Abstract
Chk1 inhibitor acts as a potent radiosensitizer in p53-deficient tumor cells by abrogating the G2/M checkpoint. However, the effects of Chk1 inhibitor on the duration of G2 arrest have not been precisely analyzed. To address this issue, we utilized a cell-cycle visualization system, fluorescent ubiquitination-based cell-cycle indicator (Fucci), to analyze the change in the first green phase duration (FGPD) after irradiation. In the Fucci system, G1 and S/G2/M cells emit red and green fluorescence, respectively; therefore, G2 arrest is reflected by an elongated FGPD. The system also allowed us to differentially analyze cells that received irradiation in the red or green phase. Cells irradiated in the green phase exhibited a significantly elongated FGPD relative to cells irradiated in the red phase. In cells irradiated in either phase, Chk1 inhibitor reduced FGPD almost to control levels. The results of this study provide the first clear information regarding the effects of Chk1 inhibition on radiation-induced G2 arrest, with special focus on the time dimension.
Highlights
Enhancement of radiation-induced cell killing by Chk1 inhibition has been observed in several studies [1]
We have shown that HeLa-fluorescent ubiquitination-based cell-cycle indicator (Fucci) cells clearly enter G2 arrest after 10-Gy irradiation, reflected by the accumulation of green cells peaking ~15 h after irradiation and their subsequent gradual disappearance [4, 5]
We examined inhibition of phosphorylation of Cdc2 at Tyr15 following combined treatment with inhibitor of Chk1 or Wee1 (MK-1775) and irradiation; the latter compound resulted in stronger inhibition (Fig. 1C)
Summary
Enhancement of radiation-induced cell killing by Chk inhibition has been observed in several studies [1]. Sakaue-Sawano et al developed a cell-cycle visualization system called fluorescent ubiquitination-based cell-cycle indicator (Fucci), which takes advantage of the cell cycle-dependent ubiquitination of Cdt and Geminin [3]. In this system, G1 and S/G2/M cells emit red and green fluorescence, respectively; more precisely, early G1-phase cells emit no fluorescence, and early S-phase cells emit both fluorescence types [3]. We predicted that application of this system would allow us to obtain precise information regarding the duration of the G2 arrest This system has made it possible to perform differential analysis of cells receiving irradiation in the red or green phase. We demonstrate for the first time that Chk inhibitor remarkably reduces the duration of radiationinduced G2 arrest, regardless of cell-cycle phase at the time of irradiation
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