In vivo photoreaction of a chiral cobalt complex: DNA cleavage by Co(DIP)_3^(3+) in mammalian cells

Abstract

The chiral complex tris (diphenylphenanthroline) cobalt (III) (Co(DIP)_3^(3+) provides a photoreactive probe for chromatin structure in mammalian cells. The complex, which upon photoactivation cleaves DNA in a conformation-specific fashion in vitro, is shown also to cleave DNA in vivo upon irradiation with ultraviolet light (>300 nm). Δ- and Λ-Co (DIP)_3^(3+) isomers are taken up efficiently into cultured Chinese hamster ovary cells and concentrate within cell nuclei. In the absence of light the complexes are toxic to the cells (10% survival at ∼ 300 nM), but after ultraviolet irradiation, the toxicity is markedly (>10-fold) increased. The synergism between irradiation and Co(DIP)_3^(3+) administration may lie in photoreactions with DNA elicited by the cobalt complex. Alkaline sucrose gradient of DNA from cells exposed to Λ-Co(DIP)_3^(3+) and irradiation show single-stranded DNA fragmentation under conditions where little cleavage is seen in cells either incubated with Λ-Co(DIP)_3^(3+) or irradiated with > 300 nm A ultraviolet light. Cellular DNA is cleaved with lower efficiency than naked DNA, likely due to decreased accessibility of sites in vivo. Hybridization of fragments obtained from the alkaline sucrose gradients to a probe specific for the amplified dihydrofolate reductase gene reveals a similar distribution of dhfr sequences and total DNA, indicating that the family of conformations recognized by Λ-Co(DIP)_3^(3+) are dispersed throughout the genome.