title: DNA damage and p53 induction do not cause ZD1694-induced cell cycle arrest in human colon carcinoma cells.
abstract: Using four complementary approaches, ie., cell synchronization, bromodeoxyuridine labeling, and DNA and Western blot analyses, we investigated the underlying mechanism of cell cycle perturbation in response to ZD1694, a quinazoline-based antifolate thymidylate synthase inhibitor. With a single exposure at a concentration of 1 microM for 2 h, ZD1694 completely inhibits thymidylate synthase over 72 h and causes a sustained growth for at least 120 h, DNA damage, and p53 induction in human carcinoma cells. Although these cells displayed an S-phase block with the precise terminal arrest point depending on the timing of drug treatment in the cell cycle, their DNA-replicating machinery associated with polymerase alpha was preserved intact. When supplemented with exogenous dThd, these cells resumed an apparently normal S-phase progression for at least 4 h. Kinetic analyses based on synchronized cells indicate that S-phase arrest occurs first, preceding the induction of DNA double strand breaks and p53/p21. SW480 cells, in which p53mu failed to transduce p21, also exhibited the mode of S-phase arrest, essentially indistinguishable from that displayed by HCT-8 cells expressing the functional p53 (p53wt). That the DNA replication process is prerequisite for DNA double strand breaks was indicated by the following: (a) DNA damage occurred only when cells treated with ZD1694 progressed through S phase; and (b) the inhibition of DNA polymerase alpha by aphidicolin-blocked DNA damage. Based on the above, we conclude that S-phase arrest by ZD1694, with a subsequent damage of DNA double strands, is caused by the block of DNA synthesis in the middle of replication due to dTTP depletion and not by p53-mediated G1-G2 checkpoint mechanisms or p21-induced inactivation of the DNA-replicating machinery.
authors.affiliation: Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263-0001, USA.
authors.forename: S I
authors.initials: SI
authors.lastname: Matsui
authors.forename: M A
authors.initials: MA
authors.lastname: Arredondo
authors.forename: C
authors.initials: C
authors.lastname: Wrzosek
authors.forename: Y M
authors.initials: YM
authors.lastname: Rustum
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D004273:DNA, Neoplasm / Q000187:drug effects; D016193:G1 Phase / Q000187:drug effects; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D011799:Quinazolines / Q000494:pharmacology*; D016196:S Phase / Q000187:drug effects; D013876:Thiophenes / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
chemical_list: D000964:Antimetabolites, Antineoplastic; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D011799:Quinazolines; D013876:Thiophenes; D016159:Tumor Suppressor Protein p53; D013940:Thymidylate Synthase; C068874:raltitrexed