DNA replication is required to elicit cellular responses to psoralen-induced DNA interstrand cross-links

Following introduction of DNA interstrand cross-links (ICLs), mammalian cel ls display chromosome breakage or cell cycle delay with a 4N DNA content. T o further understand the nature of the delay, previously described as a G(2 )/M arrest, we developed a protocol to generate ICLs during specific interv als of the cell cycle. Synchronous populations of G(1), S, and G(2) cells w ere treated with photoactivated 4'-hydroxymethyl-4,5',8- trimethylpsoralen (HMT) and scored for normal passage into mitosis, In contrast to what was f ound for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage a nd arrest in the next cell cycle. Similarly, ICLs introduced during G(1) di d not cause a G(1)/S arrest. We conclude that DNA replication is required t o elicit the cellular responses of cell cycle arrest and genomic instabilit y after psoralen-induced ICLs, In primary human fibroblasts, the 4N DNA con tent cell cycle arrest triggered by ICLs was long lasting but reversible. K inetic analysis suggested that these cells could remove up to similar to 2, 500 ICLs/genome at an average rate of 11 ICLs/genome/h.