**2. History of bystander effect phenomenon**

336 Radioisotopes – Applications in Physical Sciences

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

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

Intercellular Communication in Response to Radiation Induced Stress:

response may be mutually exclusive to the bystander effect.

**4. The mechanisms of radiation induced bystander effect** 

reactive oxygen species (ROS), namely, hydroxyl radical (OH•

•

and superoxide radical anion (O2

The ionizing radiation acts through direct ionization of organic macromolecules or through

(Marnett, 2000, Matsumoto et al., 2007). Half-life of ROS is extremely short and penetration distance is expressed in micrometers. Therefore, these factors may not reach non-irradiated cells. Electron spin resonance studies have shown, however, that long-lived radicals with a period of half-lives ca. 20 hours may appear in cells after irradiation, even at room temperature (Koyama et al., 1998); if transferred to the surroundings, they may be the factors inducing DNA damage in non-irradiated cells. The long-lived secondary radicals are likely to be less active in damaging DNA than the extremely active primary radicals generated during irradiation time. Therefore, DNA damage induced by secondary radicals may not be a sufficient barrier to stop the replication of DNA and can lead to duplication of altered DNA through generations of cells, and finally to mutation and neoplastic transformation (Azzam et al., 2003, Clutton et al., 1996, Iyer and Lehnert, 2000, Lala and Chakraborty, 2001). DMSO, a radical scavenger, reduced the level of DNA damage in irradiated cells and inhibited the bystander effect which seems to confirm the role of reactive forms of oxygen in initiating signaling molecules (Hussain et al., 2003, Kashino et al., 2007). Also, the use of vitamin C as a scavenger of long-lived radicals compromised the level of micronuclei in human fibroblasts co-incubated with irradiated cells (Harada et al., 2008), as well as in K562 myelogenous lukemia cells treated with medium from irradiated cultures of the same cell line collected one hour post irradiation (Konopacka and Rzeszowska-Wolny, 2006). However, not only DNA is the target for ROS; no less important are the fatty acid molecules, in which the peroxidation chain reactions lead, through shortlived lipid radicals, to stable end-products such as malondialdehyde (MDA), 4-

), hydrogen peroxide (H2O2)

־(, the effect of which is primarily oxidative DNA damage

Bystander Effects in Vitro and in Vivo and Their Possible Clinical Implications 339

was observed in human glioblastoma A-172 cells with functional *TP53* gene when they were co-incubated with irradiated (1-10 Gy X-rays) cells of the same line transfected with mutated *TP53* gene (A-172/mp53), or incubated in the presence of conditioned medium from irradiated cells (Matsumoto et al. 2001). The sign of radioresistance was the accumulation of HSP72 and p53 protein which had declined in the presence of nitrogen oxide scavenger or inducible nitrogen oxide synthase inhibitor. Another probable mechanism thought to be a cellular adaptive response is the low-dose enhancement of DNA repair ability and antioxidant activity, resulting in more proficient cellular responses to the subsequent challenge. Sawant et al. (2001) observed that the exposure of C3H 10T91/2 cells to single alpha particle radiation, which hit only 10% of cells, caused the death of a much larger number of cells. However, the use of 2cGy gamma rays 6 hours before exposure to the alpha particles continuously reduced the bystander effect expressed as increased surviving cell fraction. Increased resistance induced by large dose of gamma radiation was also observed in cells of the same line if they were pre-exposed to a cGy dose of 60-Co (Azzam et al., 1996), and the reduction in the percentage of micronuclei was accompanied by an increase in the repair of DNA double-strand breaks (Azzam et al. 1994). Recently, it was presented that different cell lines can show different pattern of response to low priming dose (Ryan et al. 2009). An adaptive response was detected in cell lines known to produce hypersensitive response, and was inversely correlated with the bystander effect suggesting that an adaptive

secondary cancers (Hendry, 2001). Not always, however, radiation induced bystander effect has a damaging action. The signals emitted to the microenvironment by irradiated cells seem to induce in cells unexposed to radiation more complex effects, inter alia their differentiation, probably as a comprehensive response in order to preserve the integrity of the tissue (Belyakov et al., 2006, Vines et al. 2009).

