**Intercellular Communication in Response to Radiation Induced Stress: Bystander Effects in Vitro and in Vivo and Their Possible Clinical Implications**

Maria Widel *Institute of Automatics, Electronics and Informatics Silesian University of Technology Poland* 

#### **1. Introduction**

334 Radioisotopes – Applications in Physical Sciences

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Communication between cells is important for maintaining homeostasis, the physiological regulatory processes that keep the internal environment of a system in a constant state. A disease can disturb the internal equilibrium of cells, and this can be further disrupted by various therapies. Malignances are the diseases that need to be treated by highly aggressive methods, such as radiotherapy, which affects not only tumor cells but also normal cells adjacent to the tumor and usually included in the radiation field. This treatment may interfere with normal intercellular communication. It has been a central radiobiological dogma for decades that damaging effects of ionizing radiation are the result of direct ionization of cell structures, particularly DNA, or are due to indirect damage *via* water radiolysis products. Indeed, DNA damage such as chromosomal aberrations, micronuclei, sister chromatid exchange and mutagenesis result from ionizing radiation. All of these types of damage, if unrepaired, can lead to cell death or, if misrepaired, can lead to genomic instability and carcinogenesis. Recently however, the attention was focused on the third mechanism, a phenomenon termed "radiation induced bystander effect" (RIBE). This phenomenon is a non-targeted effect where molecular signal(s) produced by directly irradiated cells elicit subsequent responses in unirradiated neighbors. These responses are manifested as decreased survival, increased sister chromatid exchanges (SCE), chromosomal aberrations (CA), micronucleus (MN) formation, gene mutations, apoptosis, genomic instability, neoplastic transformation and a variety of damage-inducible stress responses (reviewed in Morthersill and Seymour, 2001, Lorimore et al., 2003, Morgan, 2003a, 2003b, Little, 2006a,b, Chapman et al. 2008, Rzeszowska-Wolny et al., 2009a). Bystander effect accompanies very low doses of alpha particles (mGy and cGy), (Nagasawa and Little, 1992, Lorimore et al., 1998), as well as irradiation of cells with a low LET radiation (X- and gamma rays), even at conventionally used higher clinical doses (Morthersill and Seymour, 1997, 1998, 2002b, Przybyszewski et al., 2004). The mechanisms responsible for RIBE are complex and not quite well-known. Mechanisms by which bystander signals may be transmitted from irradiated to non-irradiated cells involve direct cell-to-cell contact mediated by gap

Intercellular Communication in Response to Radiation Induced Stress:

knowledge and our own experimental results.

**2. History of bystander effect phenomenon** 

Bystander Effects in Vitro and in Vivo and Their Possible Clinical Implications 337

review of the various aspects of radiation-induced bystander effect, based on the current

First observations of the bystander effect phenomenon appeared in the nineties of the last century. Using a low-dose of alpha particles which targeted only 1% of cultured Chinese hamster ovary cells (CHO), Nagasawa and Little (1992) noticed cell damage in the form of sister chromatid exchanges (SCE) appearing in about 30% of cells. The level of damage increased with 0.3-2.5 mGy dose, but not with higher ones. Subsequent experiments showed an increase in the number of cells with overexpression of *TP53* gene after 6 mGy alpha irradiation, but not after exposure to the same dose of X-rays (Hickman et al., 1994). Very soon, it appeared that this effect also occurs in cells exposed to radiation with a low LET radiation. It was observed that the factors inducing the observed effects in non-irradiated cells are soluble and can be passed through the growth medium (Deshpande et al., 1996, Morthersill and Seymour, 1997), or by an intercellular connection slot (Azam et al., 1998). Morthersill and Seymour (1997) showed that factors present in the culture medium collected from epithelial cells exposed to gamma radiation decreased survival of clonogenic nonirradiated cancer and epithelial cells in culture; therefore for the bystander effect to occur the contact of irradiated cells with non-irradiated is not necessary. Furthermore, reduced cell survival did not occur when medium harvested from irradiated fibroblasts was used. The cytotoxic effect of irradiation-conditioned medium (ICM) has been observed in several experimental systems following both particle (Deshpande et al., 1996, Lorimore et al., 1998) and photon irradiation (Clutton et al., 1996, Matsumoto et al., 2001). It was found that the bystander effect-signaling molecules may include tumor necrosis factor beta (TGFβ) and interleukin-8 (Narayanan et al., 1999) secreted to the medium or transferred through GJIC. Closing these connections by *lindane*, an inhibitor of gap junction, lead to the inhibition of bystander effect, evidenced as the reduced expression of *TP53*, *CDKN1A* (p21) and *CDC2*  genes (Azzam et al., 1998), or increased survival of clonogens (Bishayee et al., 1999). Several studies have demonstrated that the radiation-induced bystander effect triggers apoptosis (Prise et al., 1998, 2006, Morthersill and Seymour, 2001, Przybyszewski et al., 2004) and increase of micronucleus frequency, DNA double-strand breaks (DSBs) measured as histone H2AX phosphorylation (Sokolov et al., 2007, Burdak-Rothkam et al., 2007), accumulation of p53 (Tartier et al., 2007) and ATM and ATR proteins (Burdak-Rothkam et al., 2008), epigenetic changes, such as DNA hypomethylation, as well as the expression of other genes (Chaudry, 2006, Iwakawa et al., 2008, Rzeszowska-Wolny et al., 2009b). Many of these experiments showed that higher doses of radiation, including those used in conventional radiotherapy, also induce bystander effects in non-irradiated cells. They confirmed the quantitative biophysical model of Nikjoo and Kvostunov (2003, 2006) which assumes that RIBE may be a component of neighborhood responses to radiation, both at low and high doses. The results obtained in tissue explant culture (Belyakov et al., 2002, 2006, Mothersill and Seymour, 2002b), tri-dimensional cell culture, *in vivo*-like models (Bishayee et al., 1999, 2001, Belyakov et al., 2005), and in animal studies (Koturbash et al., 2006, 2007, 2008) all point out to the bystander phenomenon relevance to clinical radiotherapy. Therefore, one cannot exclude that the intensity of side effects in healthy tissues following fractionated radiotherapy may be partly related to bystander effect. It is suspected that this effect may also lead to genetic instability, the consequence of which can involve development of

