title: The CHO XRCC1 mutant, EM9, deficient in DNA ligase III activity, exhibits hypersensitivity to camptothecin independent of DNA replication.
abstract: We have analyzed the X-ray-sensitive CHO mutant cell line EM9 for sensitivity to the topoisomerase I inhibitor comptothecin. These cells exhibit defective repair of single strand DNA breaks. Recently, EM9 were complemented the DNA ligase III interactive protein, XRCC1. Defective XRCC1 apparently accounts for the low DNA ligase III activity that may explain the single-strand break repair deficiency of EM9 cells. Here, we demonstrate cytotoxic hypersensitivity of EM9 cells following a brief camptothecin treatment. Both the S-phase and non-S-phase populations of EM9 exhibited camptothecin sensitivity relative to the parent cell line AA8. In AA8 cells, only the 55% of the population corresponding to the S-phase subpopulation were sensitive to camptothecin, while the remainder of the population were totally resistant to doses as high as 10 microM. The role of DNA replication in the camptothecin sensitivity was studied using the DNA polymerase inhibitor aphidicolin in co-treatment with camptothecin. Aphidicolin treatment fully protected AA8 cells from camptothecin cytotoxicity. In EM9 cells, aphidicolin protected the S-phase fraction to some degree but all the cells remained sensitive to camptothecin cytotoxicity. These results suggest that EM9 cells are sensitized to camptothecin by a mechanism that is independent of DNA replication and may be a consequence of the XRCC1 mutation or the associated deficiency in DNA ligase III activity. Mechanistic models for the replication-independent cytotoxicity of camptothecin in EM9 cells are discussed.
authors.affiliation: Department of Pharmacology and Toxicology, University of Utah, Salt Lake City 84112, USA. lbarrows@deans.pharm.utah.edu
authors.forename: L R
authors.initials: LR
authors.lastname: Barrows
authors.forename: J A
authors.initials: JA
authors.lastname: Holden
authors.forename: M
authors.initials: M
authors.lastname: Anderson
authors.forename: P
authors.initials: P
authors.lastname: D'Arpa
mesh_terms: D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D015153:Blotting, Western; D016466:CHO Cells; D002166:Camptothecin / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D017353:Gene Deletion; D009154:Mutation; D000075223:Poly-ADP-Ribose Binding Proteins; D016196:S Phase / Q000187:drug effects; D059004:Topoisomerase I Inhibitors; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins
chemical_list: D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D059004:Topoisomerase I Inhibitors; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins; D016590:Aphidicolin; D004264:DNA Topoisomerases, Type I; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; D002166:Camptothecin