Superresolution imaging of individual replication forks reveals unexpected prodrug resistance mechanism

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.

CTCF-mediated Chromatin Structures Dictate the Spatio-temporal Propagation of Replication Foci

“…S1a) revealed a punctate distribution as well as changing physical morphology across S-phase ( Fig. 1a), similar to previous observations using immuno-gold electron microscopy 26 , conventional optical microscopy 16,27 and different super-resolution microscopy 21,[28][29][30][31] . However, the superior spatial resolution afforded by STORM (~20 nm) enabled us to perform more accurate quantitative characterization of RFi, particularly for findings based on multi-color colocalization analysis.…”

Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging

“…This median value and size heterogeneity are very similar to what has been attributed in earlier studies to the replication domains in human cells using optical super-resolution ( 16 ) and electron microscopy ( 15 ). In eukaryotes, very recent studies have suggested that on average these replication units of 150 nm correspond to the sites of DNA replication with four co-replicating DNA segments of approximately 20 kb ( 18 , 58 ). As H. volcanii replication origins are separated by several hundreds of kilobases ( 24 ), we find it unlikely that several clustered cis -acting replication origins would be simultaneously processed.…”

Biomedical applications of copper-free click chemistry: in vitro, in vivo, and ex vivo

“…They conrmed the equal biological prole of AzC in cell culture and in vivo as ara-C. Further studies of the AzC with classical azide-alkyne click chemistry, including the gSTED imaging experiment, allowed them to understand the contradiction of cell-type selectivity of nucleoside-based drugs. 65…”