Chinese Hamster ovary (CHO10B2) cells (wild-type) were kindly supplied by Dr Joel Bedford of Colorado State University (Fort Collins, CO, USA). Cells were grown in αMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated (56°C for 30 min) fetal bovine serum (FBS, Sigma, St. Louis, MO, USA) and 1% antibiotics and antimycotics (Invitrogen) in a humidified 5% CO₂ atmosphere at 37°C. Cell doubling time was ~12 h.

Cells were cultured in a moderately toxic concentration of BrdU (10 μM, Sigma, St. Louis, MO, USA) for our experiments. Log phase cells were cultured in 10 μM BrdU for 10 or 20 h before synchronization to achieve unifilar (>95%) or bifilar (~95%) substitution, respectively. The substitution of BrdU was confirmed by immunocytochemistry against the BrdU antibody (BD, Franklin Lakes, NJ, USA) for unifilar and fluorescence plus Giemsa (FPG) differential staining on metaphase chromosomes for bifilar (29). Cell cycle synchronization was achieved by the mitotic shake-off method (30). Two hours after shake-off, >95% of cells were synchronized in the G₁ phase before they were exposed to ionizing radiation. Cell synchronization was confirmed by flow cytometry. The Titan X-ray irradiator (200 kVp, 20 mA, 0.5-mm Al and 0.5-mm Cu filters; Shimadzu, Japan) yields an X-ray dose of ~1 Gy/min at room temperature. For heavy ion exposure, accelerated ions were irradiated using the Heavy Ion Medical Accelerator in Chiba (HIMAC) at room temperature. Radiation exposure was carried out in a dark environment to prevent cellular toxicity from room light. Dosimetry and beam quality tests for heavy ions were carried out and confirmed by operators of Accelerator Engineering Corp. (Chiba, Japan) (31–34).

To achieve first metaphase arrest, post-irradiated cells were treated with 0.1 μg/ml Colcemid (Sigma) 10–16 h after irradiation. The cells were treated with 75 mM KCl for 15 min at 37°C. After hypotonic treatment, the cells were fixed with fixative [methanol:acetic acid solution (3:1)] three times and were dropped onto slides. The samples were stained with filtered 10% (v/v) Giemsa solution in Gurr solution (Invitrogen). At least 30 metaphase cells were scored in at least three separate experiments. Chromosomal aberrations were scored as dicentric, fragment, ring, and interstitial and terminal deletion and pooled as total chromosomal aberrations per cell.

Cells were trypsinized and plated into P-60 cell culture dishes immediately after the ionizing radiation exposure. Approximately one week later, cells were fixed with 100% ethanol and stained with crystal violet for colony counting. Colonies containing >50 cells were counted as survivors. Plating efficiency was ~75% for the control and 70% for both unifilar and bifilar cells. RBE was calculated from doses required to achieve 10% survival fraction, and the sensitization enhancement ratio (SER) was calculated from the doses required to achieve 37% cell survival.

Synchronized cells were grown on chamber slides for 2 h. After irradiation of 1 Gy and various incubation times (1, 2, or 3 h post-irradiation), the cells were fixed and stained as previously described (35,36). Cellular imaging was accomplished using an Olympus FV300 fluorescence confocal microscope equipped with an Olympus Fluoview three dimensional image analysis system (Olympus, Tokyo, Japan). The foci were scored in at least 50 cells per data point. Three to four independent experiments were carried out.

Statistical comparison of mean values was performed using a t-test. Differences with a P-value of <0.05 were considered to indicate a statistically significant result. Error bars indicate standard error of the means. Confidence interval values were calculated by Prism 5™ software (GraphPad, La Jolla, CA, USA).

