Development of a stable dual cell‐line GFP expression system to study estrogenic endocrine disruptors

Abstract

Environmental estrogenic endocrine disruptors are a health concern. Here we constructed a dual cell-line green fluorescence protein (GFP) expression system to identify and study endocrine disrupting compounds with activities of estrogen receptor agonists or antagonists. Human breast cancer MCF-7 cells and endometrial carcinoma Ishikawa cells were infected with a two tandem estrogen response elements--E4 promoter-GFP reporter gene construct. The use of GFP reporter enabled direct and simple evaluations of cell responses. GFP intensity in stably transfected MCF7-GFP and Ishikawa-GFP cells was dose-responsive to 17-beta-estradiol, diethylstilbestrol, 2-hydroxyestradiol, and environmental toxins bisphenol A, genistein and o-p'-DDT. Raloxifene and tamoxifen were effective antiestrogens in MCF7-GFP cells, but acted as partial estrogen receptor agonists in Ishikawa-GFP cells at concentrations of 0.1 nM and above. No synergistic effect was observed in chemical combinations between organochlorine pesticides methoxychlor, o-p'-DDT, p-p'-DDT, nor between estradiol and estrone. In summary, for the first time the effects of estrogen receptor agonists or antagonists were compared between mammary and endometrial cancer cells both stably expressing identical plasmids with GFP reporter genes under the control of tandem estrogen response elements. This dual cell-line system provides a rapid method and sensitive assay to identify environmental estrogens, antiestrogens, selective estrogen receptor modulators and to study their tissue specific effects and chemical interactions. Such a system is especially useful for direct and parallel toxicity assessments with a microfluidic cell culture device.

Could in-vitro studies on Ishikawa cell lines explain the endometrial safety of raloxifene? Systematic literature review and starting points for future oncological research

“…TAM and RAL acted as estrogenic agonists and generated significant estrogenic responses in Ishikawa-GFP cells. Data suggested TAM and RAL stimulatory effects on ERERE-mediated gene transcriptions in Ishikawa cells (Xu et al, 2008). Flamini et al (2009) reported that TAM activates an ER/Ga13/RhoA/ROCK/moesin signaling pathway in Ishikawa cells and in primary cultured stromal cells.…”

“…E2 increased VEGF gene transcription and protein secretion, but RAL did not influence the level of VEGF (Dai et al, 2009). Xu et al (2008) used TAM and RAL to treat human breast cancer MCF7 cells and Ishikawa cells that were infected with a two tandem estrogenic response element (E4 promoter-GFP reporter gene construct). TAM and RAL acted as estrogenic agonists and generated significant estrogenic responses in Ishikawa-GFP cells.…”

‘Body-on-a-Chip’ Technology and Supporting Microfluidics

“…18,19,21,22 Advances in genetic manipulation technology, particularly using stem cells, 23 also holds exciting possibilities for 'body-on-achip' devices because uorescent reporters bound to promoters for specic biochemical markers can be used to study whether drug treatment causes alterations in phenotypic expression patterns. 24 Integration of microelectrode arrays into multi-organ devices provides the means to drastically increase the analytical power of such systems. Measurement of the electrophysiological properties of cell types such as neurons, cardiomyocytes and skeletal muscle permits direct functional assessment of these cells in real time.…”

Body-on-a chip: Using microfluidic systems to predict human responses to drugs

“…[6,8,9] for examples). Optical measurements have also been made of glutathione levels [6,10,11], uptake of a drug [8], and expression levels of protein using green fluorescent protein (GFP) [18]. In other systems, electrical measurements have been used [3,[19][20][21] in combination with optical measurements.…”