Association of redox-active iron bound to high molecular weight structures in nuclei with inhibition of cell growth by H2O2

Perturbations to Fe species contributing to generation of DNA single-strand breaks (SSBs) and inhibition of growth by H2O2 were studied in HL-60 cells made Fe-deficient by 24 h pretreatment with 144 μM bathophenanthroline disulfonic acid and 400 μM ascorbic acid (Free Radic. Biol. Med. 20: 399; 1996). The diffusion distance for SSB generation (d) in Fe-deficient cells, measured via inhibition with the square, top filled0OH scavenger Me2SO using alkaline elution, was 6.5 nm. This is similar to the d for Fe-normal cells reported previously. After 1 and 3 h in fresh RPMI 1640 medium containing 10% serum, SSB generation increased from 29 to 56 and 93% of control Fe-normal cells, respectively. The d of the major contributor to SSB generation at these two treatment times was 1.9 nm. This d resembled the d for Fe-ATP as determined in isolated Ehrlich cell nuclei. The association of ATP with Fe2+ was further supported by decreased SSB generation in cells in which ATP synthesis was inhibited. In contrast to SSB generation, H2O2-induced inhibition of growth of Fe-deficient cells treated immediately after placing in fresh medium was not appreciably different from Fe-normal cells. However, after 3 h, an approximately 70% greater concentration of H2O2 than for control, Fe-normal cells was required to inhibit growth. This increase in H2O2 concentration was associated with decreased generation of SSBs by H2O2 in isolated HL-60 cell nuclei. Thus, Fe bound to nuclear structures is more closely associated with inhibition of cell growth than apparent Fe-ATP species. In parallel experiments, changes in total cellular Fe assayed by ashing and complexing with ferrozine were consistent with a non-transferrin mode of acquisition. These short-term changes appear due to processes accompanying reestablishment of the Fe content and distribution normally observed during long-term growth.