BYSTANDER EFFECT INDUCED BY RADIOTHERAPEUTIC DOSES IN BREAST CARCINOMA CELL LINE Zubiría, M. G.1,2; Martínez, M.2; Dodat D.1,2; Sánchez G.1,2; Casas, O.2 and Güerci A.1,2,3 1

Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Argentina. 2CITOMA. Centro de Investigaciones de Transferencia en Oncología Molecular. Argentina. 3IGEVET

E-mail: [email protected] Abstract. BYSTANDER EFFECT (BSE) refers to the observed effects in cells that have not been directly crossed by radiation and which are near the radiated cells. It is suggested that different communication and signaling cellular pathways, such as gap junctions and the release of diverse clastogenic factors (reactive oxygen species and citokynes), take place in this phenomenon. Delayed Genomic Instability (GI) could be explained by means of BSE and that could be showed by the cloning efficiency decrease, the apoptosis increase, point mutations, and micronuclei frequency. The relevance of this phenomenon is discussed in the carcinogenic process. Besides, BSE is also important because of its impact on the present paradigm of the ionizing effect of radiation, essentially based on the Theory of the Target. The importance of this research on radiotherapy could be the optimization of the therapeutic effects of radiation. In the present study, the BSE was evaluated in breast carcinoma cell line MCF-7 by the application of both techniques: Comet Assay (alcaline version) and Clonogenic Assay. Conclusions: - ROS involvement in BSE was corroborated by Comet Assay in this cell line; ICM treatment decreased cloning efficiency, checked by Clonogenic Assay. These results are very important for the reconsideration of the current paradigm of Target Theory and its application in radiobiology.

KEYWORDS: Bystander Effect - Radiotherapeutic dose - Target Theory 1. Introduction BYSTANDER EFFECT (BSE) refers to observed effects in cells that have not been directly crossed by radiation. These un-hit cells (bystander cells) demonstrating effects may or may not be in direct contact with irradiated cells. It is suggested that different communication and signaling cellular pathways, such as gap junctions and the diverse factor release, take place in this phenomenon [1, 2]. Many different end points have been described in bystander cells. They include reduced clonogenicity, increased point mutations, increased apoptosis, increased micronuclei, and delayed effect characteristics of radiation induced genomic instability [3]. Typically, BSE is usually studied in vitro, using cell cultures in one of three approaches. One of the approaches is to use a particle accelerator that is focused using microscope optics to deliver high-energy particles to a specific cell and then to measure the effects in other cells in the culture. Another method is to harvest culture medium from irradiated cells (Irradiated Conditioned Medium, ICM) and controls and to measure the effects of medium-borne signals on unexposed recipient cells. The third approach is based on probability and it involves exposing cultures to very low doses of α particles, such that every cell could not receive an α particle ‘hit’[4]. Considering the range of doses in which stochastic effects are important, the nature of the response to radiation is determined by a combination of genetic and epigenetic factors. It is now considered to be as important as dose by many radiobiologists. Radiation protection is a scientific field that attempts to predict biological effects of low doses of radiation by extrapolating from known epidemiological studies to high-dose effects. The main source of information comes from data collected on the Japanese atomic bomb survivors. This is one of the most

reliable information of radiation-induced cancer risk for exposures to low-LET radiation in the dose range 20-250 cGy [3, 4]. In this way, standards and guidelines regarding acceptable doses to the general public and to radiation workers have been developed by assuming a linear no-threshold (LNT) hypothesis, which relates dose to biological effect [5]. This hypothesis states that the dose–effect relationship is linear even at low doses, meaning that, in theory, the lowest imaginable dose has a finite probability of causing a biological effect. All types of radiation are regulated in this way and all individual exposures are limited using these guidelines, except medical exposures [4]. However, the effect of a radiation dose no longer depends on the amount of energy deposition, but, on the cellular response to that energy deposition; that is, how much and what kind of signal is generated and, therefore, how the bystander cells respond to this signal. Consequently, BSE challenges some precepts of the LNT model and being very important in this context, because their existence (so important at low doses) means that there is no direct correlation between the number of cells that are exposed to radiation and the number of cells that are at risk at low doses [4, 5]. Thus, last effect of radiation depends on complex interactions between the irradiated cells and the bystander cells (Fig. 1). Therefore, the consequence of the BSE is that the target model of radiation damage is not applicable to low doses, even to the LNT hypothesis. In fact, direct cellular damage and BSE would have the same importance at a limit dose of 2 Gy [6]. The purpose of this paper is to consider and to discuss how the new concepts in radiobiology, which are emerging as consequence of BSE, impact on radioprotection. Figure 1: Two different mechanisms of BSE. A) By means of gap junctions; B) By extra cellular signals.

