title: A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation.
abstract: Chk1 is the major mediator in the activation of cell-cycle checkpoints in response to a variety of genotoxic stresses. We have previously shown that inhibition of Chk1 sensitizes tumor cells to topoisomerase inhibitors such as camptothecin and doxorubicin through abrogation of cell-cycle arrest (S or G2/M checkpoints). However, it was not clear whether inhibition of Chk1 could potentiate antimetabolites, a mainstay of cancer therapy, which confer genotoxic stress through a different mechanism than topoisomerase inhibitors. 5-Fluorouracil (5-FU) is the most widely used antimetabolite in the treatment of colorectal, breast and other major types of cancers. Here we demonstrate that 5-FU activates Chk1 and induces an early S-phase arrest. Chk1 downregulation abrogates this arrest and dramatically sensitizes tumor cells to the cytotoxic effects of 5-FU. 5-FU confers S-phase arrest through Chk1-mediated Cdc25A proteolysis leading to inhibition of Cdk2. Chk1 elimination stabilizes the Cdc25A protein and results in the abrogation of the S checkpoint and resumption of DNA synthesis, which leads to excessive accumulation of double-stranded DNA breaks. As a result, downregulation of Chk1 potentiates 5-FU efficacy through induction of premature chromosomal condensation followed by apoptosis. Interestingly, the profiles of various cell-cycle markers indicate that cells progress to early M phase to induce apoptosis after checkpoint abrogation. Yet, cells fail to increase their DNA content to 4N as revealed by FACS analysis, probably due to the dramatic induction of double-stranded DNA breaks and chromosomal fragmentation. This is significantly different from the cell-cycle profiles observed in the potentiation of topoisomerase inhibitors by Chk1 siRNA, which showed mitotic progression with 4N DNA content leading to mitotic catastrophe after abrogation of the S or G2 checkpoint. Thus, our results illustrate a novel mode of checkpoint abrogation and cell death conferred by Chk1 inhibition. Additionally, we show that Chk1 deficiency potentiates 5-FU efficacy through the preferential induction of the caspase-8 pathway and subsequent caspase-3 activation. In conclusion, we have clearly demonstrated that inhibition of Chk1 not only potentiates the toxicity of conventional DNA-damaging agents such as ionizing radiation and topoisomerase inhibitors, but also enhances the toxicity of antimetabolites in cancer cell lines. This discovery reveals novel scope of checkpoint abrogation and will significantly broaden the potential application of Chk1 inhibitors in cancer therapy if they do not potentiate the toxicity of 5-FU in normal cells.
authors.affiliation: Cancer Research, Abbott Laboratories, 100 Abbott Park Rd., Abbott Park, IL 60064-6101, USA.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Xiao
authors.forename: John
authors.initials: J
authors.lastname: Xue
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Sowin
authors.forename: Saul H
authors.initials: SH
authors.lastname: Rosenberg
authors.forename: Haiying
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000633:toxicity*; D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000502:physiology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000032:analysis / Q000187:drug effects; D004249:DNA Damage; D004261:DNA Replication / Q000502:physiology; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D005472:Fluorouracil / Q000633:toxicity*; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D000964:Antimetabolites, Antineoplastic; D034741:RNA, Small Interfering; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases; D005472:Fluorouracil