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

The ionizing radiation acts through direct ionization of organic macromolecules or through reactive oxygen species (ROS), namely, hydroxyl radical (OH• ), hydrogen peroxide (H2O2) and superoxide radical anion (O2 • ־(, the effect of which is primarily oxidative DNA damage (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-

Intercellular Communication in Response to Radiation Induced Stress:

Bystander Effects in Vitro and in Vivo and Their Possible Clinical Implications 341

involved in the transcription of proteins engaged in cell proliferation and differentiation, immunomodulation, cell-cycle control and apoptosis (Massague and Chen, 2000). The use of inducible nitric oxide synthase inhibitor, or anti-TGF antibodies which compromise micronuclei in cells directly irradiated with alpha particles and adjacent non-irradiated cells indicates a positive feedback. However, NO role as a mediator of the bystander effect has not been observed in all tested glioma cell lines (Matsumoto et al., 2001). In several types of cancer (colon, lung, throat) expression of inducible nitric oxide synthase (iNOS) was also linked to the *TP53* gene mutation (Lala and Chakraborty, 2001) indicating that the correct protein of p53 gene may negatively regulate the accumulation of iNOS. Many other factors were proposed as the bystander effect mediators, among them interleukin 8 (Narayanan et al., 1999), soluble tumor necrosis factor (TNFα) as well as Fas and TRAIL death ligands (Lucen et al. 2009). Also, multiple pathways are activated that take part in transmitting the bystander effect signals. Those induced in human fibroblasts by alpha particles (0.3-3 cGy) and transmitted through the GJIC or surrounding environment activated in adjacent cells various proteins such as MAP- kinase, NFκB, Raf-1, ERK1/2, JNK, AP-1 and others (Azzam et al. 2002, Lyng et al., 2006). Since application of SOD and catalase neutralizes the resulting oxygen radicals and hydrogen peroxide and hampers the bystander effect (reduction in the level of micronuclei, inhibition of nuclear factor κB and p38 MAPK activation), the mediators of these processes appear to be reactive oxygen and nitrogen species (Azzam et al., 2002). Targeting the nucleus or cytoplasm of HeLa cells by single helium ions induced expression of 53BP1, the protein which marks double-stand breaks in DNA (Tartier et al., 2007). The use of aminoguanidine, an inducible NO synthase inhibitor, or radical scavenger DMSO, cause inhibition of 53BP1 protein expression in both irradiated and co-incubated non-irradiated cells, pointing to the NO and ROS as the mediators of these lesions. At the same time, it was observed that antibiotic *filipin*, which damages the glycosphingolipid microdomains in cellular membrane, inhibited cellular signals from irradiated cells and led to a drastic reduction in the 53BP1 foci in neighboring cells. This reveals that transmission of bystander signals is dependent on the integrity of the cellular membranes, whereas membrane integrity was not necessary to generate the damage in irradiated cells. Also, the presence of mitochondria was necessary to generate bystander signals by irradiated cells, but was not necessary to their reception (Tartier et al., 2007). Calcium ion channels seem to play a role in the transmission of bystander signals. It was observed that biogenic amines, such as serotonin (5-hydroxytryptamine, 5-HT) and dopamine, may be the transducers of signals emitted by irradiated cells. The level of 5-HT neurotransmitter in culture medium decreased after irradiation of cells, likely due to its binding to the receptors which form the calcium channels, and leads to increased level of micronuclei (Poon et al., 2007). These effects were abolished after treatment of cells with calcium channel blockers calcicludin or rezerpin, which are the natural antagonists for serotonin (Poon et al., 2007, Shao et al., 2006). The study of transcript levels using DNA microchips may indicate signaling pathways and genes that are involved in the radiation-induced bystander effect. Gandhi et al. (2008), when examining the overall gene expression (global genome expression), after irradiation of human lung fibroblasts with alpha particles (0.5 Gy and 4-hour co-incubation with nonirradiated cells), observed that the expression of over 300 genes in both groups (hit and nonhit) was changed, and that 165 genes were common to both groups. Among them were genes mainly over-expressed in irradiated cells (CDKN1) and those that were overexpressed equally in irradiated and neighboring cells, namely NFκB–regulated PTGS2 (cyclooxygenase 2), IL8 and BCL2A1. However, Chaudhry (2006) observed that gene

