title: Replication-dependent irreversible topoisomerase 1 poisoning is responsible for FdUMP[10] anti-leukemic activity.
abstract: Previous studies have indicated that 5-Fluoro-2'-deoxyuridine-5'-O-monophosphate 10mer (FdUMP[10]) displays strong antileukemic activity through the dual targeting of thymidylate synthase (TS) and DNA topoisomerase 1 (Top1). The present studies were undertaken to clarify the relationship between the induction of a thymineless state and the formation of Top1 cleavage complexes (Top1CC) for inducing cell death and to clarify the role of DNA replication for induction of lethal DNA double-strand breaks (DSBs) in FdUMP[10]-treated acute myeloid leukemia (AML) cells. Human promyelocytic (HL60) and AML (KG1a, Molm13, THP-1) cells were synchronized by serum starvation and treated with FdUMP[10] with thymidine (Thy) rescue. Cells were assayed for TS inhibition, DNA DSBs, Top1CC, and apoptosis to clarify the interrelationship of TS inhibition and Top1CC for cell death. FdUMP[10] induced a thymineless state in AML cells and exogenous Thy administered within the first 18 hours of treatment rescued FdUMP[10]-induced Top1CC formation, γH2AX phosphorylation, and apoptosis induction. Exogenous Thy was not effective after cells had committed to mitosis and undergone cell division in the presence of FdUMP[10]. FdUMP[10] treatment resulted in Chk1 activation, and Chk1 inhibition enhanced FdUMP[10]-induced DNA damage and apoptosis. Jnk-signaling was required for FdUMP[10]-induced apoptosis in promyelocytic HL60 cells and in THP1 cells, but was antiapoptotic in Molm13 and to a lesser extent KG1a AML cells. The results are consistent with FdUMP[10] inducing a thymineless state, leading to misincorporation of FdU into genomic DNA of proliferating cells. Top1CC form in cells upon re-entry into S-phase, resulting in DNA double-strand breaks, and initiating apoptotic signaling that can be either muted or enhanced by Jnk-signaling depending on cell type.
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA.
authors.forename: Jamie
authors.initials: J
authors.lastname: Jennings-Gee
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Pardee
authors.forename: William H
authors.initials: WH
authors.lastname: Gmeiner
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects*; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D004273:DNA, Neoplasm / Q000378:metabolism; D005468:Fluorodeoxyuridylate / Q000494:pharmacology*; D018922:HL-60 Cells / Q000187:drug effects; D006801:Humans; D007948:Leukemia, Monocytic, Acute / Q000473:pathology; D015473:Leukemia, Promyelocytic, Acute / Q000473:pathology; D016196:S Phase / Q000187:drug effects; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D013941:Thymine / Q000378:metabolism / Q000494:pharmacology; D059003:Topoisomerase Inhibitors / Q000494:pharmacology*
chemical_list: D000470:Alkaloids; D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D059003:Topoisomerase Inhibitors; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D013941:Thymine; C404329:SB 218078