Fig. 10.

The signature of primary singlet oxygen directly generated by a photosensitizer. A. MKN-45 tumor cells received 1 μg/ml or 10 μg/ml photofrin, in the absence or presence of 100 μM AEBSF. The cells were illuminated with visible light for 20 min and were then subjected to three cycles of washing. Increasing percentages of pretreated cells were added to untreated cells and further incubated. The percentages of apoptotic cells were determined 4 h after the beginning of the coculture. B, C. Cells at the indicated densities were pretreated with 10 μg/ml photofrin, 20 min light, in the absence or presence of 100 μM AEBSF, washed in three cycles and added at increasing percentages to untreated cells. All assays had a density of 12 500 cells/100 μl assays during coculture. The percentages of apoptotic cells were determined after 5 h. The results from A show that the bystander inducing potential of directly applied singlet oxygen from a photosensitizer depends on the signature by the primary singlet oxygen plus secondary singlet oxygen. The signature of secondary singlet oxygen can be prevented by AEBSF. Part B and C shows that imprinting the signature of primary singlet oxygen is independent of cell density, whereas the induction of bystander effect inducing potential by secondary singlet oxygen requires a high cell density. Statistical analysis: A: The differences between the groups with respect to dependence on the percentage of pretreated cells are highly significant (p < 0.001). B: The differences between the two groups of curves are highly significant (p < 0.001). C: The differences between the assays containing 12 500 cells and the other three assays are highly significant (p < 0.001).