junction intercellular communication (GJIC), and indirect communication by means of soluble factors secreted by irradiated cells into the surrounding medium. It is believed that molecular signaling factors released by cells irradiated and dispatched to the medium or transferred through GJIC induce various signaling pathways in neighboring cells, leading to the observed effects. The nature of these factors may be different and they have not been definitely defined. In addition to short-lived oxygen and nitrogen free radicals (Matsumoto et al., 2001, Azzam et al., 2002), long-lived radicals (Koyama et al., 1998), interleukin 8 (Narayanan et al., 1999), TGF-β (Shao et al., 2008 a, b, Massague and Chen, 2000) and other agents can be included. Potentially, bystander phenomenon could play an important role in the appearance of undesirable localized or systemic radiotherapeutic effects in tissues not included in the irradiation field. Furthermore, the effect may appear after low-dose irradiation during diagnostic radiology procedures and following application of a radioisotope for diagnosis or treatment (Prise and O'Sullivan, 2009). Factors emitted by irradiated cells may have impact on risk of genetic instability and the induction of mutation. However, the radiation-induced bystander effect may have both detrimental and potentially beneficial consequences. If cells directly hit by ionizing energy will, through their signals (secreted or transmitted through the gap junction) damage adjacent cancer cells, or will initiate differentiation of these cells, it is desirable. However, if normal cells are damaged (epithelial and endothelial cells, fibroblasts, leucocytes, etc.), then the effect may be a disadvantage that increases the unwanted effects of radiotherapy such as late complications and second primary tumors. Bystander effect can be particularly important in the case of the use of current techniques of irradiation, such as 3D conformal radiation therapy (3D-CRT) and intensively modulated radiotherapy (IMRT), the purpose of which is to reduce the irradiation dose in healthy tissues (Followill et al., 1997). Some data indicate that bystander effect also occurs *in vivo* (Koturbash et al., 2006, 2007, Ilnytskyy et al. 2009). The studies of bystander effect in *in vivo* animal models show that the post- radiation damage can appear in tissues distant from the place of irradiation, and the effect may vary depending on the type of tissue. However, recent experimental results (Mackonis et al., 2007), including our own (Widel et al. 2008, and unpublished), show that cross-talk between irradiated and unirradiated cells may be sometimes protective and non-irradiated cells, which are in the vicinity of irradiated cells can hamper the effects caused by their irradiation. Furthermore, a radioprotective bystander effect has been observed in several studies with low-dose exposure in the form of increased cell redioresistance to subsequent higher doses (e.g. Sawant et al., 2001, Prise et al., 2006). Less known are the consequences of bystander effect in the case of dose fractionation during external irradiation. Our preliminary results from *in vitro* fractionation dose experiments, presented in this Chapter indicate that apoptosis is even more effectively induced in human melanoma radiation-targeted and bystander cells when the same dose is delivered in 3 fractions than in one single dose. A growing body of experimental *in vitro* and *in vivo* data indicate the occurrence of bystander phenomenon in radionuclide-based radiotherapy (Xue et al., 2002, Gerashchenko and Howell, 2004, Boyd et al., 2006, Mairs et al. 2007). However, studies of radionuclide-induced bystander effect demonstrate varying responses (compared to low LET radiation-induced ones), being either damaging or protective depending on dose and type of emitters. The practical consequences, as well as capacities of the bystander effect, in terms of modulating radiotherapeutic approaches, are therefore still uncertain and are the subject of intensive research. It is possible that the impact of bystander signaling on both cancer and healthy tissue responses is more relevant than it is believed at present. Below is a comprehensive review of the various aspects of radiation-induced bystander effect, based on the current knowledge and our own experimental results.