hydroxynonenal (4HNE) and other with mutagenic and carcinogenic properties and which can form massive DNA adducts (Marnet, 2000, Zhong et al., 2001). The end-products of lipid peroxidation have secondary signaling molecule properties and can activate a cascade of signals leading to either DNA damage repair, or to damage stabilization or apoptosis (Hu et al., 2006). In our research we found increased MDA concentration in irradiated Me45 human melanoma cells growing in the form of megacolonies, as well as in the neighboring megacolonies growing in the same flask but protected against irradiation with a lead shield (Przybyszewski et al., 2004). At the same time, we found in both the irradiated and shielded megacolonies, decreased glutathione peroxidase (GSH-Pox) and mitochondrial superoxide dismutase (MnSOD), as well as elevated numbers of single- and double-strand DNA breaks (SSBs and DSBs), as assessed by single cell gel electrophoresis. The level of DNA breaks in non-irradiated cells was lower and appeared with several-hour delay compared to that observed in irradiated cells, which may suggest participation of long-lived radicals in the bystander effect induction (Przybyszewski et al., 2004). Time-shifted appearance of DSBs in neighboring cells estimated as the expression of phosphorylated histone H2AX (γH2AX foci) has been observed in the *in vitro* (Hu et al., 2006, Sokolov et al. 2007) as well as in *ex vivo* (Sedelnikova et al., 2007) conditions. While the phosphorylation of histone H2AX at serine 139 is a very early-stage event in cells directly exposed to radiation, the appearance of gamma-H2AX foci in cells co-cultured with irradiated ones, or treated with ICM only, may even take several hours. The gamma-H2AX foci, which indicate the presence of DNA DSBs in cells exposed to the signals transmitted by irradiated cells, co-localize with other proteins involved in the cell cycle control and DNA damage repair, such as ATM, MRE11, NBS1, Rad50 and 53BP1 (Sokolov et al., 2007). It is worth noticing that, based on ATM foci enumeration, Ojima et al. (2009) found that DSBs induced by the radiation-induced bystander effect persist for long periods (over 24 h), whereas DSBs induced by direct radiation effects are repaired relatively quickly. However, ATM foci persisted even longer (48 h) if bystander fibroblasts were co-incubated with very low (1.2 mGy) irradiated counterparts. This indicates that bystander signals coming from irradiated cells *induce* chromatin damage which differs from that induced by direct irradiation. It has been shown that not exclusively irradiation of DNA but irradiation of cytoplasm induces cytogenetic damage in both irradiated and bystander glioma cells and fibroblasts to a comparable extent (Shao et al., 2004) The bystander responses were completely eliminated when the populations were treated with nitric oxide scavenger or agent which disrupt membrane rafts. This finding shows that direct DNA damage is not required for induction of important cell-signaling mechanisms after low-dose irradiation and that, the whole cell should be considered a sensor of radiation exposure. The use of compounds that compromise the level of nitrogen oxide abolishes the bystander effect elicited as γH2AX expression. Nitric oxide (NO) seems to be an important signaling molecule transmitted by irradiated cells, which initiates the changes in cells not exposed to radiation (Matsumoto et al., 2001, 2007, Shao et al., 2008a, b). This small molecule is also a free radical which is synthesized from the Larginine with the participation of nitric oxide synthase (NOS). It plays important, often contradictory roles in many biological processes, stimulating either the proliferation or apoptosis, which primarily depends on its concentration (Shao et al., 2008b). Nitric oxide is vasodilatator, neurotransmitter and an immunomodulatory agent, but it may also cause damage to DNA by generating peroxynitrite anion (ONOO-), which may cause oxidation or nitration of DNA (Xu et al., 2002). Shao et al. (2008a, b) demonstrated that radiationgenerated NO induced in glioma cells TGFβ1, the multifunctional transcription factor

