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Contrasting patterns of DNA fragmentation induced by thymidylate synthase inhibitors, ZD1694 and AG-331
1Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Abstract:
The patterns of DNA fragmentation were evaluated following a brief exposure (2 h) of the human ileocecal adenocarcinoma cell line, HCT-8, to several specific thymidylate synthase inhibitors, a quinazoline (ZD1694) and benz[cd]indole-containing molecule (AG-331). The magnitude and 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 (dsbs). Both agents induced dose-dependent DNA dsbs. While AG-331 induced ssbs and dsbs only in nascent DNA, ZD1694 affected both genomic and nascent DNA. The fragments of newly synthesized and genomic DNA, estimated by pulsed-field gel electrophoresis assay, were associated with the bands in the range of 0.05 to 1.1 and 1.1 to 5.7 megabases, respectively. 5-fluoro-2'-deoxyuridine (FdUrd), like ZD1694, produced both mature 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 FdUrd appears to correlate with the higher, but similar, potency of these 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 investigated herein were developed and extensively evaluated as specific and potent thymidylate synthase inhibitors, DNA damage appears to be an important additional determinant of drug effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Specific thymidylate synthase inhibitors, ZD1694 and AG-331, induce DNA double-strand breaks in HCT-8 cells. DNA damage, particularly in nascent DNA, correlates with drug potency and cell growth arrest.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Thymidylate synthase inhibitors are crucial in cancer therapy.
- Understanding drug-induced DNA damage is key to optimizing cancer treatments.
Purpose of the Study:
- To investigate DNA fragmentation patterns induced by thymidylate synthase inhibitors ZD1694 and AG-331 in HCT-8 cells.
- To assess the role of replicative DNA synthesis in drug-induced DNA damage and cytotoxicity.
Main Methods:
- Alkaline elution for DNA single-strand breaks (ssbs).
- Pulsed- and constant-field gel electrophoresis for DNA double-strand breaks (dsbs).
- Treatment with ZD1694, AG-331, 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorouracil (FUra), and aphidicolin.
Main Results:
- Both ZD1694 and AG-331 induced dose-dependent DNA dsbs.
- ZD1694 fragmented both genomic and nascent DNA, while AG-331 primarily affected nascent DNA.
- FdUrd mimicked ZD1694's effect, and FUra mimicked AG-331's effect.
- Aphidicolin protected against DNA dsbs and cytotoxicity, indicating the role of DNA polymerase.
Conclusions:
- Replicative DNA synthesis is critical for ZD1694- and AG-331-induced DNA fragmentation and cell growth arrest.
- DNA damage is a significant determinant of drug effect, beyond thymidylate synthase inhibition.