Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

Available online at www.sciencedirect.com NIM B Beam Interactions with Materials & Atoms Nuclear Instruments and Methods in Physics Research B 266 (...

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Available online at www.sciencedirect.com

NIM B Beam Interactions with Materials & Atoms

Nuclear Instruments and Methods in Physics Research B 266 (2008) 262–266 www.elsevier.com/locate/nimb

Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay Ping Li a, Li-bin Zhou a, Xiao-dong Jin a, Jing He a, Zhong-ying Dai a, Guang-ming Zhou a, Qing-xiang Gao b, Sha Li c, Qiang Li a,* a Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000, People’s Republic of China Institute of Cell Biology, School of Life Sciences, Lanzhou University, 222 Southern Tianshui Road, Lanzhou 730000, People’s Republic of China c Department of Radiotherapy, General Hospital of Lanzhou Military Area, 98 Western Xiaoxihu Road, Lanzhou 730050, People’s Republic of China b

Received 19 June 2007; received in revised form 7 November 2007 Available online 23 November 2007

Abstract DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of ‘‘Tail DNA (%)” (TD) and ‘‘Olive tail moment” (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radiosensitivity. Ó 2007 Elsevier B.V. All rights reserved. PACS: 87.53.j Keywords: DNA damage; Carbon ion beam; X-ray; Alkaline comet assay

1. Introduction Heavy ion beam may produce better clinical results in radiotherapy than conventional radiations such as low linear energy transfer (LET) X- and c-rays, because of its excellent dose localization (Bragg peak) and high relative biological effectiveness (RBE) within the dose peak. However, different types of cell lines show various radiosensitivities, even for tumors from the same histopathological origin [1]. Thus, the clinical value of a radiation sensitivity predictive assay is very important for radiation therapy, as *

Corresponding author. Tel.: +86 931 4969316; fax: +86 931 8272100. E-mail address: [email protected] (Q. Li).

0168-583X/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2007.11.021

it could potentially lead to better selection of patients for radiotherapy protocols, improving probability to predict prognosis and even a decrease in therapy-related side effects. Therefore, it is essential to develop a predictive assay for tumor radiosensitivity in medical practice. Clonogenic survival assay has become one of the most commonly used radiobiological techniques for assessing cell radiosensitivity. However, the direct determination of the radiosensitivity of cells derived from patients by clonogenic assay is rather complicated and time-consuming and then not feasible for the routine clinical use of the individual patient’s sensitivity prior to radiotherapy. Until now, the relationship between radiosensitivity and DNA damage in many tumor cell lines has been studied

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[2–6]. Various experimental approaches have been used for the detection of DNA strand breaks such as filter elution, constant field gel electrophoresis, pulsed-field gel electrophoresis (PFGE) and comet assay [2]. Comet assay is a sensitive, simple and rapid method for visualizing and measuring DNA strand breaks in single cell samples. This assay can potentially measure DNA lesions in any organ or tissue even in the absence of mitotic activity. Alkaline comet assay (ACA) is a highly sensitive method for the assessment of single strand breaks (SSBs), double strand breaks (DSBs) and alkali labile sites (ALSs) and can readily detect levels of damage induced by relevant doses of radiation. In a previous study, an inverse correlation, which was obtained between clonogenic survival and mean tail moment (TM) for comet formation, suggested that ACA could potentially be used to predict the radioresponsiveness of the single cell lines [7]. It is well known that lung cancer is one of the top killers all over the world and its radioresistance to low-LET radiations makes the conventional radiotherapy difficult and vain. Probably, lung cancers may benefit from heavy ion radiotherapy. For this reason, a malignant tumor cell, Lewis lung carcinoma cell was exposed to carbon ion beams and the cell viability together with DNA damage was detected by clonogenic and comet assays, respectively. The main aim of our experiment was to investigate the feasibility and advantages of heavy ion beams on radiotherapy prior to the start of clinical radiotherapy. The data were expected to be available for tumor radiosensitivity in medical practice. 2. Materials and methods 2.1. Cell culture Lewis lung carcinoma cell line, which was bought from the cell bank of Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, was used in this study. Cells were incubated in Dulecco’s Modified Eagle medium (DMEM, Gibco, Life Technologies, Inc., Grand Island, NY, USA) with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 100 U/ml penicillin, 100 U/ml streptomycin and 10% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37 °C. 2.2. Irradiation A carbon ion beam of 100 MeV/u was supplied by the Heavy Ion Research Facility in Lanzhou (HIRFL) at the Institute of Modern Physics, Chinese Academy of Sciences (IMP-CAS). Cell exposures were conducted at the therapy terminal of the HIRFL, which has a vertical beam line. Due to the energy degradation by the vacuum window, air gap, Petri dish cover and medium, the energy of the ion beam on cell samples was calculated to be 89.63 MeV/u, corresponding to a linear energy transfer (LET)