hydroxynonenal (4HNE) and other with mutagenic and carcinogenic properties and which can form massive DNA adducts (Marnet, 2000, Zhong et al., 2001). The end-products of lipid peroxidation have secondary signaling molecule properties and can activate a cascade of signals leading to either DNA damage repair, or to damage stabilization or apoptosis (Hu et al., 2006). In our research we found increased MDA concentration in irradiated Me45 human melanoma cells growing in the form of megacolonies, as well as in the neighboring megacolonies growing in the same flask but protected against irradiation with a lead shield (Przybyszewski et al., 2004). At the same time, we found in both the irradiated and shielded megacolonies, decreased glutathione peroxidase (GSH-Pox) and mitochondrial superoxide dismutase (MnSOD), as well as elevated numbers of single- and double-strand DNA breaks (SSBs and DSBs), as assessed by single cell gel electrophoresis. The level of DNA breaks in non-irradiated cells was lower and appeared with several-hour delay compared to that observed in irradiated cells, which may suggest participation of long-lived radicals in the bystander effect induction (Przybyszewski et al., 2004). Time-shifted appearance of DSBs in neighboring cells estimated as the expression of phosphorylated histone H2AX (γH2AX foci) has been observed in the *in vitro* (Hu et al., 2006, Sokolov et al. 2007) as well as in *ex vivo* (Sedelnikova et al., 2007) conditions. While the phosphorylation of histone H2AX at serine 139 is a very early-stage event in cells directly exposed to radiation, the appearance of gamma-H2AX foci in cells co-cultured with irradiated ones, or treated with ICM only, may even take several hours. The gamma-H2AX foci, which indicate the presence of DNA DSBs in cells exposed to the signals transmitted by irradiated cells, co-localize with other proteins involved in the cell cycle control and DNA damage repair, such as ATM, MRE11, NBS1, Rad50 and 53BP1 (Sokolov et al., 2007). It is worth noticing that, based on ATM foci enumeration, Ojima et al. (2009) found that DSBs induced by the radiation-induced bystander effect persist for long periods (over 24 h), whereas DSBs induced by direct radiation effects are repaired relatively quickly. However, ATM foci persisted even longer (48 h) if bystander fibroblasts were co-incubated with very low (1.2 mGy) irradiated counterparts. This indicates that bystander signals coming from irradiated cells *induce* chromatin damage which differs from that induced by direct irradiation. It has been shown that not exclusively irradiation of DNA but irradiation of cytoplasm induces cytogenetic damage in both irradiated and bystander glioma cells and fibroblasts to a comparable extent (Shao et al., 2004) The bystander responses were completely eliminated when the populations were treated with nitric oxide scavenger or agent which disrupt membrane rafts. This finding shows that direct DNA damage is not required for induction of important cell-signaling mechanisms after low-dose irradiation and that, the whole cell should be considered a sensor of radiation exposure. The use of compounds that compromise the level of nitrogen oxide abolishes the bystander effect elicited as γH2AX expression. Nitric oxide (NO) seems to be an important signaling molecule transmitted by irradiated cells, which initiates the changes in cells not exposed to radiation (Matsumoto et al., 2001, 2007, Shao et al., 2008a, b). This small molecule is also a free radical which is synthesized from the Larginine with the participation of nitric oxide synthase (NOS). It plays important, often contradictory roles in many biological processes, stimulating either the proliferation or apoptosis, which primarily depends on its concentration (Shao et al., 2008b). Nitric oxide is vasodilatator, neurotransmitter and an immunomodulatory agent, but it may also cause damage to DNA by generating peroxynitrite anion (ONOO-), which may cause oxidation or nitration of DNA (Xu et al., 2002). Shao et al. (2008a, b) demonstrated that radiationgenerated NO induced in glioma cells TGFβ1, the multifunctional transcription factor

