Superresolution imaging of individual replication forks reveals unexpected prodrug resistance mechanism.

Autor:	Triemer T; Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland., Messikommer A; Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland., Glasauer SMK; Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland., Alzeer J; Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland., Paulisch MH; Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland., Luedtke NW; Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland nathan.luedtke@chem.uzh.ch.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2018 Feb 13; Vol. 115 (7), pp. E1366-E1373. Date of Electronic Publication: 2018 Jan 29.
DOI:	10.1073/pnas.1714790115
Abstrakt:	Many drugs require extensive metabolism en route to their targets. High-resolution visualization of prodrug metabolism should therefore utilize analogs containing a small modification that does not interfere with its metabolism or mode of action. In addition to serving as mechanistic probes, such analogs provide candidates for theranostics when applied in both therapeutic and diagnostic modalities. Here a traceable mimic of the widely used anticancer prodrug cytarabine (ara-C) was generated by converting a single hydroxyl group to azide, giving "AzC." This compound exhibited the same biological profile as ara-C in cell cultures and zebrafish larvae. Using azide-alkyne "click" reactions, we uncovered an apparent contradiction: drug-resistant cells incorporated relatively large quantities of AzC into their genomes and entered S-phase arrest, whereas drug-sensitive cells incorporated only small quantities of AzC. Fluorescence microscopy was used to elucidate structural features associated with drug resistance by characterizing the architectures of stalled DNA replication foci containing AzC, EdU, γH2AX, and proliferating cell nuclear antigen (PCNA). Three-color superresolution imaging revealed replication foci containing one, two, or three partially resolved replication forks. Upon removing AzC from the media, resumption of DNA synthesis and completion of the cell cycle occurred before complete removal of AzC from genomes in vitro and in vivo. These results revealed an important mechanism for the low toxicity of ara-C toward normal tissues and drug-resistant cancer cells, where its efficient incorporation into DNA gives rise to highly stable, stalled replication forks that limit further incorporation of the drug, yet allow for the resumption of DNA synthesis and cellular division following treatment. Competing Interests: The authors declare no conflict of interest.
Databáze:	MEDLINE
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