#### **3. Radiation induced bystander effect, genetic instability and adaptive response**

Bystander effect, genetic instability and adaptive response seem to be related. Known as the genetic instability are the delayed effects such as lethal mutation, unstable chromosome aberrations, and delayed reproductive death (DRD) in distant generations of cells previously exposed to radiation (Gorgojo et al., 1989, Mendonca et al. 1989), or arising *de novo* chromosome aberrations (Kadim et al., 1995, Marder and Morgan 1993, Weissenborn and Streffer, 1989) and gene mutations (Little et al., 1997). Delayed reproductive death (DRD), manifested as diminution of clonogenic cell survival, appears to be caused neither by apoptosis nor by necrosis. DRD is mainly observed in cells with uninterrupted mechanisms of DNA double-strand breaks repair (Little et al., 1990, Little, 1999), but is not observed in cells with impairment of these mechanisms (Chang and Little, 1992). It was demonstrated that cell clones with post-radiation genetic instability evolve through many generations of descendants, the cytotoxic factors affecting non-irradiated cells (Kadim et al., 1995) and, the effect being independent of intercellular gap junctions (Nagasawa et al., 2003). Studies of genetic instability in which only some mouse marrow stem cells were targeted by alpha particles showed higher numbers of cells with chromosome aberrations than those of irradiated cells. These lesions are transferred to the descendant cells forming colonies (Loroimore et al., 1998). In addition, the surviving fraction of clonogenic cells decreases deeper with the dose than would result from the dose absorbed, provided the damage resulted from communication of lethally-irradiated cells with non-irradiated cells. Increased mutation frequency of hypoxanthine-guanine-phosphoribosyl transferase gene (*HPRT*) in distant generations of murine hematopoietic stem cells irradiated *in vitro* with both the X-rays and neutrons was also observed (Harper et al., 1997). Furthermore, human T-lymphocytes showed chromosome aberrations transferred through generations of their progenitor cells that had been irradiated with 3Gy X-rays dose (Holmberg et al., 1995). Factors inducing the bystander effects can be passed through gap junctions (Zhou et al., 2000, Azzam et al., 2002], or secreted to the surroundings (Lyng et al., 2000, Morthersill and Seymour, 1998). Some of them are clastogenic and can induce chromosomal damage in nonirradiated cells, analogous to that in directly-hit cells. Huang et al. (2007) observed that growth medium conditioned by some chromosomally unstable RKO derivatives induced genomic instability, indicating that these cells can secrete factor(s) that elicit responses in non- irradiated cells. Furthermore, low radiation doses suppressing the induction of delayed genomic instability by a subsequent high dose, are indicative of an adaptive response for radiation-induced genomic instability. Adaptive response is a phenomenon by which cells irradiated with a sub-lethal radiation dose (mGy or cGy) may become less susceptible to subsequent high-dose (a few Gys) radiation exposure (Wolff, 1996, Marples and Skov, 1996). The mechanism of this phenomenon is not sufficiently known. Irradiation leads to disturbances of the balance between pro-oxidant and anti-oxidant signaling molecules; one of such molecules can be nitric oxide (NO) (Spitz et al., 2004). An increase of radioresistance

secondary cancers (Hendry, 2001). Not always, however, radiation induced bystander effect has a damaging action. The signals emitted to the microenvironment by irradiated cells seem to induce in cells unexposed to radiation more complex effects, inter alia their differentiation, probably as a comprehensive response in order to preserve the integrity of

