Fig. 8. Cell culture model to study ferritin and DNA damage. To demonstrate that SW1088 astrocytoma cells in culture are susceptible to DNA damage when nuclear ferritin is not present, we used the following model. Intracellular ferritin was depleted by deferoxamine and then the cells were reincubated with standard media, which results in a reappearance of ferritin in the nucleus. However, one group of cells was exposed to wheat germ agglutinin (WGA), to block the translocation of ferritin to the nucleus, and the other group was not exposed to WGA. Both sets of SW1088 cells were treated with digitonin, which is necessary to allow WGA to enter the cells. DNA strand breaks are detected using the TUNEL assay (green; Boehringer Mannheim). DAPI staining (blue) was used to locate cell nuclei. The same microscopic field is represented in both right and left panels. (A) In this experiment, 100 µM H₂O₂ was added to the reincubation media after DFO pretreatment. If WGA was included (+WGA) in the reincubation media (to block ferritin translocation to the nucleus), most of the cells became TUNEL positive (green). If WGA was excluded from the reincubation media (-WGA) (permitting ferritin translocation to the nucleus), no TUNEL-positive cells were present. (B) In this experiment, ferric ammonium citrate (100 µM) was added to the reincubation media. There were no TUNEL-positive cells regardless of whether or not WGA was absent (-WGA) or present (+WGA) in the reincubation media. The micrographs have been chosen as representative. Experiments were repeated three times each.