Fig. 1. Adv infection sensitizes human epithelial cells to TNF-mediated apoptosis. (A) HT-29 cells were infected with either Adv.ß-gal or Adv.empty (1.4x10¹⁰ v.p./ml) or treated with buffer only and then stimulated with either 100 ng/ml TNF or carrier only. Cells were collected 48 hours after TNF addition, stained for either caspase-cleaved cytokeratin and cleaved (active) caspase 3 (white bars) or annexin V/PI (gray bars), and analyzed by flow cytometry. Adv.ß-gal was used for all subsequent experiments. (B) Sample flow cytometry plots for anti-caspase-cleaved cytokeratin and anti-cleaved (active) caspase 3 in Adv-sensitized and control cells treated with TNF. (C) HT-29 cells were infected with two concentrations of Adv (0.7x10¹⁰ and 1.4x10¹⁰ v.p./ml) for 0, 1, 3, 6 and 12 hours. The total number of Adv copies per cell was quantified by PCR. Values represent mean total ß-gal DNA normalized to mean total ß-actin DNA for duplicate samples. Variance in the Adv copy values ranged from 10 to 30% of the mean value. In parallel, HT-29 cells subject to the same infection conditions were treated with 100 ng/ml TNF and collected after 48 hours. Cleaved caspase 3-cytokeratin measurements are plotted as the mean apoptosis (above control level) of three biological replicates. (D) HeLa cells and HT-29 cells were infected with increasing doses of Adv (expressed as multiplicities of infection, p.f.u./cell) and stimulated with TNF or carrier, similar to panel A. Cells were collected 24 hours (HeLa) or 48 hours (HT-29) post-TNF treatment. Cleaved caspase 3-cytokeratin measurements are plotted as the mean of three biological replicates ± s.e.m.