30. Effect of Heavy Ion Exposure on Cell Cycle Checkpoints

Kiyomi Eguchi-Kasai, Hiromi Itsukaichi, Masahiro Murakami, Tatsuaki Kanai

Keywords: cell cycle check point, heavy ion beam, cultured cells

For cancer therapy using heavy ion beams, it is important to know the cell cycle progression for different cell types after irradiation because daily fractionated doses are given to both normal and tumor tissues. We studied the cell cycle progression in cells of normal human fibroblasts (NB1RGB) or rodent cells (V79) after irradiation with carbon ion beams.

Cells were irradiated by carbon ion beams at the Heavy Ion Medical Accelerator in Chiba (HIMAC) and the Cyclotron Facility. Initial accelerated energies of carbon ions were 135 and 290 MeV/nucleon at the HIMAC and 12 MeV/nucleon at the Cyclotron Facility. Linear energy transfer (LET) of the beams at the target was changed by inserting the plastic plate in front of the target. The LET range was 30 - 250 keV/m at the sample center. The dose rate ranged from about 1 - 5 Gy/m. The reference radiation was 200 kVp X-rays filtered through 0.5 mm Al and 0.5 mm Cu with a dose rate of 0.9 Vy/m. After irradiation, cells were incubated with 5 M bromodeoxyuridine (BrdU) if needed. Cells were then trypsinized and fixed with 70% ethanol. Cells were stained both with propidiumiodide for the DNA and anti-bromodeoxyuridine-fluorescein, and analyzed with a flowcytometer. Cell cycle distribution was analyzed with the ModiFit LT (Becton Dickinson).

For the asynchronous V79, more than 80% of the cells accumulated in the G2/M phase from 4 to 12 h with a peak at around 6h after irradiation. The maximum value and the width of the peak increased for both LET and radiation dose. Relative biological effectiveness (RBE) of G2 arrest was calculated using the width of the peak at the half values. RBE increased with LET up to 180 keV/m. RBE values for G2 arrest above 30 keV/m were bigger than those for the cell inactivation for which the RBE-LET curve had a peak around the LET of about 100 keV/m. More precise analyses were done using BrdU. Because cells were continuously labeled with BrdU after irradiation, cells at the G1 and the S phases at the time of irradiation incorporated BrdU. Labeled cells showed G2 arrest at 6 to 16 h, depending on LET and dose. In contrast, unlabeled cells showed little G2 arrest. Therefore, the large G2 arrest in V79 cells was due to the populations at the G1 and the S phases. There was no G1 arrest for V79 cells. This is natural because p53 of the V79 cells was mutated. In contrast, there was a big arrest at the G1 phase and a small arrest at the G2/M phase for the NB1RGB cells which have the normal p53.