We previously reported an indirect effect of adenoviral interferon α (Ad-IFNα) that is mediated by secreting bystander proteins (BSPs) and causing cytotoxic responses in both interferon α (IFNα)-sensitive and –resistant cancer cells. Here, we investigated whether glycosylation of BSPs is necessary for the inhibition of IFNα-resistant human bladder cancer cell line (KU7). The condition medium (CM) from the normal human urothelial cell line, TERT-NHUC, was collected as described previously and incubated with and without N-glycosidase F at 37°C (overnight). The growth inhibition of KU7 was compared between the control and experimental conditions. The proliferation of KU7 cells treated with interferon a protein (Intron A) is comparable to the cells treated with control TERT-NHUC-CM (without Ad-IFNα infection) after 72 hr incubation. The growth of KU7 was significantly inhibited by the CM from Ad-IFNα–infected TERT-NHUC culture. N-glycosidase F incubation effectively deglycosylated the BSPs in the CM (Ad-IFNα TERT-NHUC) as demonstrated by the band shifting in the PAGE gel verification. The deglycosylated CM (Ad-IFNα TERT-NHUC) produced a same degree of inhibition on the proliferation of KU7 cells (Fig 1). In conclusion, the cytotoxic effects of BSPs is independent of its status of glycosylation.