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of 28.3 keV/lm and the dose rate was adjusted to be about 4 Gy/min. The doses of 0, 0.5, 1, 2, 3, 4, 8 and 12 Gy were applied in this study and each dose had three independent irradiation experiments. All irradiations were performed at room temperature. X-ray irradiations (6 MV) to cells were performed at room temperature in the therapy unit (an electron linear accelerator, CL2100, Varian, Germany) of the General Hospital of the Lanzhou Military Area at a dose rate of 4 Gy/min. The doses applied were the same as those used for the carbon ion beam and each dose had three independent irradiation experiments. 2.3. Clonogenic assay Cell survival was determined by the standard colonyforming assay. Briefly, cells were re-plated at a density of about 100 surviving cells into 60 mm Petri dishes supplemented with DMEM medium including 10% fetal calf serum after irradiation. After incubation for 7 days, the cells were fixed and stained with 5% Giemsa solution. Colonies with more than 50 cells were counted as survivors. 2.4. Comet assay The ACA was performed as described by Singh et al. [8] one hour after irradiations. Two solutions, 0.5% normal melting point agarose (NMA) and 0.75% low melting point agarose (LMA) were prepared with phosphate buffered saline (PBS, pH 7.4). Briefly, 0.5% NMA solution of 80 ll was dropped onto each frosted slide and the slide was dried after 2 h. Cell suspension containing 5  105 cells was mixed with 0.75% LMA solution (1:3) and then the mixture of 80 ll was pipetted onto the prepared slide. Next, a coverslip was placed on the mixture. After the slide was placed on an iced surface for at least 5 min, the coverslip was removed. Then all of the sandwiched slides were immersed in cold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 1% N-lauroylsarcosine, pH 10; 10% DMSO, 1% Triton X-100 were added just before use) for 1.5 h at 4 °C in darkness. The slides were put in a submarine-type electrophoresis tank containing 300 mM NaOH, 1 mM Na2EDTA (pH > 13) for 15 min to allow DNA unwinding. Electrophoresis was conducted at 4 °C in darkness for 25 min at 1 V/cm and approximately 300 mA. After electrophoresis, the slides were rinsed gently three times with 400 mM Tris–HCl (pH 7.5) to neutralize the excess alkali and stained with 2 lg/ml ethidium bromide solution. Using Olympus BX51 fluorescence microscope equipped with an excitation filter of 515560 nm and an emission filter of 590 nm, about 50 cells per slide at 400 magnification were examined. Comet images were analyzed using the Casp1.2.2 software (Institute of Theoretical Physics, University of Wroclaw, Wroclaw, Poland). In this study, the indexes of DNA damage are ‘‘Tail DNA (%)” (TD)

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and ‘‘Olive tail moment” (OTM), respectively. The mean values of TD and OTM were derived from about 50 comet images at each dose point with the imaging software. 2.5. Data analysis The data came from triplet duplication and are given as mean ± standard error. Statistical analysis and data fitting were conducted with Origin 7.0 software (OriginLab Corporation, Northampton, MA, USA). The surviving data were fitted with weight according to the linear-quadratic (LQ) model, that is 2

SF ¼ eaDbD ;

3. Results Shown in Fig. 1 are the survival curves of Lewis lung carcinoma cells exposed to the carbon ion and X-ray irradiations respectively, where the experimental data points were fitted according to the LQ model as described in Section 2.5. The survival curve for the high-LET carbon ion beam displays nearly linear attenuation in the semi-logarithmic plot. However, an obvious shoulder is presented in the survival curve for the X-ray irradiation, which represents the repair of sub-lethal damage. The D10 values, indicating the dose required to reduce the surviving fraction to

ð1Þ

where SF is the survival fraction, D is the radiation dose, a and b are the coefficients in the linear and quadratic terms of the LQ model. The TD and OTM data points for the carbon ion beam were all fitted with weight according to the following exponentially-modified linear equation: TDðOTMÞ ¼ a  ð1  ebD Þ þ c;

ð2Þ

where TD is the tail DNA (%), OTM is the Olive tail moment, D is the radiation dose, a and b are the coefficients in the linear and exponential terms of the equation above, c is offset. Due to the data fitting suitability, the dose response curves regarding TD and OTM for the X-ray irradiation were plotted with linear regression as follows: TDðOTMÞ ¼ a þ b  D;

ð3Þ

where TD is the tail DNA (%), OTM is the Olive tail moment, D is the radiation dose, a is the initial tail DNA (%) and b is the slope value.

