title: Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links.
abstract: Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity.
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and University College Medical School, 91 Riding House Street, London, W1W 7BS, UK. p.clingen@ucl.ac.uk <p.clingen@ucl.ac.uk>
authors.forename: Peter H
authors.initials: PH
authors.lastname: Clingen
authors.forename: Colin F
authors.initials: CF
authors.lastname: Arlett
authors.forename: John A
authors.initials: JA
authors.lastname: Hartley
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Parris
mesh_terms: D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000187:drug effects*; D004260:DNA Repair; D005347:Fibroblasts / Q000187:drug effects*; D006801:Humans; D008466:Mechlorethamine / Q000494:pharmacology; D014466:Ultraviolet Rays / Q000009:adverse effects; D014983:Xeroderma Pigmentosum / Q000473:pathology*
chemical_list: D003432:Cross-Linking Reagents; D008466:Mechlorethamine; D004247:DNA