involved in the transcription of proteins engaged in cell proliferation and differentiation, immunomodulation, cell-cycle control and apoptosis (Massague and Chen, 2000). The use of inducible nitric oxide synthase inhibitor, or anti-TGF antibodies which compromise micronuclei in cells directly irradiated with alpha particles and adjacent non-irradiated cells indicates a positive feedback. However, NO role as a mediator of the bystander effect has not been observed in all tested glioma cell lines (Matsumoto et al., 2001). In several types of cancer (colon, lung, throat) expression of inducible nitric oxide synthase (iNOS) was also linked to the *TP53* gene mutation (Lala and Chakraborty, 2001) indicating that the correct protein of p53 gene may negatively regulate the accumulation of iNOS. Many other factors were proposed as the bystander effect mediators, among them interleukin 8 (Narayanan et al., 1999), soluble tumor necrosis factor (TNFα) as well as Fas and TRAIL death ligands (Lucen et al. 2009). Also, multiple pathways are activated that take part in transmitting the bystander effect signals. Those induced in human fibroblasts by alpha particles (0.3-3 cGy) and transmitted through the GJIC or surrounding environment activated in adjacent cells various proteins such as MAP- kinase, NFκB, Raf-1, ERK1/2, JNK, AP-1 and others (Azzam et al. 2002, Lyng et al., 2006). Since application of SOD and catalase neutralizes the resulting oxygen radicals and hydrogen peroxide and hampers the bystander effect (reduction in the level of micronuclei, inhibition of nuclear factor κB and p38 MAPK activation), the mediators of these processes appear to be reactive oxygen and nitrogen species (Azzam et al., 2002). Targeting the nucleus or cytoplasm of HeLa cells by single helium ions induced expression of 53BP1, the protein which marks double-stand breaks in DNA (Tartier et al., 2007). The use of aminoguanidine, an inducible NO synthase inhibitor, or radical scavenger DMSO, cause inhibition of 53BP1 protein expression in both irradiated and co-incubated non-irradiated cells, pointing to the NO and ROS as the mediators of these lesions. At the same time, it was observed that antibiotic *filipin*, which damages the glycosphingolipid microdomains in cellular membrane, inhibited cellular signals from irradiated cells and led to a drastic reduction in the 53BP1 foci in neighboring cells. This reveals that transmission of bystander signals is dependent on the integrity of the cellular membranes, whereas membrane integrity was not necessary to generate the damage in irradiated cells. Also, the presence of mitochondria was necessary to generate bystander signals by irradiated cells, but was not necessary to their reception (Tartier et al., 2007). Calcium ion channels seem to play a role in the transmission of bystander signals. It was observed that biogenic amines, such as serotonin (5-hydroxytryptamine, 5-HT) and dopamine, may be the transducers of signals emitted by irradiated cells. The level of 5-HT neurotransmitter in culture medium decreased after irradiation of cells, likely due to its binding to the receptors which form the calcium channels, and leads to increased level of micronuclei (Poon et al., 2007). These effects were abolished after treatment of cells with calcium channel blockers calcicludin or rezerpin, which are the natural antagonists for serotonin (Poon et al., 2007, Shao et al., 2006). The study of transcript levels using DNA microchips may indicate signaling pathways and genes that are involved in the radiation-induced bystander effect. Gandhi et al. (2008), when examining the overall gene expression (global genome expression), after irradiation of human lung fibroblasts with alpha particles (0.5 Gy and 4-hour co-incubation with nonirradiated cells), observed that the expression of over 300 genes in both groups (hit and nonhit) was changed, and that 165 genes were common to both groups. Among them were genes mainly over-expressed in irradiated cells (CDKN1) and those that were overexpressed equally in irradiated and neighboring cells, namely NFκB–regulated PTGS2 (cyclooxygenase 2), IL8 and BCL2A1. However, Chaudhry (2006) observed that gene

Intercellular Communication in Response to Radiation Induced Stress:

response to bystander effect signals.

