Measuring soluble forms of extracellular cytokeratin 18 identifies both apoptotic and necrotic mechanisms of cell death produced by adenoviral-mediated interferon alpha: possible use as a surrogate marker.

Adenoviral transduction of human bladder cancer cells with human interferon alpha-2b (Ad-IFN) produces cancer-specific cell death through direct and indirect mechanisms. The indirect mechanisms involve the secreted IFN produced, which kill, IFN protein-sensitive cancer cells, as well as yet unidentified bystander factors, which are cytotoxic to neighboring cancer cells. The direct cell kill results from transfection and expression of Ad-IFN in the cancer cells. As the molecular forms of cytokeratin 18, either caspase cleaved or not, have been associated with apoptotic or necrotic cell death, respectively, we determined if increases in either or both cytokeratin 18 forms could be observed following IFNalpha protein or Ad-IFN treatment of bladder carcinoma cells. Quantification of M30 and M65 enzyme-linked immunosorbent assays (assays for cytokeratin 18 associated apoptotic and necrotic cell death, respectively) were used as surrogate markers of the cell death produced. In the IFN protein-sensitive RT4 bladder cancer cells, IFN produced primarily M30-related cell death, whereas Ad-IFN treatment resulted in high levels of both M30 and M65. In contrast, conditioned medium from Ad-IFN-treated cells whether from normal human urothelial cells or bladder cancer cells caused increases mainly in M30 levels when added to IFN protein resistant KU7 or UC9 bladder cancer cells, suggesting that the bystander factors present in the conditioned medium produced primarily apoptotic cell death. In addition, a significant increase in M65 levels above that observed for M30 was seen when the IFN protein resistant KU7 and UC9 cells were treated with Ad-IFN, again indicating there is additional necrotic-related cell death produced by Ad-IFN as well. Normal urothelial cells showed no cytotoxicity nor increases in M30 or M65 after Ad-IFN treatment. As intravesical Ad-IFN treatment is presently being evaluated for its efficacy in superficial bladder cancer measurement of M30 and M65 levels in the urine at various time points before and after Ad-IFN treatment may provide not only a biomarker of efficacy but also evidence for the different types and proportion of cell kill produced by the various mechanisms of cell kill in the tumors of individual patients.