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Survival and initial chromatid breakage in normal and tumour cells exposed in vitro to gamma rays and carbon ions at the HIRFL

¹ Institute of Modern Physics, Chinese Academy of Sciences, ² Life Science School of North West Normal University, ³ Life Science School of Lanzhou University, Lanzhou, China

	Abstract

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Human hepatoma and normal liver cells were irradiated with ¹²C⁶⁺ ion beams (linear energy transfer (LET) = 96 keV µm^–1) and -rays at the Heavy Ion Research Facility in Lanzhou (HIRFL). The numbers and types of chromatid breaks were detected using the premature chromosome condensation technique. Irradiation with ¹²C⁶⁺ ions produced a majority of isochromatid break types, while chromatid breaks were dominant for irradiation with -rays. Experimental results showed that the initial level of chromatid breaks is clearly related to the absorbed dose from ¹²C⁶⁺ ions and -rays. The ¹²C⁶⁺ ions are relatively more effective at inducing initial chromatid breaks when compared with the -rays. A relative biological effectiveness (RBE) of about 2.5 resulted for the induction of initial chromatid breaks by ¹²C⁶⁺ ions relative to -rays in both cell lines.

	Introduction

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Heavy charged particle beams have been applied in tumour radiotherapy because of their clear radiobiological response in the destruction of malignant cells [1]. The induction of chromatid breaks in the cell by radiation is considered to be a major cause of damage, which can lead to cell death. It has been reported by Kawata et al [2] that low linear energy transfer (LET) radiation induced mostly chromatid breaks, whereas the most isochromatid breaks were generated by high LET radiation. Previous studies [3] by the present authors have shown that the chromatid break repaired more easily than the isochromatid break. A major project involving tumour radiotherapy using heavy ions is currently being conducted at the Heavy Ion Research Facility in Lanzhou (HIRFL). In the present communication, the use of the premature chromosome condensation technique to measure the level of G₂ initial chromatid breaks of human hepatoma cells and normal liver cells is reported.

It is a well known fact that the risk of liver cancer is high in Asia, especially in China. For this reason, one normal and one tumour human liver cell line were selected for exposure to carbon ions generated by the HIRFL. The main aim was to investigate and quantify the relationship between the radiation dose and the level of initial chromatid breakage. Such information is needed for predicting the radiosensitivity of these two liver cell lines, and for evaluating the relative biological effectiveness (RBE) of carbon ions for inducing the chromatid breaks. The experiments provided necessary and valuable data on liver cell radiosensitivity prior to the start of clinical radiotherapy. It was found that the two cell lines have moderate radiosensitivity when exposed to -rays [3].

	Materials and methods

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Cell culture and irradiation
The human hepatoma cell line SMMC-7721 and the normal liver cell line L02 (obtained from the Chinese Center for Type Culture Collection, CCTCC) with moderate radiosensitivity, were grown in RPMI-1640 medium supplemented with 10% fetal calf serum at 37°C in 5% CO₂; additionally, 0.25 U ml^–1 of insulin was added to the L02 culture medium.

Exponentially growing cells were irradiated with either -rays obtained from a ⁶⁰Co source (dose rate 0.2 Gy min^–1) or ¹²C⁶⁺ ion beams generated by the HIRFL facility with a dose range from 0 to 8 Gy. The initial energy of the ¹²C⁶⁺ ions was 80.55 MeV u^–1, which was attenuated by the 13.58 mm Lucite ( = 1.2 g cm^–3) to 20 MeV u^–1 before it reached the cells. The LET was 96 keV µm^–1 when the carbon ions interacted with the cells located in the region of the Bragg peak and the LET was calculated by the Trim Program 92. Dosimetry was performed with an air ionizing chamber and the uniformity of the carbon ion beams was found to be 85%, as measured with CR39 track etch plastic.

Colony assay
After exposure to radiation, cells were washed three times using PBS (pH = 7.0), then trypsinized and put into 5 ml culture medium, and the cell density counted by using the light microscope. At each dose point 200, 400, or 600 cells were plated into 35 mm diameter culture dishes. 5 ml RPMI-1640 medium supplemented with 10% fetal calf serum was added and cells were incubated at 37°C in 5% CO₂ until colonies formed (7–14 days). Two dishes were plated per dose point and the experiments were repeated three times. All the data are the mean± standard deviation. Data were fitted using Origin 7.0 (Original Lab, America); the data from the cells exposed to -rays were fitted using the non-linear fit program, while the data from the cells exposed to carbon ions were fitted using the linear fit program. The linear-quadratic formula

was used for the non-linear fit, where and represent the probability of a single or double photon interaction with the chromatids.

Chromosome preparation
Calyculin-A (BIOMOL America) was used as the PCC inducer, which was dissolved in 100% ethanol as a 1 mmol l^–1 stock solution. In order to achieve the chromatid breaks, 50 nmol l^–1 of Calyculin-A was added to cell cultures 5 min before irradiation. Cells were then incubated for a further 30 min at 37°C in 5% CO₂. Chromosome spread was harvested by swelling the cells in 75 mmol l^–1 KCl for 20 min at 37°C and fixed with Carnoy's fixation. A final wash and fixation in the same fixative agent was completed before dropping the cells onto a glass slide and drying them in hot humidity.