**6. Bystander effect can function bi-directionally** 

Bystander Effects in Vitro and in Vivo and Their Possible Clinical Implications 343

protein in the spleen of animals, pointing to the involvement of the TP53 gene in the bystander effect *in vivo* (Koturbash et al., 2008). However, in our own research using HCT116 colon cancer cells lines differing in *TP53* status, and the transwell system of cocultivation, we observed that *TP53 gene* is not required to uncover the bystander effect. Nonirradiated *TP53-*knockout cells (HCT116p53 -/-) were even more sensitive to apoptosis induced by signals sent by irradiated (2 Gy) cells than wild-type cells (HCT116p53+/+) (Widel et al., 2009). In the same experiments we noticed that the level of micronuclei induced in cells co-cultured with non-irradiated ones did not differ between both lines. Recently, He et al (2010) found that the bystander effect after irradiation can be modulated by the p53 status of irradiated hepatoma cells and that a p53-dependent release of cytochrome c may be involved in the RIBE. Following irradiation cytochrome c was released from mitochondria into the cytoplasm only in HepG2 (wild-type p53) cells, but not in PLC/PRF/5 (p53 mutated) or Hep3B (p53-null) cells. Only irradiated HepG2 cells induced bystander effect elicited as micronuclei (MN) formation in the neighboring Chang liver cells. In conclusion, the various criteria for assessing the role of *TP53* gene reveal differences in its

Recent studies have shown an interplay between adjacent irradiated and non-irradiated cell populations. Thus, signals leading to damage in non-irradiated cells, sent by the irradiated ones, are answered by non-hit cells affecting in turn the directly-irradiated ones. Experiments performed on MM576 melanoma cells, the goal of which was to investigate the impact of modulating irradiation fields in a way to resemble the intensity-modulated radiotherapy technique (IMRT) on survival showed, that the mutual communication works in three different manners (Mackonis et al., 2007). The first type of this communication, the classic "bystander effect", occurs when irradiated cells growing in one part of the field damage the adjacent nonirradiated cells growing in another part of the field. The second type of communication, causes an increase in the survival of non-irradiated cells, when they are co-cultured with cells exposed to high doses (6-20 Gy) or even a lethal dose. One of the factors responsible for this process is, according to these authors, the eruption of "death-burst signals", which promotes proliferation of the non-irradiated cells, although the authors do not specify the chemical nature of these signals. The third type of communication causes increased survival of cells that have received a high dose of radiation, through signaling from neighboring cells exposed to low-dose in another part of the field (Mackonis et al., 2007). Also, the irradiation of human fibroblasts with low doses of alpha-particles resulted in an increased proliferation, reduction of the level of p53 and CDKN1 (p21Waf-) proteins and an increase in the level of the CDC2 kinase. The promitogenic effect was associated with an increase in the level of the TGFβ1-induced by reactive oxygen species (Iyer and Lehnert, 2002). Our recent study revealed bystander effect of the third type, similar to that described by the Mackonis, indicating the bilateral signaling of irradiated and non-irradiated cells (Widel et al., 2008, and unpublished). Using the transwell system of coincubated irradiated mouse lung cancer cells (LLC) with non-irradiated fibroblasts (NIH3T3) growing in inserts we studied the mutual interaction of cells in terms of micronuclei and apoptosis induction. The membrane of insert bottom with 0.4 µm pores separates both types of cells but enables free circulation of medium between them. LLC cells growing in 6-well plates were irradiated with doses of 2 and 4 Gy X-rays generated by a therapeutic accelerator (Clinac 600). Immediately after irradiation the inserts with non-irradiated (bystander) fibroblasts were

