Conditioned medium from Ad-IFN-α-infected bladder cancer and normal urothelial cells is cytotoxic to cancer cells but not normal cells: further evidence for a strong bystander effect

We have reported earlier that a bystander effect is seen in cancer cells that are resistant to high concentrations of the interferon-alpha protein (Intron A) when treated with adenoviral-mediated interferon-alpha (Ad-IFN-alpha). We now provide further evidence for this bystander effect using conditioned medium (CM) collected from Ad-IFN-alpha-infected cancer and normal urothelial cells. The CMs collected from UC-9 and KU7 bladder cancer cells as well as normal urothelial cells following transfection with Ad-IFN produce cell death when added to various cancer cell types in culture but not to normal urothelial cells. The CM could be filtered, frozen and thawed, and diluted to at least one part Ad-IFN CM to five parts fresh control medium and the diluted CM still shows a similar cytotoxicity as a 100% concentration of Ad-IFN CM. This cytotoxicity was observed by both flow cytometry and MTT assays as well as by phase microscopy, and a significant sub-G1 population was seen whether the CM was collected 48, 72 or 96 h after initial Ad-IFN treatment. In addition, the CM could be partially inactivated by exposure to 65 degrees C for 30 min and totally inactivated by placement at 92 degrees C for 3 min, whereas Intron A was not inactivated under the same conditions. Importantly, although significant caspase 8 and caspase 9 cleavage occurred in Ad-IFN-treated cells as a direct effect of Ad-IFN transfection, the Ad-IFN CM produced no activation of caspase 8 and caspase 9, indicating that a different mechanism of cell death was produced by the bystander factor(s) than the direct effect of Ad-IFN. This bystander effect in turn may play an important role in the efficacy of the current Ad-IFN clinical trial for superficial bladder cancer now underway.