Visualizing cell-cycle kinetics after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci).
Hypoxia induces G1 arrest in many cancer cell types. Tumor cells are often exposed to hypoxia/reoxygenation, especially under acute hypoxic conditions in vivo. In this study, we investigated cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). Hypoxic treatment halted cell-cycle progression during mid-S to G2 phase, as determined by the cell cycle-regulated E3 ligase activities of SCF(Skp2) and APC/C(Cdh1), which are regulators of the Fucci probes; however, the DNA content of the arrested cells was equivalent to that in G1 phase. After reoxygenation, time-lapse imaging and DNA content analysis revealed that all cells reached G2 phase, and that Fucci fluorescence was distinctly separated into two fractions 24h after reoxygenation: red cells that released from G2 arrest after repairing DNA double-strand breaks (DSBs) exhibited higher clonogenic survival, whereas most cells that stayed green contained many DSBs and exhibited lower survival. We conclude that hypoxia disrupts coordination of DNA synthesis and E3 ligase activities associated with cell-cycle progression, and that DSB repair could greatly influence cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation.
Department of Oral Radiation Oncology, Division of Oral Health Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
Tatsuaki
T
Goto
Department of Oral Radiation Oncology, Division of Oral Health Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
Atsushi
A
Kaida
Department of Oral Radiation Oncology, Division of Oral Health Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Electronic address: masa.mdth@tmd.ac.jp.
Masahiko
M
Miura
D002453:Cell Cycle; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D005456:Fluorescent Dyes / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
D005456:Fluorescent Dyes; D044767:Ubiquitin-Protein Ligases; D010100:Oxygen
Double-strand break repair; Fluorescent ubiquitination-based cell cycle indicator (Fucci); G2 arrest; Hypoxia; Reoxygenation