Fig. 1. Survival curves for Lewis lung carcinoma cell line after X-ray and carbon ion beam (LET = 28.3 keV/lm) irradiations. Data points show the mean ± standard error (SE) of three independent experiments.

Fig. 2. Comet images of Lewis lung carcinoma cells irradiated with the X-ray and carbon ion irradiations at different doses. (A) Comets induced by X-rays (top, left to right: 0, 0.5, 1, 2; bottom, left to right: 3, 4, 8, 12 Gy), (B) comets induced by carbon ion beams (top, left to right: 0, 0.5, 1, 2; bottom, left to right: 3, 4, 8, 12 Gy).

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10%, are 4.29 Gy and 2.42 Gy for the X-ray and carbon ion irradiations, respectively. Therefore, the RBE of the carbon ion beam at 10% survival level is 1.77. Thus, the DNA damage induced by high-LET carbon ions was more severe than those induced by X-rays. Fig. 2(A) and (B) show the DNA damage effects of the X-ray and carbon ion irradiations on individual cells respectively. The length and intensity of comet tail in individual cell gradually increased not only with increasing dose but also with the increasing LET value. Two DNA damage parameters, TD and OTM, were evaluated and the dependences of TD and OTM on dose are shown in Fig. 3(A) and (B), respectively. The dose response curves in terms of TD and OTM for the X-rays show linear dose-effects and the slope values of the TD and OTM curves are 2.21 ± 0.19 and 2.47 ± 0.14, respectively. However, the carbon ion beams induced a biphasic dose response compared with the X-rays, in which the response curves comprise linear and exponential parts. The linear parts are steeper than those of the X-ray irradiation. At doses higher than 8 Gy, the dose response for the carbon

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ion beam shows a saturation effect with more than 90% level of confidence (see Fig. 3 and Table 1). Compared with those for the X-ray irradiation, the TD and OTM values for the carbon ion beam increased by a factor of 1.9 and 2.5 at 8 Gy, respectively. The correlation between clonogenic cellular radiosensitivity and DNA damage was studies as well. The surviving fraction at 2 Gy (SF2) was used as the index of intrinsic cellular radiosensitivity and the value of OTM at 2 Gy (OTM2) was adopted as the index of initial DNA damage. The SF2 and OTM2 values are listed in Table 2. Clearly,

Table 2 Parameters of the cellular radiosensitivity and initial DNA damage after the X-ray and carbon ion irradiations

X-rays Carbon ion beam t-test

SF2

OTM2

0.463 ± 0.181 0.174 ± 0.032 p < 0.05

2.867 ± 0.492 15.833 ± 3.826 p < 0.01

SF2, survival fraction at 2 Gy; OTM2, olive tail moment at 2 Gy.

Fig. 3. Initial DNA damages in Lewis lung carcinoma cell immediately after X-ray and carbon ion beam irradiations. Best fits for the DNA damage parameters ‘‘Tail DNA (%)” and ‘‘Olive tail moment” are demonstrated. left: represents ‘‘Tail DNA (%)”, right: shows ‘‘Olive tail moment”. Each data point represents the mean ± standard error (SE) of about 50 cells/slide of three independent examinations.

Table 1 Parameters of the weighted fits for the mean ‘‘Tail DNA (%)” and ‘‘Olive tail moment” dose response to the data points shown in Fig. 2 Endpoint

Radiation type

Linear dose response

Exponentially linear dose response

Slope values (±SE)

Coefficient of determination

a (±SE)

b (±SE)

c (±SE)

Coefficient of determination

Tail DNA (%)

