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.

WEE1 inhibition enhances sensitivity to hypoxia/reoxygenation in HeLa cells.

Hypoxia/reoxygenation (H/R) treatment reportedly induces DNA damage response (DDR), including DNA double-strand break (DSB) repair and G2 arrest, resulting in reduction of clonogenic survival. Because WEE1 plays a key role in the G2/M checkpoint along with CHK1/2, we investigated the effect of WEE1 inhibition on H/R-induced DDR using HeLa cells. The H/R treatment combined with WEE1 inhibitor abrogated G2 arrest, subsequently leading to the cells entering the M phase, and finally resulting in mitotic catastrophe after prolonged mitosis. Colony-forming assay showed an enhanced decrease in the surviving fraction and the focus formation of BRCA1 was significantly reduced. We demonstrate for the first time that WEE1 inhibition enhances H/R-induced cell death accompanied by mitotic catastrophe and that the process may be mediated by homologous recombination.

Temporo-spatial cell-cycle kinetics in HeLa cells irradiated by Ir-192 high dose-rate remote afterloading system (HDR-RALS).

BACKGROUND: Intracavitary irradiation plays a pivotal role in definitive radiotherapy for cervical cancer, and the Ir-192 high dose-rate remote afterloading system (HDR-RALS) is often used for this purpose. Under this condition, tumor tissues receive remarkably different absorption doses, with a steep gradient, depending on distance from the radiation source. To obtain temporo-spatial information regarding cell-cycle kinetics in cervical cancer following irradiation by Ir-192 HDR-RALS, we examined HeLa cells expressing the fluorescence ubiquitination-based cell cycle indicator (Fucci), which allowed us to visualize cell-cycle progression. METHODS: HeLa-Fucci cells, which emit red and green fluorescence in G1 and S/G2/M phases, respectively, were grown on 35-mm dishes and irradiated by Ir-192 HDR-RALS under normoxic and hypoxic conditions. A 6 French (Fr) catheter was used as an applicator. A radiation dose of 6 Gy was prescribed at hypothetical treatment point A, located 20 mm from the radiation source. Changes in Fucci fluorescence after irradiation were visualized for cells from 5 to 20 mm from the Ir-192 source. Several indices, including first green phase duration after irradiation (FGPD), were measured by analysis of time-lapse images. RESULTS: Cells located 5 to 20 mm from the Ir-192 source became green, reflecting arrest in G2, in a similar manner up to 12 h after irradiation; at more distant positions, however, cells were gradually released from the G2 arrest and became red. This could be explained by the observation that the FGPD was longer for cells closer to the radiation source. Detailed observation revealed that FGPD was significantly longer in cells irradiated in the green phase than in the red phase at positions closer to the Ir-192 source. Unexpectedly, the FGPD was significantly longer after irradiation under hypoxia than normoxia, due in large part to the elongation of FGPD in cells irradiated in the red phase. CONCLUSION: Using HeLa-Fucci cells, we obtained the first temporo-spatial information about cell-cycle kinetics following irradiation by Ir-192 HDR-RALS. Our findings suggest that the potentially surviving hypoxic cells, especially those arising from positions around point A, exhibit different cell-cycle kinetics from normoxic cells destined to be eradicated.

Effects of Chk1 inhibition on the temporal duration of radiation-induced G2 arrest in HeLa cells.

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.