B) A)

2. Materials and Methods 2.1. Cells and cell culture The human breast cancer cell line MCF-7 was employed in this study. Cells were routinely cultured as monolayers in D-MEM F12 nutrient medium, supplemented with 10% (v/v) fetal bovine serum, 2 mM Lglutamine, 0,01 mg/ml insulin ,100 IU/mL penicillin G and 100 µg/mL streptomycin sulfate as antibiotics in a 37 °C chamber incubator, providing a humidified atmosphere of 5% CO2 in air. All cultures were free of Micoplasma contamination. Cells of an exponentially-growing monolayer culture were then used for different treatments. 2.2. Experimental design In order to evaluate the BSE a treatment with ICM was carried out. Using this approach, we confirmed the presence of clastogenic factors in the medium of irradiated cells. It consists of the irradiation of a culture

cell and the following transfer of its medium to cells that have not been irradiated. Simultaneously, a negative control (non irradiated cells) was performed, the direct damage (irradiated cells) and the medium control (irradiated medium only). All the irradiations were carried out as we describe below (see irradiation procedure). After that, we analyzed genotoxic damage using Comet Assay to detect SSBs, DSBs and LAS [7, 8]; and cytotoxic damage using Clonogenic Assay to evaluate cellular survival (Fig 2). Each assay was repeated three times independently. Figure 2: Experimental Design. 1) Direct Damage: irradiated cells. 2) BSE: treatment with ICM. 3) Control: None irradiated cells. 4) Medium Control: irradiated medium.

1)

Direct Damage

2)

120´

3)

IC ICM M

BSE 60´

Negative Control

4)

Medium Control

2.3. Irradiation procedure The cells were irradiated with 2 Gy (Absorbed Dose) using a Varian Clinac ® electron lineal accelerator of nominal accelerating potential of 4 MeV. The irradiation procedure was performed considering a monolayer cells growth on the bottom surface of a culture flask, immersed in nutrient medium. Under these conditions, the monolayer was covered by 4 mm of medium. The flasks were placed inside a bigger recipient, which was partially filled with water, up to the medium top. The container had a flat acrylic base (22 mm) to allow the monolayer to be located beyond the electronic equilibrium’s depth. The irradiation was performed at the container bottom (Fig. 3). Figure 3: Irradiation procedure diagram.

The monitor units (MU) necessary to give the desired absorbed dose were calculated using isocentric formalism (see equation 1).

MU =

Dcel (d , Leq ) •

Do (d ref , Lref ) ⋅ S cp ( Leq ) ⋅ TMR (d , Leq ) ⋅ ISL ⋅ MU

,

(1)