expression profile differs in irradiated human fibroblasts and in non-irradiated cells treated only with radiation-conditioned medium. In the former, over-expressed were the genes of early response to radiation, while in the bystander cells the over-expressed ones included genes involved in the intercellular communication. In our genome-wide microarray study, we compared transcript profile changes in Me45 human melanoma cells grown in culture medium from irradiated cells with those which occurred after irradiation and we also observed the bystander effect at the genome level (Rzeszowska-Wolny et al., 2009). Using the criterion of a greater than ±10% change, transcripts of >10,000 genes were shown to be expressed at increased or decreased levels under both conditions, and almost 90% of these were common to ICM-treated and X-rays-treated cells. Among them were genes involved in the neuronal receptor-ligand interactions, oxidative phosphorylation, cytokine–cytokine receptor interactions, proteasomes, ribosomes and cell cycle regulation. All these tests indicate a very complex mechanism of cell response to both ionizing radiation and for signals transmitted by them to communicate with the neighboring cells.

#### **5. The role of the p53 protein in the response to bystander signals**

The *TP53* gene is a tumor suppressor gene which participates in the regulation of cell cycle and apoptosis. Its main role is to prevent the transmission of genetic disorders in cells to daughter cells by extending G1 phase, which allows the cell to repair DNA damage induced by various egzo-and endogenous agents, mainly the oxidative stress. When the damage is too bulky or the repair is ineffective, *TP53* initiates apoptosis through its own product, p53 protein, which is a transcription factor for multiple genes involved in DNA repair, regulation of cell cycle and apoptosis (Chipuk and Green, 2006, Tlsty, 2002). The role of p53 protein in the bystander effect is debatable, however. Research carried out using human fibroblasts cell lines, where only a small fraction of cells was exposed to alpha particles has shown a significant increase in p53, as well as p21Waf1 protein, not only in the targeted, but also in the non-targeted cells (Azzam et al., 1998). The effect disappears after inhibition of the gap junction intercellular communication. Similarly, expression of p53 protein was observed in the rat lung epithelial cells adjacent to alpha particle-targeted cells (Hickman et al, 1994). However, survival of clonogenic fibroblasts after 2 and 4 Gy was increased when they were exposed to the medium from fibroblasts gamma-irradiated with a dose of just 1cGy. This was accompanied by the reduction of p53 protein level in addition to the increase in intracellular pool of reactive oxygen radicals and DNA-repair protein nuclease APE (Iyer and Lehnert, 2000). The appearance of DSBs is accompanied by DNA binding protein 53BP1 which may be detected immunochemically using fluorescent-labeled antibodies. It was shown that the irradiation of cell cytoplasm with single alpha particles, induced increased numbers of 53BP1 foci not only in nuclei of irradiated cell, but also in adjacent to them nonirradiated cells (Tartier et al., 2007). The use of inhibitors targeting reactive oxygen radicals and nitric oxide prevented the formation of DNA breaks in irradiated and adjacent cells. This indicates that the bystander effect signals are transmitted not only between cells but even between cell compartments. Also, the use of membrane specific antibiotic (*filipin*) to disrupt membrane-dependent signaling has resulted in lowering the number of clusters of 53BP1 foci an important sensors of DNA double strand breaks, in cells co-incubated with irradiated ones, indicating that reception of bystander effect signaling molecules requires the integrity of the cellular membranes (Tartier et al., 2007). The tests in rats which were given 1Gy doses of X-rays, both whole-body or head-area-only, revealed expression of p53

expression profile differs in irradiated human fibroblasts and in non-irradiated cells treated only with radiation-conditioned medium. In the former, over-expressed were the genes of early response to radiation, while in the bystander cells the over-expressed ones included genes involved in the intercellular communication. In our genome-wide microarray study, we compared transcript profile changes in Me45 human melanoma cells grown in culture medium from irradiated cells with those which occurred after irradiation and we also observed the bystander effect at the genome level (Rzeszowska-Wolny et al., 2009). Using the criterion of a greater than ±10% change, transcripts of >10,000 genes were shown to be expressed at increased or decreased levels under both conditions, and almost 90% of these were common to ICM-treated and X-rays-treated cells. Among them were genes involved in the neuronal receptor-ligand interactions, oxidative phosphorylation, cytokine–cytokine receptor interactions, proteasomes, ribosomes and cell cycle regulation. All these tests indicate a very complex mechanism of cell response to both ionizing radiation and for

signals transmitted by them to communicate with the neighboring cells.