For X-rays and carbon ions with LET of 13 keV/μm, BrdU-incorporated cells were more sensitive to ionizing radiation when compared with the BrdU-negative controls (Fig. 1A and B). Both the initial shoulder and slope of the survival curves were affected by BrdU incorporation. The sensitization effect of BrdU bifilar substituted cells was stronger than unifilar incorporation. In contrast, for higher LET using heavy ions such as carbon ions at LET of 70 keV/μm or iron ions at LET of 200 keV/μm, BrdU substitution did not induce synergistic sensitization with ionizing radiation (Fig. 1C and D). The D₁₀ value, the dose which resulted in 10% cell survival and the D₃₇, value, the dose which resulted in 37% cell survival, decreased depending on the level of BrdU incorporation (Table I). For example, the D₁₀ value of 5.4 for X-irradiated unlabeled cells was reduced to 3.6 in bifilarly labeled cells, and that of 4.9 for 13 keV/μm LET carbon ion-exposed unlabeled cells was reduced to 3.4 in bifilarly labeled cells. For high-LET radiation, the change in D₁₀ value of LET 70 keV/μm carbon ions was from 2.7 to 2.3, and that of LET 200 keV/μm iron ions was from 2.4 to 2.1 for unlabeled to bifilar BrdU substitution. Differences between sets of D₁₀ values were statistically significant. On the other hand, for the D₃₇ values of unlabeled and bifilar BrdU substitution for high-LET carbon and iron ions, differences between them were regarded as statistically not significant.

As previously shown in the colony formation assay, BrdU-mediated radiosensitization was impaired as LET increased (Fig. 1C and D). To further investigate the mechanism of cell killing, we analyzed first post-irradiation metaphase chromosomes with a chromosomal aberration assay. As predicted from the survival data, no additional chromosomal aberrations were observed after BrdU substitution and subsequent high-LET radiation exposure (Table II). After X-ray or LET 13 keV/μm carbon ion exposure, BrdU substitution-mediated radiosensitization was observed at each dose point (1 and 2 Gy) with BrdU incorporation. For instance, bifilar incorporation at 2 Gy increased chromosomal aberrations by 1.71- (from 0.75 to 1.33) and 1.82-fold (from 1.06 to 1.93) for X-rays and LET 13 keV/μm carbon ions, respectively. In contrast, there was almost no radiosensitization at any dose or incorporation time as a result of carbon (70 keV/μm) or iron (200 keV/μm) ions.

DNA DSBs result in chromosomal aberrations and cell killing. To investigate the initial amount of DNA damage and DNA repair efficiency following various radiation exposures, we performed a γ-H2AX foci formation assay. One hour after X-ray exposure 22.6 foci per cell for controls, 25.5 foci per cell for unifilar substitution (13% increase), and 27.8 foci per cell for bifilar incorporation (23% increase) were scored. At 3 h after irradiation, the numbers of foci remaining were 9.2 for controls, 12.1 for unifilar incorporation (32% increase), and 14.5 foci for bifilar incorporation (58% increase) (Fig. 2). For LET 13 keV/μm carbon ions, the number of γ-H2AX foci was 18.2 and 6.6 for controls, 21.5 (18% increase) and 9.3 (41% increase) for single BrdU incorporation, and 24.1 (52% increase) and 12.1 (83% increase) for double incorporation at 1 and 3 h, respectively (Fig. 2). Low-LET radiation resulted in an increased number of initial BrdU-induced DNA DSBs and an increased amount of damage remaining in BrdU-incorporated cells. In contrast, high-LET radiation resulted in no significant difference in the number of initial BrdU-induced DNA DSBs or damage remaining in the BrdU-incorporated cells (Fig. 2).

To estimate the effects on repair capacity resulting from BrdU substitution, we calculated half-lives for the reduction of γ-H2AX foci between 1 and 3 h post-irradiation (Fig. 2). Half lives for low-LET exposures increased with the amount of BrdU substitution (from 1.54 to 2.12 h for X-ray bifilar and 1.35 to 1.97 h for bifilar LET 13 keV/μm carbon-ions). In contrast, high-LET exposures (iron-ions in particular) showed no significant difference in γ-H2AX foci half-lives with or without BrdU substitution (from 2.69 to 2.78 h, P<0.05).

In order to assess BrdU substitution effects on radiosensitization, RBE and SER values were obtained. Without BrdU substitution, CHO10B2 cells showed a maximum of an ~2.1 RBE value at LET 200 keV/μm. The LET values were decreased when cells were incorporated with unifilar or bifilar BrdU. The degree of reduction was stronger for bifilar BrdU substitution than unifilar BrdU substitution (Fig. 3). SER values showed that unifilar BrdU substitutions are ~1.4 more effective in cell killing when compared to values without substitution with X-ray exposure, while there was only a 1.1 increase in effectiveness with high LET radiation such as carbon 70 keV/μm and iron ions 200 keV/μm (Fig. 4). The same trend was observed for bifilar substitution. The SER values were decreased from 1.6 to 1.2.