De Silva, Inusha Udani; (2002) Repair of DNA interstrand cross-links induced by anticancer agents in mammalian cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bifunctional agents, which produce DNA interstrand cross-links (ICLs), are among the most effective anitumour agents in clinical use. The cellular mechanisms that act to eliminate DNA ICLs in mammalian cells are still not well understood. Studies in bacteria and yeast indicate that both nucleotide excision repair (NER) and recombination are required for ICL removal and that double-strand breaks (DSBs) are produced as repair intermediates in yeast cells. To elucidate the mechanism of ICL repair in mammalian cells, repair of ICLs induced by major groove binding bifunctional anti-cancer agents nitrogen mustards, cisplatin and the novel pyrrolobenzodiazepine (PBD) based minor groove binding agent SJG-136 was investigated. The role of NER, homologous recombination (HR) and non-homologous end joining (NHEJ) pathways in the repair of ICLs was studied using a panel of Chinese Hamster Ovary (CHO) mutant cell lines. Results presented indicate that there is no unique mechanism for the repair of DNA ICLs, but the pathway involved depends on the type of ICL. Nitrogen mustard induced ICLs are incised or 'unhooked' by XPF and ERCC1 which are involved in NER and some types of recombination, but other NER factors are not required. DSBs are induced during processing of these ICLs and are subsequently repaired by a homologous recombination pathway involving the XRCC2 and XRCC3 genes. In contrast, unhooking of cisplatin induced ICLs required other NER factors in addition to XPF and ERCC1, DSBs were not induced and repair was dependent on functional XRCC2 and XRCC3. Compared to major groove binding agents SJG-136 was highly cytotoxic in the parent CHO cell line AA8, and the ICLs induced were poorly repaired. Repair of SJG-136 induced ICLs required the whole NER system in addition to XPF and ERCC1 and DSBs were not induced. XRCC2 and XRCC3 appear to play a role in the elimination of these adducts. The NHEJ pathway did not seem to play a major role in the repair of DNA lesions induced by any of the drugs studied.

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