**5. The role of the p53 protein in the response to bystander signals** 

The *TP53* gene is a tumor suppressor gene which participates in the regulation of cell cycle and apoptosis. Its main role is to prevent the transmission of genetic disorders in cells to daughter cells by extending G1 phase, which allows the cell to repair DNA damage induced by various egzo-and endogenous agents, mainly the oxidative stress. When the damage is too bulky or the repair is ineffective, *TP53* initiates apoptosis through its own product, p53 protein, which is a transcription factor for multiple genes involved in DNA repair, regulation of cell cycle and apoptosis (Chipuk and Green, 2006, Tlsty, 2002). The role of p53 protein in the bystander effect is debatable, however. Research carried out using human fibroblasts cell lines, where only a small fraction of cells was exposed to alpha particles has shown a significant increase in p53, as well as p21Waf1 protein, not only in the targeted, but also in the non-targeted cells (Azzam et al., 1998). The effect disappears after inhibition of the gap junction intercellular communication. Similarly, expression of p53 protein was observed in the rat lung epithelial cells adjacent to alpha particle-targeted cells (Hickman et al, 1994). However, survival of clonogenic fibroblasts after 2 and 4 Gy was increased when they were exposed to the medium from fibroblasts gamma-irradiated with a dose of just 1cGy. This was accompanied by the reduction of p53 protein level in addition to the increase in intracellular pool of reactive oxygen radicals and DNA-repair protein nuclease APE (Iyer and Lehnert, 2000). The appearance of DSBs is accompanied by DNA binding protein 53BP1 which may be detected immunochemically using fluorescent-labeled antibodies. It was shown that the irradiation of cell cytoplasm with single alpha particles, induced increased numbers of 53BP1 foci not only in nuclei of irradiated cell, but also in adjacent to them nonirradiated cells (Tartier et al., 2007). The use of inhibitors targeting reactive oxygen radicals and nitric oxide prevented the formation of DNA breaks in irradiated and adjacent cells. This indicates that the bystander effect signals are transmitted not only between cells but even between cell compartments. Also, the use of membrane specific antibiotic (*filipin*) to disrupt membrane-dependent signaling has resulted in lowering the number of clusters of 53BP1 foci an important sensors of DNA double strand breaks, in cells co-incubated with irradiated ones, indicating that reception of bystander effect signaling molecules requires the integrity of the cellular membranes (Tartier et al., 2007). The tests in rats which were given 1Gy doses of X-rays, both whole-body or head-area-only, revealed expression of p53 protein in the spleen of animals, pointing to the involvement of the TP53 gene in the bystander effect *in vivo* (Koturbash et al., 2008). However, in our own research using HCT116 colon cancer cells lines differing in *TP53* status, and the transwell system of cocultivation, we observed that *TP53 gene* is not required to uncover the bystander effect. Nonirradiated *TP53-*knockout cells (HCT116p53 -/-) were even more sensitive to apoptosis induced by signals sent by irradiated (2 Gy) cells than wild-type cells (HCT116p53+/+) (Widel et al., 2009). In the same experiments we noticed that the level of micronuclei induced in cells co-cultured with non-irradiated ones did not differ between both lines. Recently, He et al (2010) found that the bystander effect after irradiation can be modulated by the p53 status of irradiated hepatoma cells and that a p53-dependent release of cytochrome c may be involved in the RIBE. Following irradiation cytochrome c was released from mitochondria into the cytoplasm only in HepG2 (wild-type p53) cells, but not in PLC/PRF/5 (p53 mutated) or Hep3B (p53-null) cells. Only irradiated HepG2 cells induced bystander effect elicited as micronuclei (MN) formation in the neighboring Chang liver cells. In conclusion, the various criteria for assessing the role of *TP53* gene reveal differences in its response to bystander effect signals.