Where:
Dcel (d, Leq) it is the absorbed dose by the monolayer placed to depth d, for a field size Leq at this depth.
Do (dref, Lref) is the reference absorbed dose measured at isocenter position at a water depth dref for a reference radiant field size Lref (10 x 10 cm in isocenter) for each given MU.
Scp (Leq) it is the relative absorbed dose factor for a field size Leq that evaluates the absorbed dose quotient at isocenter to the depth dref for the field size Leq against the field size Lref (10 x 10 cm in the isocenter).
ISL is the term that incorporates the inverse square law distance (1 in this case).
TMR (d, Leq) it is the tissue maximum ratio to depth d and equivalent radiant field size Leq to depth d.
MU is a value that provides the treatment machine dosimetric chain and is proportional to the given radiant energy for each irradiation configuration. This value is pre selected in the accelerator command console to establish the absorbed dose that we want to administer to the irradiated cells. The calculated obtained MU was then selected in the accelerator command console to irradiate cells with the desired absorbed dose. 2.4. Clonogenic survival assay After applying the different treatments, cells of an exponentially-growing monolayer culture were harvested by the use of 0.04% trypsin in phosphate-buffered saline (PBS) (4 min incubation at 37 °C) and suspended in 3 ml of medium. Using a 1-mL micropipette, the cells were pipetted up and down several times, forcing them through the tip of the pipette to break up the clumps. The cells were counted using a Neubauer chamber. After microscopic examination to ensure a reasonable quality of single-cell suspension, the cells were diluted in growth medium to yield ~300 cells/dish (by serial dilutions) and they were plated out in 60-mm dishes [9]. Cultures were incubated at 37ºC in a humidified atmosphere containing 5% CO2 for 10-14 days. The media was changed every 48 hours. The cultures were microscopically examinated every day to follow the growth conditions. The cells were then fixed and stained with 1 % p/v crystal violet, and the number of survivors (i.e., colonies containing >50 cells) scored [10]. Survival graphics were constructed by plotting CFA (expressed as cloning efficiency referred to the control cultures). 2.5. Comet Assay A single-cell gel electrophoresis was performed using the alkaline version described by Singh et al, [11] with some modifications [12]. Briefly, slides were covered with a first layer of 180 µl of 0.5% normal agarose (Invitrogen, Carlsbad, CA). 75 µl of 0.5% low-melting-point agarose (Invitrogen) were mixed with approximately 15,000 cells suspended in 15 µl and layered onto the slides, which were then immediately covered with coverslips. After performing the agarose solidification at 4°C for 15 minutes, the coverslips were removed and the slides were immersed overnight at 4°C in fresh lysis solution. The slides were equilibrated in alkaline solution for 20 minutes. Electrophoresis was carried out for 20 minutes at 20 V and 250 mA. Afterwards, the slides were neutralized by washing them three times, every 5 minutes with Tris buffer (pH 7.5) and subsequently washing them in absolute ethanol. The slides were

stained with a solution of 1/1000 SYBR Green I nucleic acid stain (Molecular Probes, Eugene, OR) [13]. 200 randomly selected comet images were analyzed per experimental point. The comet score was carried out blind. Based on the extent of strand breakage, cells were classified according to their tail length in five categories, ranging from 0 (no visible tail) to 4 (still a detectable head of the comet but most of the DNA in the tail). In such a way the quantification was made through Damage Index parameter (see equation 2) [14].

DI =

Dd = 4

∑P

Dd = 0

( Dd )

× Dd

(2)

Where:
Dd is Damage degree (from 0 to 4).
P is percentage of cells with certain damage degree (Dd). Considering 100 analyzed cells, the DI ranged from 0 to 400 arbitrary units [7]. 2.6. Image Analysis The scoring was made at a magnification of 400 X by using a fluorescent microscope (Olympus BX40 equipped with a 515- to 560-nm excitation filter) connected through a Sony 3 CCD-IRIS color video camera (Sony Electronics, Fort Myers, FL). The image for each single cell was acquired immediately after opening the microscope shutter to the computer monitor. The analysis was made by using the Image-Pro Plus 3.2 Program. 2.7. Statistical Analysis Cells were irradiated or treated with ICM at least three times in independent experiments with two replicates flasks per experiment. Chi Square Test (χ2) or Student´s t-test (n=3) was used to determine the significance in Comet Assay and Cloning Assay, respectively. 3. Results 3.1. Comet Assay The results obtained in this assay have demonstrated genotoxic damage induced by ionizing radiation. It was generally shown by a high percentage of damaged cells and, using specifically the Damage Index parameter (Fig 4). We classified the damage into two categories: severe damage (comet degree 3 and 4) and slight damage (comet degree 1 and 2). On this way, the direct irradiated cells have shown, as we supposed, severe DNA damage (65.22% from total damaged cells), while the bystander cells have shown slight DNA damage (82.94% from total damaged cells). With the used dose (2Gy), both treatments (Direct Irradiation and BSE) have shown significant differences (p<0,001) regarding control.

3.2. Cloning Assay The results of cloning assay were sufficient to ensure a reproductive failure in treated cells (Direct Irradiation and ICM). The Survival Fraction was significant lower in both treatments (p<0.001) regarding control. A diminished cloning efficiency could demonstrate citotoxic damage in BSE cells; it would be caused by borne factors medium. On the other hand, comparing the bystander cells´survival fraction was twice as much as direct irradiated cells´ survival fraction (Fig. 5). Figure 4: DNA damage. A) Percentage of cells with and without damage. B) Damage Index. (*** p<0.001). A)

B) 100

80

250

***

Damage Index

Cells (%)

300

Without Damage With Damage

***

60

40

200

150

100

20 50

0

0

Control

DD

BSE

Medium

Control

DD

BSE

Medium

Figure 5: Survival Fraction. (*** p<0.001).

