was added to each well. The luciferase activities were measured using a SYNERGY 2 microplate reader (Bio‐Tek, USA) as described previously . The results were expressed as relative luciferase activity versus control (ratio of the luciferase activities [test sample/control group]).…”

Section: Methodsmentioning

confidence: 99%

“…The cell viability was determined by using the CCK‐8 kit as described previously . Cells were seeded in 96‐well culture plates (8 × 10 3 cells per 100 µL growth medium/well) followed by an overnight incubation.…”

Section: Methodsmentioning

confidence: 99%

“…The amount of released Ag + in DMEM culture medium was determined by SF‐ICP‐MS . Different concentrations of AgNPs (12.5, 25, 50, 100, 200 µg/mL) were prepared in complete cell culture medium and kept at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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