DNA-DAMAGE AND P53 INDUCTION DO NOT CAUSE ZD1694-INDUCED CELL-CYCLE ARREST IN HUMAN COLON-CARCINOMA CELLS

Using four complementary approaches, i.e., cell synchronization, bromo deoxyuridine labeling, and DNA and Western blot analyses, we investiga ted 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 mu M for 2 h, ZD1694 completely inhibits thymidylate synthase over 72 h and causes a sustai ned 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 trea tment in the cell cycle, their DNA-replicating machinery associated wi th polymerase alpha was preserved intact. When supplemented with exoge nous dThd, these cells resumed an apparently normal S-phase progressio n for at least 4 h. Kinetic analyses based on synchronized cells indic ate that S-phase arrest occurs first, preceding the induction of DNA d ouble strand breaks and p53/p21. SW480 cells, in which p53(mu) failed to transduce p21, also exhibited the mode of S-phase arrest, essential ly indistinguishable from that displayed by HCT-8 cells expressing the functional p53 (p53(wt)). That the DNA replication process is prerequ isite for DNA double strand breaks was indicated by the following: (a) DNA damage occurred only when cells treated with ZD1694 progressed th rough S phase; and (b) the inhibition of DNA polymerase alpha by aphid icolin-blocked DNA damage. Based on the above, we conclude that S-phas e 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 G(1)-G(2) checkpoint mechan isms or p21-induced inactivation of the DNA-replicating machinery.