The chromosome was stained with 5% Giemsa for 20 min. According to the standard criteria [4], more than 40 G₂ phase cells were scored for each dose level investigated. Briefly, a chromatid discontinuity or misalignment of the distal part to the lesion, or a non-stained region longer than the chromatid width was considered to be a chromatid break. Isochromatid breaks were considered to be two breaks occurring at the same position on the two sister chromatids, i.e. a penetrated lesion through the two q arms or p arms of the chromosome was regarded to be an isochromatid break. The total chromatid breaks were calculated by summing the numbers of chromatid and isochromatid breaks. 20 non-irradiated cells were scored and the mean of these chromatid breaks was recorded. There were very few spontaneous chromatid breaks. The number of chromatid breaks observed in the irradiated cells was reduced by the mean number of chromatid breaks observed in the non-irradiated cells, and this number forms the experimental data from which the results were evaluated. A small number of chromatid exchanges were also noticed, but these were not considered to be significant and did not have a linear relationship with absorbed dose.

	Results

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Survival fraction of two cell lines exposed to gamma rays and carbon ions
Survival curves for SMMC7721 and L02 cells exposed to -rays and carbon ions are shown in Figure 1. Several trends can be seen: (1) the two cell lines have different survival curves for -rays and carbon ion irradiation; (2) the cells irradiated with -rays have a linear quadratic survival curve, while it is almost linear for carbon ion irradiation; (3) the surviving fraction of L02 cells was higher than that of the SMMC7721 cells for both exposure scenarios. For the cells exposed to -rays, the fit parameters and were 0.03 and 0.06 for SMMC7721 cells and 0.04 and 0.05 for L02 cells, respectively.

View larger version (12K): [in this window] [in a new window]   Figure 1. Survival curve of SMMC7721 and L02 cells exposed to gamma rays and carbon ions. Survival fraction value is the mean±standard deviation. The survival curve of cells exposed to gamma rays was linear-quadratic, and was almost linear when cells were exposed to carbon ions. The survival fraction of SMMC7721 cells and L02 cells are significantly different when exposed to either gamma rays or carbon ions.

View larger version (14K): [in this window] [in a new window]   Figure 2. Correlation between absorbed dose and chromatid breaks. In both(a) L02 and (b) SMMC7721 cell lines, more isochromatid than chromatid breaks were seen when cells were exposed to carbon ions, and fewer isochromatid than chromatid breaks seen when cells were exposed to gamma rays. Fewer breaks were induced in L02 cells than in SMMC7721 cells after either carbon ion or gamma ray exposure. All the data were the mean±standard deviation.

The proportion of chromatid breaks in two cell lines exposed to -rays and ¹²C⁶⁺ ions
Figure 3 shows the proportion of two types of chromatid breaks of two cell lines exposed to -rays and ¹²C⁶⁺ ion beams. It can be seen in Figure 3 that for ¹²C⁶⁺ and -ray irradiation of cells, the dominant breakage types were isochromatid and chromatid, respectively.

View larger version (20K): [in this window] [in a new window]   Figure 3. Proportion of two types of chromatid breaks. For cells irradiated with gamma rays, the number of chromatid breaks in both cell lines was much more than that of isochromatid breaks; for cells exposed to carbon ions, the isochromatid breaks were much more frequent than the chromatid breaks. They were significantly different.

	Discussion

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After irradiation, G₂ chromatid breaks and isochromatid breaks increased linearly with the absorbed dose for both cell lines, the increased ratio of L02 and SMMC7721 cell lines were 2.5 and 3.5 for chromatid breaks and 4.5 and 7 for isochromatid breaks, which was consistent with data from Kawata et al [2]. The yields of the two types of chromosome breaks irradiated with ¹²C⁶⁺ ion beams were more than that of -rays. The relative biological effectiveness (RBE) of ¹²C⁶⁺ ion beams in inducing the chromatid breaks was found to be about 2.5. These results are in agreement with several previous studies [2, 5, 6, 7].

The main type of chromatid break was isochromatid for cells exposed to ¹²C⁶⁺ ion beams, while the chromatid breaks predominated in cells exposed to -rays. Kawata et al [2] reported that chromatid breaks dominated after low LET irradiation, while isochromatid breaks dominated for high LET exposures, such as heavy ions, suggesting that most isochromatid breaks resulted from two separate breaks on sister chromatids induced by independent electron tracks. For low LET radiation types, not enough energy can be deposited to penetrate sister chromatids simultaneously, so most breaks were found to be chromatid in nature. For cells exposed to heavy ions, more isochromatid breaks were induced. This explains why heavy ions have the higher RBE, which is relevant for the destruction of tumour cells.

	Conclusion

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Our results suggest that heavy ions generated by the HIRFL have a higher efficiency in killing tumour cells, and a high RBE for the induction of the chromatid breaks. Also, the chromatid breaks are tightly correlated with the cell surviving fraction and radiosensitivity.

	Acknowledgments

 
This work was supported jointly by National Natural Science Foundation of China and National Basic Research Program of China through fellowship No. 10335050 and 2003CCB00200. We express our thanks to all the workers in HIRFL, for providing us with the high quality heavy ion beams.

Received for publication June 9, 2005. Revision received November 9, 2005. Accepted for publication November 15, 2005.

	References

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1. Kraft G. Tumor therapy with heavy charged particles. Progress in Particle Nuclear Physics 2000;45:s473–544.[CrossRef]
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3. Yang JS, Li WJ, Zhou GM, et al. A comparative study on radiosensitivity of various tumor cells and human normal liver cells. World J Gastroenterol 2005;11:4098–101.[Medline]
4. Savage JR. Classification and relationships of induced chromosomal structural changes. J Med Genet 1976;13:103–22.[Abstract/Free Full Text]
5. Kawata T, Gotoh E, Durante M, et al. High-LET radiation-induced aberrations in prematurely condensed G2 chromosome of human fibroblasts. Int J Radiat Biol 2000;76:929–37.[Medline]
6. Kawata T, Durante M, Furusawa Y, et al. Rejoining of isochromatid breaks induced by heavy ions in G2-phase normal human fibroblasts. Radiat Res 2001;156:598–602.[Medline]
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