DNA damage and p53 induction do not cause ZD1694-induced cell cycle arrest in human colon carcinoma cells.
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.
Matsui
S I
SI
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263-0001, USA.
Arredondo
M A
MA
Wrzosek
C
C
Rustum
Y M
YM
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*
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