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The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV– and chemotherapeutic drug–induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinase inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU–treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell-cycle checkpoint. Cells lacking BRCA2 were sensitive to 5-FU in the presence of ATR inhibitor. Moreover, ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the nonhomologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU–based chemotherapy.

Recently, researchers have developed protocols for human cerebral organoids using human pluripotent stem cells, which mimic the structure of the developing human brain. Existing research demonstrated that human cerebral organoids which undergo short cultivation periods, contain astrocytes, neurons, and neural stem cells, but lacked mature oligodendrocytes, and mature, fully functional neurons. In this study, we analyzed organoids induced from H9 human embryonic stem (ES) cells that were cultivated for as long as six months. We observed mature oligodendrocytes, positive for MBP (myelin-basic protein), and mature GAD67 (glutamate decarboxylase 67 kDa isoform)-positive inhibitory neurons and VGLUT1 (vesicular glutamate transporter 1)-positive excitatory neurons via immunohistochemical analysis. These observations suggest that long-term cultivation of cerebral organoids can lead to the maturation of human cerebral organoids, which can be used as a tool to study the development of human brains.