DNA Damage and p53 Induction Do Not Cause ZD1694-induced Cell Cycle Arrest in Human Colon Carcinoma Cells¹

Using four complementary approaches, i.e., 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 µm 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 α 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 p53^mu 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 (p53^wt). 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 α 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.