Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke

Abstract

Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose-response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34ng/ml for TPA, 0.050mg/ml for the 2R4F, 0.044mg/ml for the Bright cigarette, and 0.060mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P<0.0001), and between the single-tobacco cigarettes and the 2R4F (P=0.0008, 2R4F vs. Burley and P<0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.

Improved multiparametric scrape loading-dye transfer assay for a simultaneous high-throughput analysis of gap junctional intercellular communication, cell density and viability

“…The obtained EC 50 values for TPA in studied cell lines were comparable to range of the values published in previous studies 51,67 . Similarly, GJIC-inhibitory activities of fluoranthene and 18-β-glycyrrhetinic acid in WB-F344 cells evaluated by our innovative assay were similar with previously reported studies 26,52,68 . By screening GJIC stimulators, we confirmed that our multiparametric assay can be also used for assessment of chemopreventive agents, such as caffeic acid phenethyl ester 69 or sodium butyrate 70 , which were previously found to restore cell-cell communication in neoplastically transformed, GJIC-deficient WB-F344-ras cells.…”

“…As summarized in the Supplementary Table S3, some of these methods for GJIC assessment have been recently adapted for HTS and/or HCA/HCS. These assays are mostly based on commonly used dye-transfer techniques such as electroporation 21 , laser perforation 22 , microinjection 23 , microfluidic loading 24,25 , parachute/preloading assay [26][27][28] , or on a luminometric or fluorimetric sensing of specific molecules exchanged via gap junctions between donor and recipient cells 29,30 . However, these methods can require a special equipment (electroporating 21 , microscopic 22,27,28 , microinjecting 23 , microfludic 24,25 ), that is not commonly available in a laboratory, and proprietary, closed source software for high-throughput image analysis 21,22,26,28 , or rely on specialized transduced cell models 29,30 .…”

“…Within the both assays, the spread of the dye from donor to recipient cells can be monitored with an epifluorescence microscope. Both techniques represent a very good platform for HTS with a high level of automation and few manual steps [26][27][28] . However, as any other method for monitoring GJIC, also these techniques possess some disadvantages ( Supplementary Table S3), including: (1) need for the trypsinization of the donor cells, which itself might be considered as a relatively invasive step, (2) the need for formation of gap junction channels between donor and recipient cells during the chemical exposures and limited opportunities to study rapid mechanisms of GJIC inhibition via gating of gap junction channels), (3) a requirement of a relatively long time lag for the reattachment of cells (15 min-5 h), (4) the ability of the cells to form gap junctions in these assays depends enormously on the cell type, (5) gap junction-nonspecific dye transfer of calcein has been observed, (6) and calcein can be actively pumped out from some cell types by multidrug resistance proteins 19,33 .…”

“…In drug and bioactive compound screening, chemicals and samples altering GJIC only at cytotoxic concentrations might not be of interest for the treatment of diseases, such as ischemia or cancer. Finally, Hoechst 33342 and propidium iodide staining to quantify total and dead cells should be compatible also with other HCA/HCS methods used for GJIC assessment, as demonstrated for parachute assay and evaluation effects of cigarette smoke 26 .…”

“…The evaluation of 216 images from a 24-well plate (24 wells × 3 cuts × 3 channels) can be accomplished within minutes compared to hours of manual analysis. Our image analysis tools were developed within an open source software package Fiji/ImageJ and they are publicly available to potential future users, which represents another benefit in contrast to other existing semi−/ automated tools for image-based GJIC analysis, which are usually based on Matlab 22,48,49 or other proprietary, closed source software 26,28 .…”