• Energy Research

In this study, whether survivin plays a direct role in mediating high-LET radiation resistance in human hepatoma cells was investigated. Small interfering RNA (siRNA) targeting survivin mRNA was designed and transfected into human hepatoma HepG2 cells. Real-time PCR and western blotting analyses revealed that survivin expression in HepG2 cells decreased at both transcriptional and post-transcriptional levels after treatment with survivin-specific siRNA. Caspase-3 activity was determined with a microplate reader assay as well. Following exposure to high-LET carbon ions, a reduced clonogenic survival effect, increased apoptotic rates and caspase-3 activity were observed in the cells treated with the siRNA compared to those untreated with the siRNA. The cells with transfection of the survivin-specific siRNA also increased the level of G₂/M arrest. These results suggest that survivin definitely plays a role in mediating the resistance of HepG2 cells to high-LET radiation and depressing survivin expression might be useful to improve the therapeutic efficacy of heavy ions for radioresistant solid tumors.

Carbon-ion radiotherapy uses spread-out Bragg peaks (SOBP) to produce uniform biological effects within a target volume. The relative biological effectiveness is determined by the in vitro cell kill after a single dose is employed to design the SOBP. A question remains as to whether biological effects for in vivo tissues after fractionated doses are also uniform within the SOBP.Mouse foot skin was irradiated with fractionated doses of carbon ions at various linear energy transfer (LET) values. A new ridge filter was designed based on alpha and beta values for each LET to cause moderate skin reaction, and was studied concerning its uniformity.The reciprocal total doses of intermediate-LET carbon ions and of reference gamma rays linearly increased with an increase of a dose per fraction in Fe-plots. As the single total dose of higher LET run off linearity, data obtained from 2 to 6 fractions were used to design a new ridge filter. The physical dose distribution of the new ridge filter was almost identical to, and indistinguishable from, the ridge filter designed based on the in vitro cell kill.The LET dependence of alpha is a principle of the biological factor to be used for designing spread-out Bragg peaks of carbon-ion radiotherapy.

Fractionated proton beam radiotherapy is spreading worldwide these days. However, biological data of sub-lethal damage recovery (SLDR) after proton irradiation is not known yet. We here conducted split-dose experiments (20-360 min intervals) to clarify SLDR kinetics, and also compared the kinetics between cells with different repairability of DNA double-strand breaks. CHO and 51D1 cell lines but not V3 cell line showed significant SLDR, which reached plateau in 4-6 h. The recovery rates and recovery halftime of SLDR after X-rays were significantly higher and shorter than proton beams for CHO and 51D1 cells, respectively. Additionally, the frequency of remaining gamma-H2AX foci after two fractions was remarkably higher for X-rays than proton beams. These data suggest that there is a difference between proton beam and X-rays in SLDR and the retained DNA double-strand breaks after split-dose irradiation.

To understand how human tumor cells respond to the combined treatment with nocodazole and high LET radiation, alterations in cell cycle, mitotic disturbances and cell death were investigated in the present study. Human cervix carcinoma HeLa cells were exposed to nocodazole for 18 h immediately followed by high LET iron ion irradiation and displayed a sequence of events leading to DNA damages, mitotic aberrations, interphase restitution and endocycle as well as cell death. A prolonged mitotic arrest more than 10 h was observed following nocodazole exposure, no matter the irradiation was present or not. The occurrence of mitotic slippage following the mitotic arrest was only drug-dependent and the irradiation did not accelerate it. The amount of polyploidy cells was increased following mitotic slippage. No detectable G(2) or G(1) arrest was observed in cells upon the combined treatment and the cells reentered the cell cycle still harboring unrepaired cellular damages. This premature entry caused an increase of multipolar mitotic spindles and amplification of centrosomes, which gave rise to lagging chromosomal material, failure of cytokinesis and polyploidization. These mitotic disturbances and their outcomes confirmed the incidence of mitotic catastrophe and delayed apoptotic features displayed by TUNEL method after the combined treatment. These results suggest that the addition of high-LET iron ion irradiation to nocodazole enhanced mitotic catastrophe and delayed apoptosis in HeLa cells. These might be important cell death mechanisms involved in tumor cells in response to the treatment of antimitotic drug combined with high LET radiation.

It is believed that the dose-rate of radiation will have an influence on cell sensitivity. The dose-rate effects on cell survival can be expressed by the change of the β term in the linear quadratic model. The value at a high-dose-rate decreases below 60 Gy/h and reaches zero at 0.2 Gy/h or less for photons. However, the effect for a high-LET ion-beam is not well known. At HIMAC, cells were exposed to 70 keV/μm carbon-ion beams at different dose-rates between 0.5 and 600 Gy/h at room temperature. The β values for all survival curves show no significant differences among the dose-rates tested for HSG, V79 and CHO cells. Changing the ion-beam dose-rate had no effect on cell survival. This suggests that high-LET particle beams, such as galactic cosmic rays, may not exhibit a dose-rate effect on cell survival. Low-dose-rate radiation showed an effect similar to high-dose-rate radiation.