Ad.TERT.Tβ₁₀-induced increase in cancer-specific FAS expression in human ovarian cancer cells, resulting in apoptosis.

<p>(A) Subconfluent 2774 human ovarian cancer cells were exposed to Ad.TERT.LacZ or Ad.TERT.Tβ₁₀ at an MOI of 10 for 48 hr and followed by next experiments such as RT-PCR and Western blotting to detect FAS (CD95) mRNA and protein expression, respectively. (B) Subconfluent primary human ovarian cancer cells co-cultured with fibroblasts were also exposed to 10 MOI of Ad.TERT.Tβ₁₀ for 24 hr and followed by immunocytochemistry. Specific FAS/CD95 antibody staining (Alexa 488, green) was merged with differential interference contrast (DIC) images and nuclear staining images (DAPI, blue) using imaging software. White and orange arrows indicate fibroblasts and ovarian cancer cells, respectively. (C) Cells were exposed to Ad.TERT.LacZ or Ad.TERT.Tβ₁₀ at an MOI of 10 with or without caspase inhibitor (zVAD, 40 µM), and actin polymerization inhibitor (cytochalasin D, 1 µg/ml) for 48 hr. The 2774 ovarian cancer cells were then stained according to the commercial caspase 3 assay kit protocol (Thermo) and imaged using a Thermo Scientific Cellomics ArrayScan high-content screening (HCS) reader. In the bar graph, the values representing a percentage of the vehicle control is followed by the ratio between the area of caspase 3 activation and the area of the nucleus. And the results are mean ± SE (bars) of three different experiments. Significant differences from vehicle group are denoted by a double asterisk (**), P<0.01 (left bar graph). Cytotoxicity under the same conditions was determined in cultured ovarian cancer cells using the alamar Blue (AB) assay as an <i>in vitro</i> alternative model. The results are expressed as a percentage of the vehicle control and are mean ± SE (bars) of three experiments. Significant differences from vehicle control group are denoted by an asterisk (*), P<0.05; a double asterisk (**), P<0.01 (right bar graph)