Bystander effect, genetic instability and adaptive response seem to be related. Known as the genetic instability are the delayed effects such as lethal mutation, unstable chromosome aberrations, and delayed reproductive death (DRD) in distant generations of cells previously exposed to radiation (Gorgojo et al., 1989, Mendonca et al. 1989), or arising *de novo* chromosome aberrations (Kadim et al., 1995, Marder and Morgan 1993, Weissenborn and Streffer, 1989) and gene mutations (Little et al., 1997). Delayed reproductive death (DRD), manifested as diminution of clonogenic cell survival, appears to be caused neither by apoptosis nor by necrosis. DRD is mainly observed in cells with uninterrupted mechanisms of DNA double-strand breaks repair (Little et al., 1990, Little, 1999), but is not observed in cells with impairment of these mechanisms (Chang and Little, 1992). It was demonstrated that cell clones with post-radiation genetic instability evolve through many generations of descendants, the cytotoxic factors affecting non-irradiated cells (Kadim et al., 1995) and, the effect being independent of intercellular gap junctions (Nagasawa et al., 2003). Studies of genetic instability in which only some mouse marrow stem cells were targeted by alpha particles showed higher numbers of cells with chromosome aberrations than those of irradiated cells. These lesions are transferred to the descendant cells forming colonies (Loroimore et al., 1998). In addition, the surviving fraction of clonogenic cells decreases deeper with the dose than would result from the dose absorbed, provided the damage resulted from communication of lethally-irradiated cells with non-irradiated cells. Increased mutation frequency of hypoxanthine-guanine-phosphoribosyl transferase gene (*HPRT*) in distant generations of murine hematopoietic stem cells irradiated *in vitro* with both the X-rays and neutrons was also observed (Harper et al., 1997). Furthermore, human T-lymphocytes showed chromosome aberrations transferred through generations of their progenitor cells that had been irradiated with 3Gy X-rays dose (Holmberg et al., 1995). Factors inducing the bystander effects can be passed through gap junctions (Zhou et al., 2000, Azzam et al., 2002], or secreted to the surroundings (Lyng et al., 2000, Morthersill and Seymour, 1998). Some of them are clastogenic and can induce chromosomal damage in nonirradiated cells, analogous to that in directly-hit cells. Huang et al. (2007) observed that growth medium conditioned by some chromosomally unstable RKO derivatives induced genomic instability, indicating that these cells can secrete factor(s) that elicit responses in non- irradiated cells. Furthermore, low radiation doses suppressing the induction of delayed genomic instability by a subsequent high dose, are indicative of an adaptive response for radiation-induced genomic instability. Adaptive response is a phenomenon by which cells irradiated with a sub-lethal radiation dose (mGy or cGy) may become less susceptible to subsequent high-dose (a few Gys) radiation exposure (Wolff, 1996, Marples and Skov, 1996). The mechanism of this phenomenon is not sufficiently known. Irradiation leads to disturbances of the balance between pro-oxidant and anti-oxidant signaling molecules; one of such molecules can be nitric oxide (NO) (Spitz et al., 2004). An increase of radioresistance

**3. Radiation induced bystander effect, genetic instability and adaptive** 

the tissue (Belyakov et al., 2006, Vines et al. 2009).

**response** 

*TP53* gene (A-172/mp53), or incubated in the presence of conditioned medium from irradiated cells (Matsumoto et al. 2001). The sign of radioresistance was the accumulation of HSP72 and p53 protein which had declined in the presence of nitrogen oxide scavenger or inducible nitrogen oxide synthase inhibitor. Another probable mechanism thought to be a cellular adaptive response is the low-dose enhancement of DNA repair ability and antioxidant activity, resulting in more proficient cellular responses to the subsequent challenge. Sawant et al. (2001) observed that the exposure of C3H 10T91/2 cells to single alpha particle radiation, which hit only 10% of cells, caused the death of a much larger number of cells. However, the use of 2cGy gamma rays 6 hours before exposure to the alpha particles continuously reduced the bystander effect expressed as increased surviving cell fraction. Increased resistance induced by large dose of gamma radiation was also observed in cells of the same line if they were pre-exposed to a cGy dose of 60-Co (Azzam et al., 1996), and the reduction in the percentage of micronuclei was accompanied by an increase in the repair of DNA double-strand breaks (Azzam et al. 1994). Recently, it was presented that different cell lines can show different pattern of response to low priming dose (Ryan et al. 2009). An adaptive response was detected in cell lines known to produce hypersensitive response, and was inversely correlated with the bystander effect suggesting that an adaptive response may be mutually exclusive to the bystander effect.
