title: Fludarabine triphosphate inhibits nucleotide excision repair of cisplatin-induced DNA adducts in vitro.
abstract: Fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clinically active against chronic lymphocytic leukemia and low-grade lymphomas. We reported previously that fludarabine nucleoside synergistically enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that the synergism was concomitant with inhibition of removal of cellular CDDP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair. To extend our work, we investigated whether fludarabine inhibits nucleotide excision repair (NER) of CDDP-induced DNA intrastrand adducts. The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-cell extracts from repair-competent cells. To prevent the cell-bound high mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group box proteins by immunoprecipitation prior to the assay. Repair synthesis, measured by the incorporation of [(32)P]dATP or [(32)P]dCTP, was inhibited by 50% at 26 or 43 microM fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-patch elongation. These results were consistent with those from pulse-chase experiments demonstrating the conversion of nicked circular plasmid to the closed circular form by cell extracts filling the repair gaps. When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at the incision/excision stage, 100 microM fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circular damaged plasmid substrate; the inhibition was dose dependent. We conclude that fludarabine triphosphate inhibited NER at the steps of incision and repair synthesis. These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor efficacies of combination regimens containing agents that induce NER.
authors.affiliation: Division of Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
authors.forename: L
authors.initials: L
authors.lastname: Li
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: A B
authors.initials: AB
authors.lastname: Glassman
authors.forename: M J
authors.initials: MJ
authors.lastname: Keating
authors.forename: M
authors.initials: M
authors.lastname: Stros
authors.forename: W
authors.initials: W
authors.lastname: Plunkett
authors.forename: L Y
authors.initials: LY
authors.lastname: Yang
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002945:Cisplatin / Q000378:metabolism; D003568:Cytidine Monophosphate / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects*; D006609:High Mobility Group Proteins / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D018736:DNA Adducts; D006609:High Mobility Group Proteins; D018809:Proliferating Cell Nuclear Antigen; C057270:cisplatin-DNA adduct; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D003568:Cytidine Monophosphate; D014740:Vidarabine; C024352:fludarabine; D002945:Cisplatin