CONTRASTING PATTERNS OF DNA FRAGMENTATION INDUCED BY THYMIDYLATE SYNTHASE INHIBITORS, ZD1694 AND AG-331

The patterns of DNA fragmentation were evaluated following a brief exp osure (2 h) of the human ileocecal adenocarcinoma cell line, HCT-8, to several specific thymidylate synthase inhibitors, a quinazoline (ZD16 94) and benz[cd]indole-containing molecule (AG-33 I). The magnitude an d size of DNA fragmentation induced by the two agents were assessed by alkaline elution for DNA single-strand breaks (ssbs), and by pulsed- and constant-field gel electrophoresis for DNA double-strand breaks (d sbs). Both agents induced dose-dependent DNA dsbs. While AG-331 induce d ssbs and dsbs only in nascent DNA, ZD1694 affected both genomic and nascent DNA. The fragments of newly synthesized and genomic DNA, estim ated by pulsed-field gel electrophoresis assay, were associated with t he bands in the range of 0.05 to 1.1 and 1.1 to 5.7 megabases, respect ively. 5-fluoro-2'-deoxyuridine (FdUrd), like ZD1694, produced both ma ture and nascent DNA fragmentation, whereas only nascent DNA breakage induced by 5-fluorouracil (FUra) was detected, similar to AG-331. The induction of both mature and nascent DNA fragmentation by ZD1694 and F dUrd appears to correlate with the higher, but similar, potency of the se agents. Aphidicolin, a DNA polymerase inhibitor, protects from DNA dsbs and cytotoxicity by ZD1694 and AG-331. These observations suggest that replicative DNA synthesis is an important factor in ZD1694- and AG-331-induced DNA fragmentation and, subsequently, cell growth arrest . The results indicate that although the new antimetabolites investiga ted herein were developed and extensively evaluated as specific and po tent thymidylate synthase inhibitors, DNA damage appears to be an impo rtant additional determinant of drug effect. The newly synthesized DNA appears to be a common sensitive target of FUra, FdUrd, ZD1694, and A G-331; whereas the induction of mature DNA fragmentation is associated exclusively with FdUrd and ZD1694.