X-rays Carbon ion beams

2.21 ± 0.19 –

0.96 –

– 41.40 ± 2.49

– 0.32 ± 0.05

– 5.72 ± 1.77

– 0.98

Olive tail moment

X-rays Carbon ion beams

2.47 ± 0.14 –

0.98 –

– 48.94 ± 5.82

– 0.27 ± 0.09

– 0.19 ± 0.09

– 0.95

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the high-LET carbon ion beam produced a lower SF2 value and a higher initial OTM2 than the X-ray irradiation. 4. Discussion Previous studies have proved that heavy ions are more effective than low-LET radiations in inducing cell reproductive death [9]. Therefore, the clinical treatment using high-LET ion beams may maximize the advantages of radiation therapy. The higher radiosensitivity of Lewis lung carcinoma cells to the carbon ion beam and higher RBE observed in this study reveal that treatment of tumors of this cell type might benefit from carbon ion therapy. According to our experimental results, a significant correlation could be obtained between clonogenic radiosensitivity (SF2) and induced initial DNA damage (OTM2) detected by comet assay under alkaline condition. According to the application of SF2 which could be as both an index of clonogenic cellular radiosensitivity and a potential prognostic factor for the radiotherapy [10], we selected OTM2 as an index of initial DNA damage of cells undertaking 2 Gy irradiations using ACA. Recently it has been reported that there is a good inverse correlation between cell survival and mean tail moment [7]. The size of the comet tail in the comet assay depends not only on the number of DNA strand breaks but also on the chromatin structure [11], indicating that the initial DNA damage detected by the comet assay is related to cellular radiosensitivity. Chromatin structure stability may be an important factor that relates not only to intercellular difference in initial DNA damage, but also to radiosensitivity [12]. At this point, several authors have reported that there is a good correlation between the initial DNA damage and cellular radiosensitivity [13], while others insist that there is no relationship between the initial DNA damage and cellular radiosensitivity [14]. The distinct correlation between cell reproductive death and initial DNA damage following X-ray and carbon ion irradiations probably depends on the difference in radiation quality. Our results indicated that the DNA fragments in cells irradiated by the carbon ions showed a saturation effect while this behavior was not found in the X-ray experiment at the present highest dose. These observations could be explained reasonably if the fragments initiated by the carbon ions are different from the fragments induced by the X-rays, either in length or in the clustering of breaks

close together, to change the migration behavior of the DNA. In conclusion, the cell reproductive death and initial DNA damage have been proved to be dependent on the radiation quality. High-LET carbon ion beam produces more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with low-LET X-ray irradiation. Due to the correlation between SF2 and OTM2, comet assay could be a significant predictive test even combining with clonogenic assay to assess the radiosensitivity of cells in the target volume prior to clinical treatment. Acknowledgements This work is jointly supported by the Century Program of the Chinese Academy of Sciences (O506120BR0) and the National High Technology Research and Development Program of China (863 Program, 2006AA02Z499). The authors are grateful to the operating crew of the HIRFL complex for supplying the carbon ion beam. References [1] M. Bergqvist, D. Brattstro¨m, M. Stalberg, H. Vaghef, O. Brodin, B. Hellman, Cancer Lett. 133 (1998) 9. [2] P.L. Olive, J.P. Banath, H.S. Macphail, Cancer Res. 54 (1994) 3939. [3] J.H. Peacock, H.A. Edwards, T.R. Mcmillan, G.G. Steel, Int. J. Radiat. Biol. 56 (1989) 543. [4] M.E. Price, V.J. Mckelvey-Martin, T. Robson, D.G. Hirst, S.R. Mckeown, Radiat. Res. 153 (2000) 788. [5] W.P. Roos, A. Binder, L. Bohm, Int. J. Radiat. Biol. 76 (2000) 1493. [6] J.M. Ruiz de Almodovar, M.I. Nunez, T.J. Mcmillan, N. Olea, C. Mort, M. Villalobos, V. Pedraza, G.G. Steel, Br. J. Cancer 69 (1994) 457. [7] V.J. McKelvey-Martin, E.T.S. Ho, S.R. McKeown, S.R. Johnston, P.J. McCarthy, N.F. Rajab, C.S. Downes, Mutagenesis 13 (1998) 1. [8] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, Exp. Cell Res. 175 (1988) 184. [9] J. Carlsson, B. Stenerlo¨w, K. Russell, E. Grusell, B. Larsson, E. Blomquist, Anticancer Res. 15 (1995) 273. [10] C.M.L. West, S.E. Davidson, R.D. Hunter, Int. J. Radiat. Biol. 56 (1989) 761. [11] I. Brammer, M. Zoller, E. Dikomey, Int. J. Radiat. Biol. 77 (2001) 929. [12] R.S. Malyapa, W.D. Wright, J.L. Roti, Radiat. Res. 145 (1996) 239. [13] G. Bacova´, L’. Huna´kova´, M. Chorva´th, M. Chorva´th, E. Boljesˇikova´, B. Chorva´th, J. Senla´, A. Ga´belova´, Neoplasma 47 (2000) 367. [14] E. Dikomey, I. Brammer, Int. J. Radiat. Biol. 76 (2000) 773.