Cellular interaction between DNA topoisomerase I and DNA-PKcs and its potential role in the sensitivity to camptothecin. Free

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Topoisomerase I (top1) is a nuclear enzyme involved in the removal of torsional constraints associated with DNA replication and transcription. It cleaves the DNA on one strand and forms covalent top1-DNA cleavage complexes which are the target of top1 inhibitors such as camptothecins (CPT) which are widely used in the treatment of human cancers. It is known that cleavage complexes are not toxic per se and that their conversion into lethal DNA double-strand breaks (DSBs) involves a collision with advancing replication forks. The resulting lesion contains a single free DNA double-strand end. In mammalian cells, this kind of lesions trigger Homologous Recombination rather than Non-Homologous End-Joining (NHEJ). However, previous studies have shown that M059J and CHO-V3 NHEJ-deficient cells were hypersensitive to CPT. These cells are deficient for the catalytic subunit of the DNA-dependent protein kinase complex (DNA-PKcs), which kinase activity is essential for NHEJ. Moreover, DNA-PKcs was shown to co-immunoprecipitate with top1. In light of these data, we investigated whether top1-DNA-PKcs interaction could play a role in CPT-induced cytotoxicity.

We checked by crossed immunoprecipitations that DNA-PKcs could indeed interact with top1 of HeLa cells and further showed that top1 and DNA-PKcs co-localized in the nuclei of cells, which indicates that this interaction could take place in vivo. We then showed that the amount of DNA-PKcs-top1 complexes was dramatically reduced when cells were treated by CPT and that pre-treatment with the replication inhibitor aphidicolin could prevent the dissociation of top1 from DNA-PKcs. This indicated that DNA-PKcs interacts predominantly with the free form of top1. Even though the mechanism by which DNA-PKcs-top1 interaction could modulate the cellular sensitivity to CPT is unknown, we can rule out the possibility that phosphorylation of top1 by DNA-PKcs is involved. This relies on previous studies showing that NU7026, a potent inhibitor of the kinase activity of DNA-PKcs, could not sensitize cells to CPT and that no difference in CPT sensitivity could be observed between CHO V3KA4 cells expressing a kinase-dead DNA-PKcs and control cells. Thus, top1-DNA-PKcs interaction rather than phosphorylation of top1 by DNA-PKcs is important for the regulation of CPT-induced cell death. Together these data also suggest that NHEJ is not involved in the repair of CPT-induced DSBs. One hypothesis might be that top1-DNA-PKcs interaction could control the levels of free nuclear top1 and regulate the amount of top1 poisoning by endogenous DNA damage or by CPT. Our preliminary data showing increased basal levels of top1-DNA cleavage complexes in M059J cells as compared to M059K control cells in the absence of drug is in favour of this hypothesis and need further investigations.