Cells and cell culture. HTori-3 cells are a human thyroid epithelial cell line immortalized by transfecting primary cultures of human thyroid epithelial cells with an origin-defective SV40 genome. This cell line is not tumorigenic and unirradiated HTori-3 cells form colonies in soft agar with a relatively low efficacy of approximately 0.3% (6
). In this study, HTori-3 cells were maintained in Dulbecco's modiﬁed Eagle's medium (DMEM) supplemented with 7% fetal bovine serum (FBS). The cells were dissociated by trypsin-EDTA treatment and sub-cultured as required.
Radiation sources and dose. The radiation experiments were performed using a Cesium-137 γ-ray source, which emits 0.662-MeV γ-rays, carbon ions (0.3-GeV/n with LET of 13.6 keV/µm) or iron ions (1-GeV/n with LET of 150 keV/µm) generated by the Alternating Gradient Synchrotron (AGS) at the Brookhaven National Laboratory (BNL).
Cell clonogenic survival assay. The effect of HZE particle radiation on cell survival was evaluated by clonogenic survival assays. HTori-3 human thyroid epithelial cells were used for these experiments. To determine the cell survival levels following radiation exposure, the cells were irradiated with γ-rays, 1-GeV/n iron ions or 0.3-GeV/n carbon ions at a single dose of 0 (sham radiation control), 10, 20, 40, 80, 125, 200 or 400 cGy. After the radiation exposure, the cells were dissociated by treatment with trypsin-EDTA, resuspended in medium, plated in T-25 tissue culture flasks at 320-800 cells per flask for the γ-ray and carbon ion radiation experiments, or 320-2,400 cells per flask for the iron ion radiation experiment, and cultured for 6 days. At the end of the incubation period, the cell colonies were fixed and stained with crystal violet and methylene blue dissolved in 90% ethanol and counted under a dissection microscope. The number of cell colonies was divided by the number of cells plated to calculate the clonogenic survival for each flask. The surviving fraction data were plotted against the radiation doses to calculate radiation sensitivity constants according to the multitarget theory (7
) using the equation S = ne-kD
, where S is the surviving fraction, n represents the number of targets, -k is the radiation sensitivity constant and D is the dose of radiation (cGy).
Soft agar colony formation assay. The transformation of HTori-3 cells (6
) irradiated with γ-ray, iron ion and carbon ion radiation was quantitated by a soft agar colony formation assay that measures the ability of the cells to grow under anchorage-independent conditions. Sham-irradiated cells were included as controls. HTori-3 cells were previously adapted for studies of radiation transformation (8
). Anchorage-independent growth is a phenotypic change associated with the ability of these cells to form tumors in animals; tumor formation was previously reported within 7-20 weeks after irradiated HTori-3 cells were transplanted into athymic nude mice (8
To carry out the experiments, HTori-3 cells were irradiated with γ-rays or carbon ions at a single dose of 10, 20, 40 or 200 cGy or iron ions at a single dose of 200 cGy. Sham-irradiated cells were included in each radiation experiment as a control. To determine the yield of anchorage-independent colonies of HTori-3 cells growing in soft agar, irradiated and sham radiation control cells were treated with trypsin and suspended in growth medium containing 0.8% methyl cellulose and plated in 24 multi-well polystyrene plates at a density of 8,000 cells/well. The bottoms of wells were pre-coated with an agar layer prepared by adding 1.8% agar to 2X DMEM without supplements to yield a final agar concentration of 0.9%. The plates were incubated at 37˚C and the medium was changed twice a week. Four or 8 wells per treatment group were stained with Neutral Red at 3-4 weeks after plating.
Data and statistical analyses. The surviving fraction was calculated by dividing the number of colonies counted in tissue culture flasks by the number of cells seeded into the tissue culture flasks. The yield of anchorage-independent colonies was calculated by dividing the number of colonies counted in soft agar by the number of viable cells plated in soft agar.
The mean surviving fraction and the yield of anchorage-independent colonies were calculated for each treatment group and compared among different treatment groups by one-way ANOVA followed by Tukey's test. The statistical analyses were performed using Prism version statistical software (version 2.0; GraphPad Software, San Diego, CA, USA).