1,2 Control Survival Fraction

1

BSE DD

0,8 0,6

*** 0,4 0,2

***

0

4. Discussion For many years a central radiobiological paradigm contended that the biological effects caused by exposure to ionizing radiation occur in the irradiated cells as a consequence of DNA damage. Thus, the genetic changes, such as mutations and chromosomal aberrations, which are important events in carcinogenesis course, would arise in the irradiated cells as a consequence of mistakes during DNA reparation [15, 16]. Nevertheless, several studies have demonstrated that this is not a strict condition. In this context, the BSE allows to explain deleterious effects that cannot be raised by the target theory in unhit cells [17, 18, 5, 19, 20, 21].

The present work demonstrates the interactions between irradiated and non irradiated cells through factors released to medium, using the approach based on the transfer of culture medium harvested from irradiated cells. However, although many candidate bystander factors and modes of action have been reported in the literature, the exact mechanism of this signaling process is not known yet. It could involucrate the participation of reactive oxygen species (ROS) and other clastogenic factors, such as cytokines [22, 23, 24]. By mean of the alkaline version of comet assay, we were able to demonstrate the genotoxic effects of this clastogenic factors. The treated cells showed slight DNA lesions, corresponding to SSBs, DSBs and labile alkaline sites [25]. Regarding clonogenic assay, we verified a decrease of the survival fraction in response to the ICM treatment. Combining both, clonogenic`s and comet`s results, we reinforced the BSE concept as a consequence of ionizing radiation. This suggests that the cellular response transcends the target impact, involving genetic and epigenetic factors. Considering the periods of time used in the experimental design and in the different techniques, we suppose that slight DNA lesions detected with comet assay in the ICM treatment would be probably repaired by cells during the incubation. Thus, these lesions would contribute little to the decrease on the survival fraction, because they were plausible of reparation. On the contrary, the direct irradiation causes severe DNA lesions and they would be the reason of the higher decrease on the survival fraction. In accordance with all exposure, the consequence of BSE is that the target model of radiation damage is not tenable at all. Considering interactions between the irradiated cells and the bystander cells, there is no longer any single cell that is at risk from radiation damage; however, the risk is spread among bystander cells [4]. Therefore, the conceptual basis for the existing methods that are used to determine radiation risk require some modifications. In this context, the radiation protection might need to be re-structured conceptually, with far more emphasis on global cellular response to radiation. Besides, the traditionally accepted linear relationship between cause and effect would not be suitable to low doses. Therefore, the current method of extrapolating risk from well-characterized high-dose data to low-dose situations, although operationally useful, might not be conceptually sound. 5. Acknowledgements This work is part of the proyect “Mecanismos involucrados en la inducción de daño genético por exposición crónica a bajas dosis de radiaciónes ionizantes. Efectos Colaterales, Inestabilidad Genómica y modulación por compuestos tiólicos” (PICT N° 1-14329, 2005-2007). The authors wish to thank Institute of “Terapia Radiante S.A.” for access to the electron lineal accelerator. M.G. Zubiría has a fellowship from “Fundación Avanzar” and National University of La Plata. We wish to thank Mariela Salvoch for her collaboration in revising this paper. 6. Referentes [1]MATSUMOTO, H. et al. Vanguards of paradigm shift in radiation biology: radiation-induced adaptive and bystander responses. J Radiation Research 48; 97 (2007). [2] KO, M., et al. Neoplastic transformation in vitro by low doses of ionizing radiation: role of adaptive response and bystander effects. Mutation Research 597; 11 (2006). [3] MOTHERSILL, C., et al. Radiotherapy and the potential explotation of bystander effects. Internacional Journal of Radiation Oncology, Biology and Physics 58; 575 (2004). [4] MOTHERSILL, C., SEYMOUR, C. Radiation-induced bystander effects— implications for cancer, Nature Reviews Cancer 4; 158 (2004).

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