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

Abstract

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.

Fig.1

Cytotoxicity and increase in M30 produced by Intron A in IFNα sensitive RT4 bladder cancer cells. A. M30 and M65 levels in the medium of the various treatments shown. No significant increase in M65 was seen compared to M30 levels with any treatment except for Ad-IFN. All values given are those in which the control M30 and M65 levels have been subtracted. Therefore both Intron A and staurosprine produced apoptotic cell death, whereas Ad-IFN caused both apoptotic and necrotic cell death as measured by M30 and M65. B. Phase-contrast micrographs illustrating cell death with numerous floating cells 48h after treatment of RT4 cells with the various concentrations of Intron A as well as Ad-IFN and staurosporine. A concentration dependent increase in cytotoxicity is seen ranging from 1000 to 10,000 IU/ml of Intron A. Magnification 400X. Ad-β-gal was not cytotoxic to the RT4 cells (not shown).

Fig.2

Cytotoxicity produced by CM from Ad-IFN treated bladder cancer and normal urothelial cells on IFNα resistant UC9 bladder cancer cells. A. Numerous floating dead cells are seen in UC9 cells 48h after plating with CM obtained from Ad-IFN treated UC9, KU7 and TERT-NHU cells. Magnification 400X B. An increase in M30 was found for each CM treatment shown in A, indicative of apoptotic cell death caused by the bystander factor(s) present in the CM. No increase in M65 levels were observed above that of M30. As expected, no increase in M30 was found following Intron A treatment, since UC9 cells are resistant to Intron A. Ad-IFN, however, produced increases in both M30 and M65 suggesting that Ad-IFN induces both an apoptotic and necrotic component of cell death.

Fig.3

CM from TERT-NHU cells obtained from Ad-IFN treatment is cytotoxic to UC9 cells and produce increases in M30. Shown in the left lower plate Ad-IFN was not cytotoxic to normal TERT-NHU cells. Magnification 400X. NHU-AdIFN-CM also produced no significant levels either of M30 or M65. However, when various preparations of NHU-AdIFN CM were added to UC9 cells for 72h or 96h, considerable cytotoxicity and increases, primarily in M30 levels were seen. Asterisks indicate results in which the M30 and M65 levels in the control CM results have been subtracted from the levels measured for each of the NHU-Ad-IFN-CM added to UC9 cells for 72 hr. The 96h results are the actual levels recorded with control levels not being subtracted since they were not determined.

Fig.4

Diagram outlining proposed mechanisms of Ad-IFN produced cancer cell death. Both normal urothelial and bladder cancer cells produce high and prolonged levels of IFNα following Ad-IFNα exposure which kills the tumor cells through an apoptotic, TRAIL-related mechanism if the cancer cells are sensitive to IFNα. If the cancer cells are resistant to IFNα they still can be killed by a TRAIL-unrelated apoptotic mechanism involving the production of bystander factor(s). Both cause an increase in M30 levels. A direct necrotic cancer cell death is also produced following Ad-IFN transfection and expression which is a reflected in an increase in M65 levels. Other mechanisms of direct cell death may also be involved such as secondary necrosis causing some of the increase in M65 levels.