Research Group for Radiation and Biomolecular Science
Group Leader: Akinari Yokoya
Members: Kentaro Fujii, Miho Noguchi, Yudai Izumi, Kiichi Kaminaga, Yukiko Kanari,
Yuka Sakamoto, Satoshi Yamamoto
The research objective of our group is to elucidate theradiation effect of living systems from molecular to cellularlevel. To explore the effects of X-ray irradiation on cycle
dynamics of human cells (HeLa), single cells were tracked using
the fluorescent ubiquitination-based cell cycle indicator (Fucci)
technique and the obtained data were interpreted based onsystems biology.
Single-cell tracking for cell cycle modulation of HeLa cells
Recent advances in microscopic live cell imaging
technologies with cell-labeling indicators have enabled us toeasily observe various cellular functions as dynamics. Using
HeLa-Fucci cells showing their cell-cycle stages with specificcolors, we have tracked individual cell cycle after X-ray
exposure by a time lapse technique.
A typical fluorescence image for a single non-irradiated(control) cell is shown in Figure Fig. 1. Both red fluorescence
for the G1 phase and green fluorescence for the G2/M phasewere alternately observed during the culture period, indicating
progression of the cell cycle.

Fig. 2 Time-dependence of green (G2) fluorescence intensity ofindividual cells. Panel 1: non-irradiated cell. Panel 2: irradiatedcells. Irradiated cells with an interval between the first andsecond cell divisions of shorter and longer than 21 h are shown inPanels 3 and 4, respectively. The points of X irradiation areindicated by the closed circles at the left of each line.

Fig. 1 The time-dependence of fluorescence intensity for a single HeLaFucci cell emitting red and green fluorescence, corresponding to
the G1 and S/G2 phases respectively, as shown in the photograph.
After cell division at 17 h of culture, as shown by the rapid
decrease of green (G2) fluorescence, only one of the daughter cells
was plotted.

Dynamical analysis of cellular population based on systems
biology
The intensity of green fluorescence in G2/M cell nuclei of 21
irradiated cells was monitored for 48 h after X-irradiation, as
well as 25 non-irradiated cells. The fluorescent profiles ofindividual cell are shown as overlapping lines in Fig. 2. To
facilitate comparison, the profiles were justified to the time of
the first cell division, which was designated as time zero and is
the minimum recorded fluorescence value. It should be noted Xirradiation of the HeLa-Fucci cells prolonged the cell cycle,
particularly the second G2 phase. Notably, the irradiated cells
could be separated into two distinct populations. One population
had a similar G2 phase as that of non-irradiated cells that the Xirradiation treatments appear at various points on thefluorescence profiles due to the normalization of cell division.
Generally cell cycle regulated by the cyclin dependent kinase(Cdc2) and cyclin B complex, protein-tyrosine phosphatase(Cdc25) has been known to be inhibited by "checkpoint
proteins", Chk1, Chk2, and a positive-feedback loop regulates
entry of HeLa cells into the M phase. Thus we propose themechanism underlying the appearance of the two types of cell
populations is that cell cycle switching from the "ON" to "OFF"state may be sharply induced by increased phosphorylation of
Cdc2 when the concentration of phosphorylated Cdc25 falls
below a certain threshold value, as shown in Fig. 3 [1].

Fig. 3 Proposed switching mechanism of cell cycle regulation.

Reference[1] K. Kaminaga, et al., Radiat. Protect. Dosim. (Epub ahead of print)
doi:10.1093/rpd/ncv168.