Effects of Proton Beams and X Rays on the Cell Cycle of Fluorescent Ubiquitination-Based Cell Cycle Indicator (Fucci)-Expressing Cells

Abstract

Our previous study demonstrated that cisplatin had more potent sensitizing effects and yielded a significantly higher percentage of apoptotic cell death when combined with proton beams than with X-ray irradiation. Although such results may be explained by differences in the DNA repair mechanisms, other factors such as effects on cell cycle and organelles may possibly be involved. In this study, we evaluated the biological effects of protons and photons in combination with cisplatin on the cell cycle of 2 cultured cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). HeLa cells expressing the Fucci (HeLa/Fucci) were provided by the RIKEN BioResource Center (Tsukuba, Japan). HSG/Fucci was established in our laboratory by introducing mCherry and AmCyan1 into human HSG cells using the PiggyBac system. First, we evaluated the relative biological effectiveness (RBE) of the 2 cells using a standard colony assay. Then, to evaluate the cell cycle, appropriate numbers of cells were plated on 96-well plates to create scaffolds 6 hours before each experiment. From 1 hour before irradiation, the cells were exposed to the medium containing 0-2.5 μmol/L cisplatin and were irradiated with single doses of 0-8 Gy with X rays or proton beams under the same biological conditions. One hour after irradiation, cisplatin was washed out from the medium. The red and green fluorescence in G1 and S/G2/M phases, respectively, were observed and counted using the live cell fluorescence imaging data. Then, the effects on the cell cycle were evaluated. The RBE (D10) was 1.04 (95% CI: 1.01-1.07) for HeLa/Fucci and 1.04 (1.03-1.05) for HSG/Fucci. These data were comparable to our previous results on HSG: 1.01 (1.00-1.03). In time-lapse imaging, both X rays and proton beams increased the percentage of green S/G2/M phase cells after irradiation and delayed the cell cycle, suggesting the G2 arrest. The proportion of green cells was increased by high-dose proton irradiation, suggesting more significant arrest. Even after single administration of cisplatin, the percentage of green cells increased depending on the concentration of cisplatin, and it occurred at a later time than the G2 arrest caused by irradiation. The percentage of green cells at that time was higher when cisplatin was combined with proton beams than with X rays. Two peaks of increased green cells were observed, suggesting the different effects of cisplatin and irradiation on the cell cycle. In Fucci-expressing cells, both proton beams and X rays delayed the cell cycle, resulting in an increase in S/G2/M phase cells probably due to G2 arrest. Considering the timing of the increase in G2 arrest, potent biological effects may be obtained by delivering proton irradiation during radiosensitive cell cycle phases in chemo-proton therapy.