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.