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Proc Natl Acad Sci U S A. 2018 Feb 13;115(7):E1366-E1373. doi: 10.1073/pnas.1714790115. Epub 2018 Jan 29.

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

Author information

1
Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland.
2
Department of Chemistry, University of Zurich, CH-8057 Zurich, Switzerland nathan.luedtke@chem.uzh.ch.

Abstract

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.

KEYWORDS:

click chemistry; in vivo imaging; nucleosides and nucleotides; superresolution microscopy; theranostics

PMID:
29378947
PMCID:
PMC5816165
DOI:
10.1073/pnas.1714790115
[Indexed for MEDLINE]
Free PMC Article

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